CN114258915B - Application of 9-phenanthrene in the control of rice blast - Google Patents

Abstract

The invention discloses the application of 9-phenanthrol in the prevention and treatment of rice blast. Experiments show that the compound can effectively inhibit the mycelial growth and spore germination of Magnaporthe grisea, can interfere with the formation of appresses of conidia, has obvious inhibitory effect on rice blast, and inhibits the pathogenicity of Magnaporthe grisea, so in rice It has good application value in the prevention and control of plague and drug development.

Description

Application of 9-phenanthrene in the control of rice blast

technical field

The invention belongs to the field of biotechnology, and in particular relates to the application of 9-phenanthrene in the prevention and treatment of rice blast.

Background technique

Rice (Oryza sativa L.) is one of the most important food crops and plays a very important role in food production. Nearly 50% of the world's population uses it as a staple food. Its safe production is crucial to ensuring social stability and economic development . Like other crops, rice will inevitably be attacked by a series of pathogenic bacteria during its growth and development, which seriously affects the yield and quality of rice. One of the serious diseases is a major problem in rice production in the world. It can reduce rice production by 10% to 30% every year. This disease can occur in all rice planting areas and in different growth stages of rice. It has the characteristics of wide distribution and great harm. This has led to a serious reduction in rice production, threatening the safety of food production in my country and the world. Therefore, research on the control of blast fungus has important economic and social benefits.

The infection process of rice blast fungus mainly includes: (1) conidia spread with wind and rain and adhere to the surface of rice leaves; (2) conidia germinate and form germ tubes; (3) germ tubes differentiate to form attached (4) appressorium differentiates to form infection nails; (5) infection nails penetrate the host cells, form infection hyphae in the host cells, and spread between cells. Magnaporthe oryzae mainly infects the above-ground tissue of rice with conidia. When the conidia contact the epidermis of the host, they germinate to form germination tubes, which then differentiate specifically to produce appresses, which form infection nails, penetrate the host epidermis and persist. Enter the host cell, causing the host plant to appear symptoms in about 5-7d. Among them, the key process for the successful infection of rice by Magnaporthe oryzae is the formation of a highly specialized infection structure - appressorium. The turgor pressure produced by the mature appressorium can make the infection pin penetrate the rice cuticle, thus successfully infecting rice cells. When M. oryzae cannot form complete appressoria, its pathogenicity is significantly weakened.

At present, the main methods of controlling rice blast are breeding resistant varieties, chemical control and field management. Most of the resistant varieties are vertically resistant varieties based on the "gene-to-gene" theory, and these varieties are prone to lose disease resistance. Chemical control has the advantages of economy, high efficiency, convenience, and rapidity, and has always been an indispensable part of the comprehensive management system of rice blast. The control of rice blast is a hot topic in global research. Chemical control occupies an important position in the integrated pest control, which has the characteristics of quick results, simple methods, strong first aid, and is not subject to regional and seasonal restrictions.

9-Phenanthrol (9-Phenanthrol, CAS No. 484-17-3) is a fused-ring aromatic hydrocarbon compound, a red solid, which is often used as a non-selective cation channel activated by Ca ²⁺ in clinic (transient receptor cation channel subfamily M member4, TRPM4) inhibitor, for the treatment of symptoms such as myocardial ischemia. At present, the application of 9-phenanthrene in agricultural production has not been reported.

Contents of the invention

In view of the above technical problems, the present invention provides the application of 9-phenanthrene in the prevention and treatment of rice blast.

The invention provides the use of 9-phenanthrene in the preparation of medicines for preventing and controlling blast fungus.

Further, the use of the 9-phenanthreneol in the preparation of drugs for inhibiting the mycelial growth and spore germination of Magnaporthe grisea.

Further, the use of the 9-phenanthreneol in the preparation of a drug that interferes with the formation of appressoria of Magnaporthe grisea.

Preferably, the concentration of said 9-phenanthrenol is 5ug/m-30ug/m.

The present invention also protects the method of using 9-phenanthreneol to prevent and control rice blast, comprising the following steps: dissolving 9-phenanthreneol in dimethyl sulfoxide, configuring it into a mother liquor of 5000ug/ml, and absorbing 9-phenanthreneol with a pipette Add the phenol mother solution to the PDA medium that has been cooled to 55°C, adjust the concentration of 9-phenanthrenol to 5ug/ml-30ug/ml; then spray the spore suspension evenly on the rice leaves, so that the leaves are covered with droplets.

Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention found that 9-phenanthrene phenol has an inhibitory effect on the growth of blast fungus, can interfere with the formation of appresses of conidia, and significantly delay the formation of appresses of spores. It has obvious inhibitory effect on rice blast, so it has good application value in the prevention and control of rice blast and drug development.

