WO1996019920A1

WO1996019920A1 - Compositions for treating nematode infections in trees

Info

Publication number: WO1996019920A1
Application number: PCT/JP1994/002244
Authority: WO
Inventors: Kinji Umehara; Kouji Uchida; Hajime Maki
Original assignee: Pfizer Pharmaceuticals Inc.
Priority date: 1994-12-27
Filing date: 1994-12-27
Publication date: 1996-07-04
Also published as: KR960020687A; CN1137345A

Abstract

The present invention provides a nematocidal composition for treatment or prevention of nematodes infections in trees, such as wilting disease in pine trees, which comprises a compound of general formula (I), wherein R when taken individually is hydrogen; R1 when taken individually is hydrogen or hydroxy; R and R1 when taken together represent a double bond; R2 is a C¿3?-C8cycloalkyl; and R?3¿ is hydrogen or methyl, and an inert carrier. The present invention also provides a method for treatment or prevention of nematodes infections in trees, which comprises treating trees to be treated with the above nematocidal composition.

Description

COMPOSITIONS FOR TREATING NEMATODE INFECTIONS IN TREES

Technical Field

This invention relates to a nematocidal composition for treatment or prevention of nematodes infections in trees, such as wilting disease in a pine tree (pine wood nematodiasis), and a method for the treatment or prevention of nematodes infections in trees, using such nematocidal composition.

Background Art The forest ecosystem is a diverse and complex system. It is subject to a variety of pests, including insects, nematodes, viruses, fungi and mites. Pine forests, and particularly Japanese red and black and Ryukyu pine forests, are subject to wilting disease caused by a pine wood nematode, Bursaphelenchus xylophilus.

The pine wood nematode is vector borne by beetles of the Cerambycid species, at least eight species of Cerambycidae being found in association with Bursaphelenchus xylophilus. Monochamus alternatus, one of the eight species, is the major carrier of

Bursaphelenchus xylophilus in Japan (Mamijya. JARO. Vol. 10, No. 4, 206-211 , 1976).

Several efforts to control the pine wood nematodes have been carried out. For example, Japanese Patent Application Publication No. S57-57442 discloses a method for control of the nematode Bursaphelenchus xylophilus. which comprises contacting said nematodes with morantel or a water soluble acid addition salt thereof, by introducing morantel or a water soluble acid addition salt thereof into the trunk of pine trees, to control wilting disease of pines. However, since the nematocidal activity of the nematocidal agent disclosed in this patent is not sufficient, the nematocidal agent should be administered to the pine trees in a large amount. Thus, when using an injection or infusion method for the administration of the active ingredient, a large number of holes should be made in the trunks of the trees for the administration. Of course, it is better to reduce the number of holes to be made in view of the health of th pine trees to be treated. Further, Japanese Patent Application Publication No. H06-37501 (U.S.

Patent 5,089,480) discloses a composition for the treatment and prevention of parasitic infections in humans and animals which comprises an antiparasitically effective amount of avermectin derivatives. However, there is no teaching or suggestion in this patent to use the compounds for the treatment or prevention of nematodes infections in trees. such as wilting disease.

In view of the above situations, it would be desirable if a nematocidal agent having higher nematocidal activity against nematodes in trees, such as pine wood nematodes, were provided.

Brief Disclosure of the Invention

The present invention provides a nematocidal composition for treatment or prevention of nematodes infections in trees, which comprises a compound of the general formula:

(I) wherein R when taken individually is hydrogen; R¹ when taken individually is hydrogen or hydroxy; R and R¹ when taken together represent a double bond; R² is a C_3.g cycloalkyl; and R³ is hydrogen or methyl, and an inert carrier.

The nematocidal compositions of this invention are useful in killing or eliminating nematodes in trees, such as pine wood nematode, i.e, nematode Bursaphelencus xylophilus which causes wilting disease in a pine tree, and thus are useful in the treatment or prevention of nematodes infections such as wilting disease in a pine tree. Accordingly, the present invention also provides a method for treatment or prevention of nematodes infections in trees, which comprises treating trees with the above- mentioned nematocidal composition.

