WO2022088912A1 - Compound for controlling plant pathogenic bacteria and use thereof - Google Patents

Abstract

A compound for controlling plant pathogenic bacteria and the use thereof. The compound is 4-allylcatechol. 4-Allylcatechol has inhibitory effects on Xanthomonas oryzae pv.oryzae, Xanthomonas oryzaepv.oryzicola, Xanthomonas citri subsp.citri, Acidovorax citrulli, Ralstonia solanacearum, Xanthomonas fragariae, Xanthomonas campestris pv.mangiferaeindicae, Xanthomonas campestris pv.campestris, and Pectobacterium carotovorum subsp.brasiliense when the test concentration is 1000 μmol/L.

Description

A compound for controlling phytopathogenic bacteria and its application technical field

The invention relates to the technical field of plant disease prevention and control, in particular to a compound for controlling plant pathogenic bacteria and its application.

Background technique

South China pepper (Piper austrosinense) is a woody climbing vine of the Piperaceae family, mainly distributed in Guangxi, Guangdong, Hainan and coastal islands. According to "Chinese Materia Medica", Huanan pepper has the effect of reducing swelling and analgesia, and mainly treats toothache and bruises. 4-allyl catechol is a component with good bactericidal activity isolated from the piperaceae plant Huanan pepper by the biological activity tracking method, and its chemical structural formula is shown in formula I.

After searching, it was found that protocatechualdehyde, the active ingredient of South China pepper, has been used in medicine for antibacterial purposes. At present, there is no report on the agricultural activity of 4-allyl catechol at home and abroad, nor has 4-allyl catechin been reported. A report on the inhibitory effect of phenols on phytopathogens.

SUMMARY OF THE INVENTION

In view of the deficiencies of the prior art, the present invention provides a compound for controlling phytopathogenic bacteria and its application.

The scheme of the present invention includes the following aspects:

The present invention provides a compound for preventing and treating phytopathogenic bacteria, the compound is 4-allyl catechol, and the compound structural formula is shown in formula I:

In one aspect, the present invention provides the application of 4-allylcatechol in preventing and treating bacterial diseases of plants. Preferably, the disease is a plant bacterial disease. More preferably, the pathogenic bacteria of the disease are: rice bacterial blight Xanthomonas oryzae pv.oryzae, rice bacterial leaf spot Xanthomonas oryzae pv.oryzicola, citrus canker Xanthomonas citri subsp.citri, melon bacterial fruit leaf spot Acidovorax citrulli, Ralstonia solanacearum, Xanthomonas fragariae, Xanthomonas campestris pv.campestris, Pectobacterium carotovorum subsp.carotovorum, Pectobacterium carotovorum subsp.brasiliense and/or Xanthomonas campestris pv.mangiferaeindicae.

The results of in vivo tests showed that 4-allyl catechol had a good inhibitory effect on the pathogenicity of potato soft rot, and also had a good control effect on mango bacterial angular spot and melon bacterial fruit spot. .

On the other hand, the present invention also provides a fungicide or bacteriostatic agent for preventing and treating plant diseases, the active ingredient of which is 4-allyl catechol, optionally supplemented with an adjuvant.

For the above preparations, according to actual needs, conventional methods in the art can be used to prepare suspensions, aqueous emulsions, microemulsions, emulsifiable concentrates, wettable powders or water-dispersible granules.

The present invention also further provides a method for extracting 4-allyl catechol from the branches and leaves of South China pepper, the method comprising the following steps:

S1: take the branches and leaves of South China pepper, dry them in the shade, pulverize and sieve to obtain dry powder, use ethanol solution for the first cold dipping, and filter to obtain the first filtrate and filter residue; The second filtrate is obtained, the first filtrate and the second filtrate are combined, and concentrated to dryness to obtain the ethanol extract of Chinese pepper;

S2: add water to suspend the ethanol extract of P. chinensis, extract with petroleum ether, and concentrate to dryness to obtain the petroleum ether extract;

S3: the petroleum ether extract is loaded onto the column by the petroleum ether wet method, and 9 fractions are obtained by elution in the petroleum ether-ethyl acetate system;

S4: Fraction 3 was eluted with petroleum ether-ethyl acetate system to obtain 4-allylcatechol.

Preferably, in step S1, the volume concentration of the ethanol solution is 90%-95%; the time of the first cold soaking is 6-7d; the mass ratio of dry powder to ethanol solution is 1:9-10; filter residue and ethanol The mass ratio of the solution is 1:(9～10).