Description of drawings

Fig. 1 is the influence of different concentrations of 9-phenanthrene on the growth of blast fungus mycelia;

Fig. 2 is the inhibition rate figure of different concentrations of 9-phenanthrol to different blast fungus bacterial strains;

Fig. 3 is the influence of different concentrations of 9-phenanthrene on the germination of blast fungus conidia;

Fig. 4 is the influence of different concentrations of 9-phenanthrene on the formation of blastocyst oryzae appressoria;

Fig. 5 is the impact of different concentrations of 9-phenanthrene on the pathogenicity of blast fungus;

Figure 6 is the effect of different concentrations of 9-phenanthrene on the biomass of Magnaporthe grisea.

detailed description

Example 1 Effect of 9-phenanthrene on the growth of blast fungus mycelia

A method utilizing 9-phenanthrene to suppress the growth of blast fungus mycelia, comprising the following steps:

S1: Dissolve 9-phenanthreneol in dimethyl sulfoxide, make it into a mother solution (5000ug/ml), add it into PDA medium so that the final concentrations of 9-phenanthreneol are 1.25μg/ml, 2.5μg/ml , 5 μg/ml, 10 μg/ml and 15 μg/ml, the temperature of the PDA medium was controlled at 55°C; meanwhile, the PDA medium without adding 9-phenanthrol was set as a blank control group.

The preparation method of above-mentioned PDA culture medium:

(1) Take weighing paper and weigh 10g of sucrose and 15g of agar powder in the balance;

(2) Peel and clean the potatoes, weigh 200g of the peeled potatoes and cut them into cubes of about 1cm with a knife on a clean gauze;

(3) Measure 1000mL of distilled water, add the distilled water and 200g of peeled potato pieces into the pot together, cook for 20-30min after the water boils, then filter the potato residue with 4 layers of clean gauze in a 1L beaker;

(4) Add 10g of sucrose to the filtered liquid, stir evenly with a glass rod and pour the filtrate into the pot, add 15g of agar powder while stirring, pay attention to rapid stirring to avoid agar agglomeration from sticking to the bottom of the pot;

(5) When the liquid is boiled to transparent, stop heating and pour it into a 1L beaker, add distilled water to adjust the volume to 1000mL, the natural pH, and divide it into Erlenmeyer flasks, which should not exceed one-third of the volume of the Erlenmeyer flasks. Sterilize in a high-pressure steam sterilizer at 121° C. for 20 minutes to obtain a PDA medium.

The specific method of adding 9-phenanthreneol to the PDA medium: use a pipette to absorb 9-phenanthreneol and add it to the PDA medium that has been cooled to 55°C, so that the concentration of 9-phenanthreneol in the final medium is 1.25 μg/ml, 2.5μg/ml, 5μg/ml, 10μg/ml and 15μg/ml, mix the culture medium and pour it into a sterile petri dish, try to avoid bubbles in the process of pouring the plate to affect the experimental results, and get different 9 - PDA medium with phenanthrol concentration.

S2: Cultivate the test bacterium Magnaporthe grisea;

The concrete method of above-mentioned cultivation test fungus blast fungus is as follows

2.1 In the ultra-clean workbench where bacteria are sterilized, light the alcohol lamp, and cool the PDA medium sterilized by high pressure steam to about 55°C;

2.2 Pour about 22mL of culture medium into each dish with a diameter of 90mm, and try to ensure that there are no air bubbles during the pouring process;

2.3 After the culture medium is completely solidified, take out the filter paper sheet of the blast fungus for testing from the refrigerator;

2.4 Put the tweezers on the burner outside the alcohol lamp. After the tweezers cool down, use the tweezers to pick up the filter paper with the blast fungus and put it on the PDA medium;

2.5 Finally, use a parafilm to seal the culture dish, and place it upside down in a constant temperature incubator at 28°C to cultivate the strains to be tested.

S3: After 7 days of culture for the test bacterium Magnaporthe grisea, use a sterile puncher to cut the bacterial block on the test bacterium Magnaporthe grisea, the diameter of the bacterial block is 6mm, put it into the PDA medium containing 9-phenanthrol and the blank control In PDA medium;

S4: The PDA medium containing 9-phenanthrene phenol and the blank control PDA medium put into the bacterial block were cultured in a constant temperature incubator, the temperature of the constant temperature incubator was 28° C., and the culture time was 7 days. The colony diameters were measured with a vernier caliper, and the mycelium growth rate and inhibition rate of the experimental group and the blank control group were calculated.

Calculated as follows

Inhibition rate=(control strain growth diameter-0.7)-(treatment strain growth diameter-0.7))/(treatment strain growth diameter-0.7) (Note: 0.7 represents the diameter of the inoculated bacteria block)

The experimental group prepared different concentrations of 9-phenanthrol PDA medium, respectively 1.25ug/ml, 2.5ug/ml, 5ug/ml, 10ug/ml and 15ug/ml, respectively, inoculated the blast fungus bacteria blocks, and counted the colony diameter after 7 days Calculate the inhibitory rate of 9-phenanthrenol to inhibit the rice blast bacterium, and the results are as shown in Figure 1:

Results: By inoculating 7 different strains of Magnaporthe oryzae in the culture medium containing 9-phenanthrene, the results showed that different concentrations of 9-phenanthrene had an effect on the mycelial growth of Magnaporthe oryzae, and the inhibition rate increased with the increase of the concentration. Significantly increased (Figure 1, Figure 2).