The present invention provides a nematocidal composition having higher nematocidal activity, paricularly against pine wood nematodes than conventional nematocidal agents. Because of the high nematocidal activity comparing with the conventional nematocidal agents, it is possible to reduce the amount of the active ingredient to be administered to the trees, thereby making possible to reduce the number of holes in the trunks of the trees to be made for the injection or infusion method. Therefore, according to the present invention, a better method for treating or preventing nematodes infections in trees, such as wilting disease in pine trees can be provided. Detailed Description of the Invention

The nematocidal composition of the this invention comprises, as active ingredient, the compound of formula (I) as indicated above. The compounds of formula (I) are fully described in Japanese Patent Application Publication No. H06-37501 (U.S. Patent No. 5,089,480), including their production process. As a brief description of the production process, the compounds of formula (I) can be prepared by a process which comprises adding a suitable cycloalkane carboxylic acid or a salt, ester or amide thereof or oxidative precursor therefor, to a fermentation of an avermectin producing organism such as Streptomyces avermitilis ATCC 31267, 31271 or 31272. Examples of suitable acids include cyclopropane carboxylic acid, cyclobutane carboxylic acid, cyclopentane carboxylic acid, cyclohexane carboxylic acid, cycloheptane carboxylic acid and cyclooctane carboxylic acid. In a preferred embodiment, the fermentation is performed in the presence of cyclohexane carboxylic acid sodium salt to yield the compound of formula (I) wherein R is hydrogen; R¹ is hydroxy; R² is cyclohexyl; and R³ is methyl. Conventional chemical modification reactions as described in Kokoku H06-37501 can be used to modify R, R¹ or R³ to the desired group. For example, the compounds of formula (I) wherein R and R¹ together represent a double bond may be prepared by subjecting a compound of formula (I) wherein R is H and R¹ is OH to a known dehydration reaction. The compounds of formula (I) wherein R³ is H may be prepared, by a known demethylation reaction, from a compound of formula (I) wherein R³ is methyl.

In a preferred embodiment of this invention, the nematocidal composition comprises a compound of formula (I) wherein R and R¹ together represent a double bond, R² is cyclohexyl; and R³ is hydrogen, and a compound of formula (I) wherein R is hydrogen; R¹ is hydroxy; R² is cyclohexyl; and R³ is hydrogen. The most preferred composition of this invention comprises a compound represented by the following formula:

The amount of the compound of formula (I) to be incorporated into the nematocidal composition, may range widely depending upon the activity of the compound, the conditions of the trees to be treated, the environments surrounding the trees to be treated and the like. In general, the amount of the compound formula (I) may be in the range of from 0.001 to 70 weight percent, more preferably 0.005 to 30 weight percent, most preferably from 0.01 to 10 weight percent, based on the total weight of the composition.

If desired, the nematocidal composition of this invention may comprise additional active ingredients such as morantel or its salts, levamizole and mesulphen. The nematocidal composition of this invention comprises an inert carrier in a liquid or solid form. By the inert carrier is meant one that will not or react with the active ingredient and one that may be administered safely to the trees to be treated. Suitable inert carriers may be in a liquid form (such as solvents) since the nematocidal composition is usually administered to the pine trees by an injection or infusion method. Suitable solvents are those which are non-toxic to the tree and have a viscosity equal to or less than that of water, and which are capable of dissolving an active ingredient to be used. Suitable solvents include, for example, water; aromatic hydrocarbons such as benzene, toluene and xylene; alcohols such as methanol, ethanol, propanol and butanol; ethers such as ethyl ether, dioxane and tetrahydrofuran; halogenated hydrocarbons such as methylene chloride, chloroform and dichloroethane; amides such

5 as N,N-dimethylformamide; and nitriles such as acetonitrile. Particularly suitable solvents include water, methanol, acetone, hexane, acetonitrile and a mixture thereof. The most suitable solvent is a mixture of water and methanol because of its availability and the solubility of a compound of formula (I) of this invention. The ratio of methanol to water may be from 0. 1 : 99.9 to 60 : 40, preferably from 5 : 95 to 50 : 50.

