CN1281609C - Compound with pine wood nematode killing activity with poison and use thereof - Google Patents

Abstract

The present invention relates to compounds with activity of poisoning pine wood nematode and applications thereof, which belongs to the technical field of biotic pesticides. The active compounds are prepared from producing strains by using the methods of conventional solid fermentation and extracting separation. The producing strains are gliocladium roseum (Gr87), and are preserved in China Committee for Culture Collection of Microorganisms on October 9th, 2002 with the preservation number of CGMCCNo. 0807. The active compounds comprises 6 compounds. The compounds can be used to effectively poison the pine wood nematode, and can be used to prepare biotic pesticides for poisoning nematode in a single mode or a combined mode.

Description

A class of compounds with poisonous and killing pine wood nematode activity and application thereof

Technical field:

The invention relates to a class of compounds with the activity of poisoning and killing pine xylophilus and applications thereof, belonging to the technical field of biological pesticides.

Background technique:

Bine wood nematode disease of pine trees is a devastating disease caused by pine wood nematode (Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle) parasitizing in the body of Pinus sp. The important quarantine object is listed as the first of forest diseases and insect pests, and is called "smokeless forest fire". Since the disease was discovered in 1905 and reported in 1934, it has been widely distributed in Japan, the United States, Korea, Canada, Mexico, Greece, Portugal and China. Japan's annual cost for the prevention and control of this disease accounts for 93.6% of the cost of forest pest control and 20% of the total forestry investment, but the annual loss of wood due to this disease still reaches about 1 million m ³ . Since the disease was discovered in China in 1982, it has now spread to Jiangsu, Zhejiang, ^Anhui , Guangdong, Shandong, Hubei, Shanghai, ^Hong Kong and Taiwan. It causes huge loss of forestry economy and forest ecology and serious damage to natural landscape, and poses a serious threat to the pine forests in the vast suitable habitat areas of our country. Since the 1980s, the control methods for pine wood nematode mainly include:

1. Apply insecticides such as fenitrothion (MEP), fenthion (MPP), naphthyl carbamate (NAC) and the like that can kill the vector insects of pine wood nematode - Monochamus alternatus. The characteristic of this control method is a passive control method that relies on killing the vector to protect the pine trees. It cannot effectively control the occurrence of the disease, and has a considerable degree of chemical pollution to the ground vegetation.

2. Inject nematicides (mesulfenphos, tartalicacid) directly into the tree to control pine wood nematodes. This method is only suitable for protecting precious trees, and the effect is not ideal, and it is highly toxic to humans and animals.

At present, the method of controlling pine wood nematode disease is mainly to control Monochamus alternatus by spraying insecticides. However, due to the long adult period and strong drug resistance of Monochamus alternata, the control effect is not ideal. So far, no effective and low-toxic special pesticides for killing pine wood nematodes that can be practically applied have been studied, and there are no research reports on medicaments or methods for both treating pine wood nematodes and Monochamus alternata.

Invention content:

The purpose of the present invention is to provide a class of compounds derived from freshwater fungi with poisonous and killing pine wood nematode activity and applications thereof.

The production bacterial strain of the present invention is Gliocladium roseum Gr87 bacterial strain, and this bacterial strain has been preserved in the General Microorganism Center of China Committee for the Collection of Microbial Cultures on October 9, 2002, and the preservation number is: CGMCC No.0807

A class of compounds with poisonous and killing pine xylophilus activity of the present invention is extracted and isolated from the fermentation culture of the production strain to obtain 6 compounds with similar structures, wherein compounds 1 and 2 are new compounds, and the structures of the 6 compounds are as follows Shown in formula 1-6 (hereinafter referred to as compound 1-6 respectively):

1 R ₁ =OH R ₂ =CH ₃ x =3

2 R ₁ =OH R ₂ =CH ₃ x =4

3 R ₁ =H R ₂ =CH ₃ x =2

4 R ₁ =OH R ₂ =CH ₃ x =2

5 R ₁ =OH R ₂ =CH ₂ CH ₃ x = 2

6 R ₁ =OH R ₂ =CH(OH)CH ₃ x=2

The physical and chemical constants, hydrogen spectrum data and carbon spectrum data of active compounds 1-6 of the present invention are shown in Table 1, Table 2 and Table 3 respectively.