Example 2 Effect of 9-phenanthreneol on spore formation and appressoria formation of Magnaporthe grisea

Implementation steps:

S1 Put the 9-phenanthrene-containing PDA medium and the blank control PDA medium that were put into the bacterial block into a constant temperature incubator and cultivate them for 7 days, then use a sterile puncher to cut the bacterial block on the blast fungus, the diameter of the bacterial block 6mm, pick three bacterial blocks and place them in the PDB medium,

The preparation method of described PDB medium:

(1) Take weighing paper and weigh 10g of sucrose in the balance;

(2) Peel and clean the potatoes, weigh 200g of the peeled potatoes and cut them into cubes of about 1cm with a knife on a clean gauze;

(3) Measure 1000mL of distilled water, add the distilled water and 200g of peeled potato pieces into the pot together, cook for 20-30min after the water boils, then filter the potato residue with 4 layers of clean gauze in a 1L beaker;

(4) Add 10g of sucrose to the filtered liquid, stir it evenly with a glass rod, pour it into a 1L beaker, add distilled water to adjust the volume to 1000mL, the natural pH, and divide into conical flasks, not exceeding the conical flask One-third of the volume, and then sterilized in a high-pressure steam sterilizer at 121°C for 20 minutes to obtain PDB medium.

S2: Put the bacterial mass into the PDB medium and place it at 120rpm. After cultivating on a shaker at 28°C for 4-5 days, pipette 400 μL of mycelium suspension into the oat medium to make the bacterial liquid evenly distributed. Place it in an incubator at 28°C for about 6 days, use a sterile cotton swab to scrape the mycelia, then place it in an incubator with light and dark alternately (12h light, 12h dark) and cultivate it for about 5d, wash the spores with 0.1% Tween water, configure conidia suspension, and adjust the spore concentration to 10 ⁵ /mL; 9-phenanthrol mother liquor was added to the conidia suspension to a final concentration of 1 μg/ml, 10 μg/ml and 20 μg/ml, mixed with 9- The conidia suspension of phenanthrene was dropped on the hydrophobic paddle, and after 12 hours of dark moisturizing at 28°C, the formation of appressoria was observed under a microscope;

S3: Make water agar medium, put the cellophane on the water agar; wash the spores cultured in the oat medium with sterile water, filter, spread evenly on the cellophane and mark it, at 2h, 4h, 6h, 8h The spore germination and appressorium formation were observed respectively. 50 spores were observed each time, for a total of 3 observations.

The preparation method of described oat culture medium:

(1) Take weighing paper and weigh 40g of oats and 15g of agar powder in the balance;

(2) Add an appropriate amount of water to the weighed oats and soak for about 10 minutes, and break them evenly with a wall breaking machine;

(3) Pour the evenly crushed oats into a 1L beaker;

(4) Add 15 grams of agar powder to a 1L beaker filled with oatmeal, add distilled water to make the volume to 1000mL, the natural pH, and divide it into blue cap bottles, which cannot exceed one-third of the volume of the blue cap bottles. Sterilize in a high-pressure steam sterilizer at 121° C. for 30 minutes to obtain oat culture medium.

Result: The results of spore germination and appressorium formation are shown in Figure 3 and Figure 4: After adding different concentrations of 9-phenanthrene to the conidia suspension, the results show that: 10ug/ml 9-phenanthrene phenol has no effect on the conidia of Magnaporthe oryzae Germination and appressorium formation have inhibitory effects, and at 20ug/ml, conidia germination and appressorial formation are significantly inhibited (as shown in Figure 3 and Figure 4).

Example 3 Effect of 9-phenanthrene on the pathogenicity of blast fungus

The experimental steps are as follows:

Add the 9-phenanthrene phenol mother solution to the conidia suspension of Magnaporthe oryzae to a final concentration of 10ug/ml and 30ug/ml, and spray the spore suspension evenly on the rice leaves so that the leaves are covered with droplets, and keep in the dark at 28°C Moisturize for 24 hours, then move to the greenhouse, investigate the blast fungus disease 7 days after inoculation, and count the mycelial biomass by fluorescence quantitative method, the specific formula is 2 ^C T ^(Os -UBQ) - ^C _T ^(Mo -Pot2).

Result: Rice was inoculated with the conidia suspension containing 9-phenanthrol, and the incidence of rice was observed 7 days after inoculation. The upper lesions were significantly reduced. After inoculating the conidia suspension treated with 30ug/ml 9-phenanthrol, only sporadic small lesions could be seen on the rice leaves (Fig. 5). By counting the mycelial biomass at the lesion, it was found that the mycelial biomass of the lesion formed by the spore suspension treated with different concentrations of 9-phenanthrol decreased significantly (Fig. 6). It shows that 9-phenanthrene phenol reduces the incidence of rice blast by inhibiting conidia germination and appressoria formation.

Claims (4)

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, characterized in that: the concentration of the 9-phenanthrol is 5ug/ml to 30ug/ml.

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