Preferably, in step S3, the volume ratio of petroleum ether-ethyl acetate is (2.5～3):1; in step S4, the volume ratio of petroleum ether-ethyl acetate is (9～10):1.

The beneficial effects obtained by the present invention:

Experiments have proved that the 4-allyl catechol provided by the present invention has a significant inhibitory effect on a variety of important crop diseases, especially several vegetable and tropical fruit diseases at a concentration of 1000 μmol/L, and has a significant inhibitory effect on rice bacterial blight. Bacteria, rice bacterial leaf spot, citrus canker, melon bacterial fruit spot, tomato bacterial wilt, strawberry bacterial keratosis, mango bacterial keratosis, cabbage black rot, potato soft rot Good bacteriostatic effect, the inhibition rate is above 90%, and has a certain bacteriostatic effect on cabbage soft rot pathogen, and the inhibition rate is 53.80%. The results of in vivo tests showed that the compound had a good inhibitory effect on the pathogenicity of potato soft rot fungus, and also had a good control effect on mango bacterial angular leaf spot and melon bacterial fruit leaf spot.

In a word, the compound provided by the present invention has a significant inhibitory effect on plant pathogenic bacteria, and provides a natural source bactericidal active substance for the prevention and control of agricultural diseases; the compound can be used for the prevention and treatment of bacterial blight of rice and bacterial black spot of mango, etc. Important agricultural diseases; it is used as a fungicide or bacteriostatic agent, and its characteristics of high efficiency, low toxicity and safety meet the requirements of current new pesticide creation.

Detailed ways

In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

The experimental methods used in the embodiments of the present invention are conventional methods unless otherwise specified.

Materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources unless otherwise specified.

Example 1 Preparation and identification of 4-allyl catechol

The preparation method of 4-allyl catechol, comprises the following steps:

S1: After taking the branches and leaves of South China pepper and drying in the shade, pulverize and pass through a 40 mesh sieve, obtain 2kg of dry powder, use the volume concentration 95% ethanol solution (the mass ratio of dry powder and 95% ethanol is 1:10) after the first cold soak for 7d, filter, The first filtrate and filter residue are obtained; the filter residue is continuously extracted by cold soaking for the second time with a volume concentration of 95% ethanol solution (the mass ratio of filter residue and 95% ethanol is 1:10), and the extraction is carried out for 3 to 5 times until the filtrate is colorless, and For the second filtrate, the first filtrate and the second filtrate were combined, and the filtrate was concentrated to dryness in a rotary thin-film evaporator under reduced pressure to obtain 302.14 g of an ethanolic extract of P. chinensis.

S2: Suspend the ethanol extract of P. chinensis with water, extract with petroleum ether and ethyl acetate in turn, and concentrate to dryness under reduced pressure to obtain 103.85 g of petroleum ether extract and 128.29 g of ethyl acetate extract, respectively.

S3: apply the petroleum ether extract to the column (silica gel column) by the petroleum ether wet method, elute in the petroleum ether-ethyl acetate (volume ratio 2.5:1) system, and combine similar fractions by TLC detection, and obtain according to the polarity. 9 fractions.

S4: wherein fraction 3 was eluted with petroleum ether-ethyl acetate (volume ratio 10:1) system to obtain compound 1 (128 mg).

The chemical structure of compound 1 was confirmed by MS, ¹ H NMR and ¹³ C NMR and was identified as 4-allylcatechol, and its spectral data were:

ESI-MS m/z: 173[M+Na] ⁺ ,149[MH] ^- ;

¹ H-NMR (500 MHz, CDCl ₃ ) δ _H : 6.75 (lH, d, J=7.8 Hz, H-5), 6.67 (IH, s, H-2), 6.57 (lH, d, J=7.8 Hz ,H-6),5.88(1H,m,H-8),5.02(2H,m,H-9),3.20(2H,d,J=6.4Hz,H-7);

¹³ C-NMR (125MHz, CDCl ₃ ) δ _C : 133.4 (C-1), 115.9 (C-2), 143.6 (C-3), 141.8 (C-4), 116.2 (C-5), 121.2 ( C-6), 39.5 (C-7), 137.8 (C-8), 115.7 (C-9).

The above data are consistent with those reported in the literature (Yoshizawa Y, Kawaii S, Kanauchi M, et al. Chavicol and related compounds as nematocides[J]. Bioscience, biotechnology, and biochemistry, 1993, 57(9): 1572-1574.), so Identified as 4-allyl catechol with a purity of >95%.

The above-mentioned 4-allylcatechol can also be obtained by chemical synthesis. Compound 1 is a brown clot (fragment), m.p.: 42-46°C, soluble in organic solvents such as acetone.