10 When the nematocidal composition is used in the liquid form, the concentration of the active ingredient in the nematocidal composition solution may vary widely, i.e. , from dilute (about 0.01 percent) to concentrated or even saturated solutions. However, from a practical standpoint of view, it is preferable to use a nematocidal composition solution having a concentration of from 0.001 to 40 percent, preferably from 0.002 to

15 20 percent, more preferably from 0.004 to 10 percent. The nematocidal composition solutions may be applied to the trees so that the concentration of the active ingredient in a tree would be in the range of from 0. 1 to 300 ppm, preferably fo 0.3 to 100 ppm, more preferably from 0.5 to 50 ppm.

Before use of the nematocidal composition, in view of ease of handling, it may

20 be in a solid formulation containing the desired amount of active compound. The solid formulation may be dissolved or dispersed in a solvent as mentioned above for use in the injection or infusion method. The solid formulations are prepared by uniformly mixing the active ingredient with suitable finely divided solid carrier such as talc, diatomaceous earth, calcium carbonate, bentonite or soybean flour, and other desired

25 additives. The additives which can be used in the present invention are any known additives to be used in agricultural chemicals such as fillers, disintegrating agents and binders including starch, lactose, talc, magnesium stearate and vegetable gums.

Then, the method for the treatment or prevention of nematodes infections in trees, will be described below.

30 According to the method of this invention, trees susceptible to nematodes infections, such as a pine tree susceptible to wilting disease can be treated with the nematocidal compositons as mentioned above. Suitable trees which can be treated by the method of this invention include pine trees such as Japanese red pine (Pinus den si flora), black pine (P. thunbergii) and Ryukyu pine (P. luchuensis).

The treatment of the trees with the nematocidal composition of this invention can be carried out by administering or introducing the nematocidal composition into the trunk of a tree to be treated. Administration into the tree trunk is generally accomplished by an injection or infusion method known to those skilled in the art. In this case, the nematocidal composition should be in a liquid form, and thus may be composed of the compound of formula (I) of this invention and a solvent capable of dissolving said compound. Gravity or pressure devices can be used to administer the nematocidal composition into the trunk of a tree. Injection or infusion at more than one site on a tree can be done. Multiple infusion sites permit administration of sufficient volume of the nematocidal composition within a minimum time period so as to avoid interference by sap exudation.

Administration of the nematocidal composition to the trees to be treated is carried out at an appropriate time, depending upon the environment of the filed where the trees to be treated are present. In Japan, it is desirable to carry out the administration of the nematocidal composition before the time of the life cycle of M. alternatus (major carrier for Bursaphelenchus xylophilus) when it attains the dauerlarvae stage of development. Annual administration is favored to insure continued protection of the pine trees. When the infusion method is used, an appropriate amount of the nematocidal composition can be infused into the tree trunk within a period of two hours or less. Infusion periods longer than this generally encounter difficulty as a result of sap exudation which interferes with the infusion process. The use of pressure techniques of administration tend to minimize or even eliminate such difficulty. The effectiveness of the nematocidal composition is readily demonstrated by determining their efficacy in killing or paralyzing nematodes as described in the following examples.

Examples The present invention will be described in more detail with reference to the following Examples and Comparative Examples.

Examples 1 to 11 Nematodes (Bursaphelenchus xylophilus) were isolated from a sample of the pine trees which has grown in Chiba-ken, Japan. 100 heads each of nematodes dropped into a petri-dish each containing 1ml of a distilled water.

A compound of formula (I) of this invention (wherein R and R¹ together represent a double bond, R² is cyclohexyl; and R³ is hydrogen; Compound Bl), was dissolved in a mixture of water and methanol to obtain 11 nematocidal composition solutions each having a concentration of from 4.6 to 0.005 g/ml (methanol concentration of from 0.94 to 0.001 percent), as indicated in Table 1.

One (1) ml of each test composition was mixed with 1 ml of the nematode suspension having 100 heads of nemadoes, and sensitized at 25°C for 24 hours. After sensitization, the mixture was warmed at 37°C, out of which 2 ml was transferred into a latticed petri dish (4.5 cm in diameter) and observed under 16-power microstereoscopy .