Table 1. Physicochemical constants of compounds 1-6 1 2 3 4 5 6 molecular weight C ₃₀ H ₂₈ N ₆ O ₆ S ₅ C ₃₀ H ₂₈ N ₆ O ₆ S ₆ C ₃₀ H ₂₈ N ₆ O ₅ S ₄ C ₃₀ H ₂₆ N ₆ O ₆ S ₄ C ₃₁ H ₃₀ N ₆ O ₆ S ₄ C ₃₁ H ₃₀ N ₆ O ₇ S ₄ FAB-MS 729 761 680 697 711 727 [α]D(CHCl ₃ orpyridine) [α] _D ^18.0 +553.6°(c0.004532g/mL inpyridine) [α] _D ^18.0 +555.8°(c0.004365g/mLin pyridine) [α] _D ^18.2 +548.9°(c0.004668g/mLin pyridine) α] _D ²² +727.5°(c 0.1 inCHCl ₃ ) α] _D ²² +688.2(c 0.1 in CHCl ₃ ) α] _D ²² +768.9(c 0.1 inCHCl ₃ ) UV (MeOHor pyridine) 261.3, 255.6, 250.1, 202.4 261.2, 255.6, 250.2, 202.4 299.0, 261.4, 255.6, 250.0, 202.4 240, 303 241,305 242,301 IR(KBr) 3441, 1664, 1608, 1557, 1490, 1480, 753 3430, 1678, 1611, 1552, 1483, 1470, 749 3451, 1676, 1484, 1379, 1095, 748 3446, 1669, 1580, 1483, 750 3390, 2923, 1690, 1668, 1610, 1469, 1349, 1302, 1247, 1203, 1093, 1065, 756 3412, 2924, 1684, 1674, 1608, 1469, 1351, 1303, 1247, 1094, 1068, 765

Table 2. ¹ HNMR (Pyridine) of Compounds 1-6 C 1 2 3 4 5 6 5a 5.72s 6.08s 5.82s 5.78s 5.77s 5.79s 7 6.67d (7.8) 6.65d(7.8) 6.58m 6.70d (7.8) 6.72d (7.8) 6.70d (7.8) 8 7.12t (7.5, 7.8) 7.05t (7.5, 7.8) 6.98m 7.12t (7.5, 7.8) 7.15t (7.5, 7.8) 7.10t (7.5, 7.8) 9 6.77t(7.5) 6.61m 6.66m 6.79t(7.5) 6.79t(7.5) 6.80t(7.5) 10 8.34(7.5) 8.37d (7.5) 6.98m 8.28d(7.5) 8.27d(7.5) 8.28d(7.5) 11 6.27s 6.38s 6.93s 6.26s 6.26s 6.29s 12 2.79s 2.86s 2.89s 2.83s 2.88s 2.82s 13 1.77s 1.73s 1.97s 1.75s 1.81s 1.74s 5a' 5.78s 5.85s 6.95s 5.78s 5.77s 5.86s 7′ 6.72d (7.8) 6.69d (7.8) 6.58m 6.70d (7.8) 6.72d (7.8) 6.68d (7.8) 8' 7.09t(7.7) 6.93t (7.7) 6.98m 7.12t (7.5, 7.8) 7.15t (7.5, 7.8) 7.07t (7.5.7.8) 9' 6.69(7.5) 6.61m 6.66m 6.79t(7.5) 6.79t(7.5) 6.80t(7.5) 10′ 8.17(7.6) 8.12(7.7) 6.98m 8.28d(7.5) 8.27d(7.5) 8.31d (7.5) 11′ 6.39s 6.08s 3.33brs(15.0)5.03brs(15.0) 6.26s 6.24s 6.33s 12′ 3.01s 2.87s 2.87s 2.83s 2.88s 2.82s 13′ 1.57s 1.61s 1.92s 1.75s 2.12m 4.99q 14′ 1.25t(7.4) 1.52d(6.2)