Example 2

The difference between this example and Example 1 is:

In step S1, the volume concentration of the ethanol solution is 90%; the time of the first cold soaking is 6d; the mass ratio of the dry powder to the ethanol solution is 1:9; the mass ratio of the filter residue to the ethanol solution is 1:9.

In step S3, the volume ratio of petroleum ether-ethyl acetate is 3:1;

In step S4, the volume ratio of petroleum ether-ethyl acetate is 9:1.

The experimental results of this example are the same as those of Example 1.

Example 3 Determination of the inhibitory activity of 4-allylcatechol against phytopathogenic bacteria

In the research on the agricultural bactericidal active substances of South China pepper, the inventor found that 4-allyl catechol has a preventive effect on plant pathogens by using the activity tracking method. The following experiments were taken as an example to verify the effect of 4-allylcatechol in controlling plant diseases.

1. Test strains

Xanthomonas oryzae, Xanthomonas oryzae, Xanthomonas citri subsp.citri, Acidovorax citrulli, Ralstonia solanacearum ), strawberry bacterial angular spot (Xanthomonas fragariae), cabbage black rot (Xanthomonas campestris), cabbage soft rot (Pectobacterium carotovorum subsp.carotovorum), potato soft rot (Pectobacterium carotovorum subsp.brasiliense), mango bacterial horn Spot fungus (Xanthomonas compestris pv. mangiferaeindicae). The above strains were provided by the Chemical Biology and Application Research Group of Pesticide, Institute of Environment and Plant Protection, Chinese Academy of Tropical Agricultural Sciences.

2. Test method

2.1 In vitro activity detection

In order to evaluate the agricultural bactericidal activity of 4-allyl catechol, the present invention adopts the 96-well plate method to determine the minimum inhibitory concentration and sub-inhibitory concentration of the compound on plant pathogenic bacteria to evaluate the inhibitory effect of the compound of the present invention on plant pathogenic bacteria. bacterial activity. The method refers to the method of Shi Chao (Shi Chao et al. Inhibitory effect and mechanism of protocatechualdehyde on Enterobacter sakazakii [J]. Modern Food Science and Technology, 2017, 33(07): 105-111, 62.) , add the prepared bacterial suspension to a 96-well microtiter plate, 10 μL per well. The sample control group was added with 10 μL of bacterial suspension and 200 μL of liquid medium (control group), and the blank control group was added with 200 μL of liquid medium. Mix the pharmaceutical solution and the bacterial suspension by blowing and beating, and place the sample in an incubator to vibrate (500 r/min) for cultivation. The inhibitory effect of the compound against pathogens was calculated by the following formula.

Bacteriostatic rate (%)={(OD ₆₀₀ of control group—OD ₆₀₀ of blank control group)—(OD ₆₀₀ of test group—OD ₆₀₀ of compound solution control)}/(OD ₆₀₀ of control group—OD ₆₀₀ of blank control group)×100%

data analysis:

The results of the antibacterial activity of 4-allyl catechol against the tested 10 phytopathogenic bacteria are shown in Table 1. Leaf blight, Rice bacterial leaf spot, Citrus canker, Melon bacterial fruit spot, Tomato bacterial wilt, Strawberry bacterial keratosis, Mango bacterial keratosis, Cabbage black rot, Potato soft rot It has a good bacteriostatic effect, and the inhibition rate is above 90%.

Table 1 Antibacterial activity of 4-allylcatechol against 10 phytopathogenic bacteria

Test strain	Inhibition rate(%)	Test strain	Inhibition rate(%)
Bacterial blight of rice	95.06	cabbage black rot fungus	98.63
Bacterial leaf spot oryzae	99.24	Soft rot fungus of cabbage	53.80
Citrus canker	99.03	Potato soft rot fungus	90.97
Bacterial fruit spot of melon	97.71	Mango bacterial keratosis	99.58
Tomato R. solanacearum	97.56	Strawberry bacterial keratosis	97.39

According to the aforementioned screening results, the minimum inhibitory concentration (MIC) and sub-inhibitory concentration (SIC) of 4-allyl catechol against 8 pathogens including B. oryzae (shown in Table 2) were determined. The results showed that , 4-allylcatechol had better activity against B. oryzae, its MIC was 340μmol/L, SIC was 100μmol/L; the sub-inhibitory concentration against potato soft rot was 6μmol/L.

Table 2 Minimum inhibitory concentration and sub-inhibitory concentration of 4-allylcatechol against 8 pathogens including B. oryzae

Test bacteria	MIC(μmol/L)	SIC(μmol/L)
Bacterial blight of rice	340	100
Bacterial leaf spot oryzae	666	260

Citrus canker	650	12.5
cabbage black rot fungus	800	100
Soft rot fungus of cabbage	1400	100
Potato soft rot fungus	1000	6
Mango bacterial keratosis	666	41.63
Strawberry bacterial keratosis	1000	80

2.2 Tissue test

The test of 4-allyl catechol on potato bacterial soft rot refers to the method of Habibeh et al. coatingsin storage[J].Postharvest Biology and Technology, 2019, 156.) The specific operation is as follows: purchase healthy and fresh untreated potatoes of uniform shape and size, wash and dry them, weigh them, and then soak them in 1% sodium hypochlorite for 30 minutes. Then rinse with water and air-dry for later use; dilute 4-allyl catechol with acetone to a sub-inhibitory concentration, mix the potato soft rot fungus with the agent for testing, use a pipette tip to poke holes on the potato, and then The bacterial solution with drug was added, and each treatment was repeated 3 times. Kasugamycin was set as drug control and clear water control was set, and the treated potatoes were stored in a wet box. After 48 hours, the rotten parts were excavated and weighed to calculate the inhibition rate.

Inhibition rate (%)=(control decay weight-treatment decay weight)/control decay weight×100%.

The results of potato biopsy showed that 4-allyl catechol showed a good inhibitory effect on the pathogenicity of potato soft rot fungus at the treatment concentration of 6 μmol/L, and the inhibition rate reached 82.79%. Compared with the control drug kasugamycin, there is no significant difference.

Table 3 The effect of 4-allylcatechol on the pathogenicity of potato soft rot at sub-inhibitory concentrations

Test sample	Liquid concentration (μmol/L)	Rotten weight (g)	Inhibition rate(%)
4-allylcatechol	6	5.02±3.87b	82.79
4% Kasugamycin	15	3.60±0.85b	87.66
Clear water control	-	29.17±7.12a	-

2.3 Pot experiment

2.3.1 Pot experiment on melon bacterial fruit spot

Dilute 2 mL of 4-ene-propyl catechol with different concentrations (1000 μmol/L, 500 μmol/L and 250 μmol/L), 4% kasugamycin aqueous solution (converted to active ingredient concentration of 500 μmol/L) and clear water 5 Each treatment was evenly sprayed on the seedlings of muskmelon (variety: Xizhoumi No. 25), and then each pot was sprayed with the inoculation of the fruit spot fungus suspension (concentration of 1×10 ⁹ CFU/mL), and each treatment was repeated 4 pots, each pot was 5 seedlings were cultivated with moisturizing light, and the incidence was observed on 7d and 14d of cultivation, and the disease index (formula 1) and control effect (formula 2) were calculated [Reference: Identification of Bacillus BJ_6 and control of melon bacterial fruit spot _ Jia Huihui]. Classification of leaf diseases [refer to "Efficacy II""Fungicides for the Control of Cucumber Bacterial Angular Spot"]:

Grade 0: no lesions;

Grade 1: The lesion area accounts for less than 5% of the entire leaf area;

Grade 3: The lesion area accounts for 6-10% of the entire leaf area;

Grade 5: The lesion area accounts for 11-20% of the entire leaf area;

Grade 7: The lesion area accounts for 21-50% of the entire leaf area;

Grade 9: The lesion area accounts for more than 51% of the entire leaf area;

The formula for calculating drug efficacy:

Disease index = ∑ (number of diseased leaves at all levels × relative grade value) / (total number of leaves under investigation × highest disease grade value) × 100 (1)

Control effect (%) = (control disease index – treatment disease index)/control disease index × 100 (2)

data analysis:

The results of pot experiments on melon bacterial fruit spot showed that 4-allyl catechol also had a strong control effect on melon bacterial fruit spot. The control effect of melon bacterial fruit spot is 71.42%, which has no significant difference with the control agent 2% kasugamycin water; when its concentration is reduced to 250 μmol/L, its control effect on melon bacterial fruit spot remains the same. can be as high as 65.44%.