Table 3. C ¹³ NMR (Pyridine) of Compounds 1-6 C 1 2 4 3 5 6 1 166.3s 166.1s 166.1s 166.6s 166.3s 166.1s 3 74.1s 74.0s 74.3s 74.6s 74.5s 74.3s 4 162.3s 162.2s 162.3s 162.7s 162.6s 162.3s 5a 83.6d 83.4d 83.3d 83.3d 83.5d 83.3d 6a 150.8s 150.9s 150.9s 150.6s 151.0s 150.9s 7 110.5d, 110.3d 110.3d 109.0d 110.5d 110.3d 8 130.9d 130.0d 130.1d 129.9d 130.3d 130.1d 9 119.5d 119.4d 119.4d 118.3d 119.5d 119.3d 10 128.8d 128.6d 128.6d 129.3d 128.7d 128.6d 10a 130.9s 130.8s 130.8s 129.6s 130.9s 130.8s 10b 67.4s 67.5s 67.5s 65.6s 67.6s 67.5s 11 82.6d, 82.8d 82.8d 81.3d 83.0d 82.8d 11a 78.5s, 78.8s 78.5s 77.7s 78.7s 78.5s 12 26.9q 27.2q 26.9q 27.2q 27.2q 26.9q 13 17.5q 17.5q 17.6q 18.3q 17.8q 17.6q 1 168.6s 167.4s 166.1s 165.5s 167.2s 167.4s 3′ 72.5s 75.3s 74.3s 74.7s 74.5s 74.3s 4′ 164.9s 164.5s 162.3s 162.5s 161.8s 162.1s 5a' 82.0d 84.2d 83.3d 82.0d 83.6d 83.5d 6a' 153.8s 151.6s 150.9s 150.8s 150.1s 150.8s 7' 110.6d 110.7d 110.3d 109.2d 110.5d 110.3d 8' 130.1d 128.8d 130.1d 129.9d 130.3d 130.1d 9' 118.8d 119.0d 119.4d 118.2d 119.7d 119.5d 10′ 129.7d 129.1d 128.6d 129.3 128.8d 128.7d 10a' 128.2s 131.1s 130.8s 131.1s 130.9s 130.9s 10b' 65.51s 67.5s 61.3s 67.6s 67.5s 11′ 85.6d 82.8d 45.8t 83.0d 82.8d 11a' 86.6d 78.5s 74.9s 79.1s 78.5s 12′ 27.9q 28.6q 26.9q 26.9q 28.2q 29.2q 13′ 21.2q 23.2q 17.6q 17.7 25.1t 67.2d 14′ 9.9q 20.0q

In the anti-nematode activity test of the active compound of the present invention in the liquid immersion method, when the treatment time was 36 hours and the concentration was 1.0 mg/mL, the lethality of compounds 1-6 to pine wood nematodes were 62.4%, 63.5%, 54.2%, 53.4%, 44.6% and 49.7%, and the mixed solution of compound 4 and 1 and the methanol crude extract of compound 1-6 are to the lethality rate of pine xylophilus 75.4% and 93.2%. It can be seen that compounds 1-6 can effectively poison and kill pine wood nematodes, and can be used alone or in combination for the preparation of pesticide preparations; at the same time, the crude extracts of active compounds or mixtures in different proportions have higher poisoning activity against pine wood nematodes than monomeric compounds The fact that these compounds have a synergistic poisonous effect on pine xylophilus.

Therefore, the compound of the present invention can be used as a pesticide nematicide.

Detailed ways:

Embodiment 1: the separation and preparation of compound 1-6

a. Seed culture of fungus Gliocladium roseum Gr87:

PDA medium: 200g of potatoes, 20g of glucose, 1.0g of yeast extract, 6.0g of glycerol, 17g of agar, 1000mL of water, make a test tube slant, pick the strains into the slant, and culture at 28°C for 5 days;

b. Solid fermentation culture of the fungus Glioma pink:

Wheat culture medium (the preparation process is: soak the wheat in tap water for 48 hours, then wash it, drain the water, put it into a 500cm Erlenmeyer flask, wrap the mouth of the bottle with cotton cloth and sterilize twice at 15psi, 40min each time).

Pick the strain cultured on the slant into the solid fermentation medium, and culture it statically at room temperature 25°C for 30 days;

c. freeze-drying the mycelia of Glioma pinka that has been cultivated above, and extracting it by cold immersion in an optional organic solvent (such as methanol, ethanol, propanol, ethyl acetate, tetrahydrofuran, acetonitrile) for 4 times, and The concentrated crude extract is absorbed on a solid absorbent material (e.g. crushed natural materials such as kaolin, clay, talc, quartz, etc.; or crushed synthetic materials such as particulate silica gel, alumina, etc.) and chromatographed on a silica gel column Separation, dry loading, gradient elution with chloroform/acetone=0-50% as eluent.

d. Collect 1%-10% chloroform/acetone eluate and concentrate under reduced pressure.

e. Chromatographically separate the above concentrated solution on a silica gel column again, load the sample by dry method, and use petroleum ether/acetone=10-80% as the eluent gradient elution.

f. Collect the 20-30% petroleum ether/acetone eluent whose sulfuric acid color is orange-yellow on the TLC plate, and concentrate to obtain a bright-yellow crystal, which is the mixture shown in the structural formula. Compounds 1-6 can be obtained through multiple separations by Sephedex-LH20 and HPLC chromatography.

Example 2:

The liquid immersion method is used to detect the anti-nematode activity of the monomer and the mixture of the compound of the present invention.

1. Test drug:

Dissolve the test sample in a small amount of organic reagent DMSO, and then add a certain concentration of Tween aqueous solution to prepare sample test solutions with the concentrations shown in Table 4. In this solution, the final concentration of DMSO is <3%, and the final concentration of Tween is <5‰. In addition, the same concentration of DMSO was dissolved in an aqueous solution containing 0.5% (V/V) Tween-20 as a control.