Table 4-Pot test of allyl catechol on melon bacterial fruit spot

2.3.2 Pot experiment on mango bacterial keratosis

When the leaf color of the potted mango shoots turns to yellow-green, dilute the kasugamycin water solution with concentration 1, concentration 2, concentration 3, and 4% (converted to the concentration of active ingredients as 500 μmol/L), and 5 treatments with clean water are evenly distributed. Spray on the leaves of mango seedlings (variety: Tainong), after natural drying, spray and inoculate mango bacterial keratosis fungus (the concentration of bacterial suspension is adjusted to 1×10 ⁹ CFU/mL), and add 1% Tween- 80. Each treatment was repeated for 4 pots, incubated with moisturizing light for 10 days and 20 days, the disease incidence was observed, the disease index was investigated (formula 1), and the control effect was calculated (formula 2). Bacterial angular spot disease classification standard [refer to "Efficacy II""Fungicides for the Control of Citrus Canker")]:

Grade 0: no lesions;

Grade 1: 1-5 diseased spots per leaf;

Grade 3: 6-10 diseased spots per leaf;

Grade 5: 11-15 disease spots per leaf;

Grade 7: 16-20 diseased spots per leaf;

Grade 9: Each leaf has more than 21 diseased spots;

The formula for calculating drug efficacy:

data analysis:

The results showed that 4-allyl catechol had a strong control effect on mango bacterial keratosis. When the tested concentration was 1000 μmol/L and 500 μmol/L, the control effects on mango bacterial keratosis were respectively. It was 76.75% and 67.73%, which was significantly better than the control agent 2% kasugamycin water; and when its test concentration was reduced to 250 μmol/L, its control effect on mango bacterial keratosis was still 60.32%, which was comparable to There was no significant difference between the control drug 2% kasugamycin aqueous solution.

Table 5 Pot test of 4-allylcatechol on mango bacterial keratosis

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.

Claims (10)

1. A compound for preventing and treating phytopathogenic bacteria, wherein the compound is 4-allyl catechol, and the compound structural formula is shown in formula I:

   
2. Application of 4-allylcatechol in the control of plant bacterial diseases.
3. The application according to claim 2 is characterized in that, the pathogenic bacteria of described disease are: rice bacterial blight Xanthomonas oryzae pv.oryzae, rice bacterial leaf spot Xanthomonas oryzae pv.oryzicola, citrus canker Xanthomonas citri subsp. citri, Acidovorax citrulli, tomato Ralstonia solanacearum, strawberry Xanthomonas fragariae, cabbage black rot Xanthomonas campestris pv.campestris, cabbage soft rot Pectobacterium carotovorum subsp.carotovorum, potato soft rot Pectobacterium carotovorum subsp.brasiliense and/or Xanthomonas campestris pv.mangiferaeindicae.
4. The application according to claim 3, wherein the pathogenic bacteria of the disease are: potato soft rot, mango bacterial angular spot and/or melon bacterial fruit spot.
5. A fungicide for preventing and treating plant diseases, characterized in that the active ingredient is 4-allyl catechol, optionally supplemented with an adjuvant.
6. A bacteriostatic agent for preventing and treating plant diseases, characterized in that the active ingredient is 4-allyl catechol, optionally supplemented with an adjuvant.
7. The bactericide according to claim 5 or the bacteriostatic agent according to claim 6, characterized in that, the dosage form of the bactericide or bacteriostatic agent is suspension, aqueous emulsion, microemulsion, emulsifiable concentrate, wettable powder or Water dispersible granules.
8. The preparation method of 4-allyl catechol, is characterized in that, comprises the following steps:

   S1: take the branches and leaves of South China pepper, dry them in the shade, pulverize and sieve to obtain dry powder, use ethanol solution for the first cold dipping, and filter to obtain the first filtrate and filter residue; The second filtrate is obtained, the first filtrate and the second filtrate are combined, and concentrated to dryness to obtain the ethanol extract of Chinese pepper;

   S2: add water to suspend the ethanol extract of P. chinensis, extract with petroleum ether, and concentrate to dryness to obtain the petroleum ether extract;

   S3: the petroleum ether extract is loaded onto the column by the petroleum ether wet method, and 9 fractions are obtained by elution in the petroleum ether-ethyl acetate system;

   S4: Fraction 3 was eluted with petroleum ether-ethyl acetate system to obtain 4-allylcatechol.
9. The preparation method according to claim 8, characterized in that, in step S1, the volume concentration of the ethanol solution is 90% to 95%; the time of the first cold soak is 6 to 7 days; , the mass ratio of dry powder and ethanol solution is 1:9～10; during the second cold soaking, the mass ratio of filter residue and ethanol solution is 1:(9～10).
10. preparation method according to claim 8, is characterized in that, in step S3, the volume ratio of petroleum ether-ethyl acetate is (2.5～3): 1; In step S4, the volume ratio of petroleum ether-ethyl acetate is (9-10): 1.