2. Cultivation and preparation of pine xylophilus

Cultivation method of pine wood nematode B.xylophilus: insert Botrytis cinerea into a PDA plate, culture at 25°C until the plate is full, insert a piece of medium with B.xylophilus, and culture at 25°C Until the mycelium disappears, the nematodes cover the plate.

When in use, pick out the medium containing the nematodes, put them into a nematode separator, wash out the nematodes with sterile water, concentrate by centrifugation, and prepare a nematode suspension (about 500 pieces per milliliter).

3. Test method

Dilute the compound of the present invention to 400ppm, 200ppm, 100ppm concentration test medicinal solution, place 2ml of the medicinal solution in a petri dish with a diameter of 5cm, then add 20ul (about 300) nematode suspensions of live nematodes, and mix gently Then place the dish in an incubator at 25°C. The nematode mortality was calculated at 12, 24 and 48 hours respectively. And observe under a binocular microscope to observe the changes in the nematode body wall.

The method for identifying nematode death is: add 1-5 drops of 5% NaCl solution to the treatment plate, observe after 2 minutes, dead worms are stiff, and live worms are curled or twisted.

Mortality % = number of dead nematodes / total number of nematodes × 100

Taking the test solution without adding the sample as the control, the whole experiment was repeated three times, and the average death rate was calculated by taking the average value of the three times.

4. Test results

Table 4. The results of the poisonous activity of test samples against pine xylophilus Sample Test No. Sample Test Concentration Lethality (%) in different treatment time (hours) 12 twenty four 36 Compound 1 1.0mg/mL0.8mg/mL0.5mg/mL 000 53.227.815.6 62.442.121.3 Compound 2 1.0mg/mL0.8mg/mL0.5mg/mL 000 57.832.121.3 63.534.727.8 Compound 3 1.0mg/mL0.8mg/mL0.5mg/mL 000 48.530.619.6 54.234.825.4 Compound 4 1.0mg/mL0.8mg/mL0.5mg/mL 000 46.736.527.9 53.434.530.1 Compound 4 and Compound 1 1.0mg/mL0.8mg/mL0.5mg/mL 000 70.452.839.1 75.454.942.3 Compound 5 1.0mg/mL0.8mg/mL0.5mg/mL 000 36.729.419.4 44.633.721.6 Compound 6 1.0mg/mL0.8mg/mL0.5mg/mL 000 46.232.822.6 49.744.528.7 methanol extract 1mg/mL0.5mg/mL 64.552.3 88.557.4 93.260.2 1%-10% Chloroform/Acetone Concentrate 1mg/mL0.5mg/mL 58.732.4 80.652.0 85.457.6 20-30% petroleum ether/acetone concentrate 1mg/mL0.5mg/mL 54.225.9 73.135.8 80.344.2 control 3.2 3.8 4.1

The result shows: compound 1-6 of the present invention all has poisonous activity preferably to pine wood nematode, can be used for the preparation of pesticide nematicide; The fact that the killing activity is higher than that of monomeric compounds shows that these compounds have synergistic poisonous effect on B. xylophilus.

Claims (4)

1, a kind of compound with pine wood nematode poisoning activity by producing bacterial strain, adopts the method for conventional solid fermentation and extraction separation to obtain, and it is characterized in that:

A. producing bacterial strain is pink sticking broom mould (Gliocladium roseum) Gr87, preserving number: CGMCCNo.0807;

B. the structural formula of this compound is:

Wherein: R ₁=OH, R ₂=CH ₃, x=3;

Or: R ₁=OH, R ₂=CH ₃, x=4.

2, a kind of composition with pine wood nematode poisoning activity by producing bacterial strain, adopts the method for conventional solid fermentation and extraction separation to obtain, and it is characterized in that:

A. the production bacterial strain of said composition is pink sticking broom mould (Gliocladium roseum) Gr87, preserving number: CGMCC No.0807;

B. said composition contains the compound 1-6 of following formula:

Compound 1:R ₁=OH, R ₂=CH ₃, x=3;

Compound 2:R ₁=OH, R ₂=CH ₃, x=4;

Compound 3:R ₁=OH, R ₂=CH ₃, x=2;

Compound 4:R ₁=H, R ₂=CH ₃, x=2;

Compound 5:R ₁=OH, R ₂=CH ₂CH ₃, x=2;

Compound 6:R ₁=OH, R ₂=CH (OH) CH ₃, x=2.

3, the application of the described active compound of claim 1 is characterized in that this active compound is used for the preparation of pine wood nematode pesticide preparation extremely.

4, the application of the described composition of claim 2 is characterized in that said composition is used for the preparation of pine wood nematode pesticide preparation extremely.