The nematodes (adults and larvae-3) treated were classified into 3 groups: no-effect , paralyzed and dead group while pricking with a needle point, and the number of the nematodes in each group was counted.

The results of the testing are as shown in Table 1. Comparative Examples 1 to 6 and Reference Examples 1 to 2

Morantel tartrate was dissolved in a mixture of water and methanol to obtain 6 nematocidal composition solutions having a concentration of from 4.6 to 0.15 μg/ml (methanol concentration of from 0.94 to 0.03 percent), as described in Table 1.

One (1) ml of each test composition was mixed with 1ml of the nematode suspension having 100 heads of nematodes, and sensitized under shade at 25°C for 24 hours. As the control (Reference Examples 1 to 2), 1 ml of water and 1 ml of 0.94% methanol were mixed with 1 ml of the nematode suspension, respectively, and sensitized at 25°C for 24 hours.

After sensitization, the same procedure as used in Example 1 was repeated to analyze the efficacy of the test compositions. The results of the testing are also as shown in Table 1. Table 1 (Efficacy Against Bursaphelenchus xylophilus)

1 Mematocidal Compositions Number of Nematodes (%)

A. I. Me thanol Cone. Survival Paralvs is Mortality

( g act/ml

Example 1 4.6 0.94 0 27.7 72.3

Example 2 2.3 0.5 0 17.2 82.8

Example 3 1.2 0.2 0 28.8 71.2

Example 4 0.6 0.1 0 63.5 36.5

Example 5 0.3 0.06 0 69.8 30.2

Example 6 0.15 0.03 0 70.6 29.4

Example 7 0.07 0.01 4.3 76.1 19.6

Example 8 0.04 0.007 20.3 71.8 7.7

Example 9 0.02 0.004 36.4 56.4 7.2

Example 1C ) 0.005 0.002 38.9 52.8 8.3

Example 1 1 0.005 0.001 62.5 37.5 0

Ref. Ex. 1 0 0.94 73.5 23.5 3.0

Ref Ex. 2 0 Water 81.4 18.6 0

Comp. Ex. 1 4.6 0.94 0 49.0 51.0

Comp. Ex. 2 2.3 0.5 0 76.9 23.1

Comp. Ex. 3 1.2 0.2 0.29 ' 88.5 8.6

Comp. Ex. 4 0.6 0. 1 5.6 83.3 11.1

Comp. Ex. 5 0.3 0.06 12.9 83.9 3.2

Comp. Ex. 6 0.15 0.03 9.7 90.3 0

As seen from Table 1 , in Examples 1 to 11 , the concentration at which no-effect nematodes were not observed was higher than 0.15 μg/ml, and the concentration at which no dead nematodes were observed was lower than 0.005 μg/ml. In Comparative Examples 1 to 6 (using morantel tartrate), the concentration at which no-effect nematodes were not observed was higher than 2.3 μg/ml as morantel (3.9 μg/ml as morantel tartrate), and the concentration at which no dead nematodes were not observed was lower than 0.15 μg/ml. Accordingly, comparing the numbers of no-effect nematodes in the Examples and those in Comparative Examples, the compositions of the present invention showed nematocidal activity at about 15 times lower concentration than the morantel tartrate compositions. Therefore, it has been proven that the nematocidal composition of the present invention is remarkably more effective than the conventional nematodicidal composition against Bursaphelenchus xylophilus.

Examples 12 and 13 In order to measure nematocidal activity of a compound of formula (I) of this invention (wherein R is hydrogen, R ¹ is hydroxy; R² is cyclohexyl and R³ is hydrogen; Compund B2), the following experiment was carried out.

A nematode suspension used was prepared in the following manner. A population of Bursaphelenchus xylophilus obtained from the General Forest Laboratory of the Ministry of Agriculture, Forestry and Fisheries was used as the seed nematodes. Botrytis cinerea was incubated in barley medium at about 22°C for about 10 days to form colonies. The seed nematodes were transferred to the colonies and cultured at about 27°C for about 10 days. The mixture of medium and nematode was suspended in sterilized water, and isolated by the Baermann funnel technique for 30 to 60 minutes to collect a nematode suspension. Only the non-virgin female imagines were fished by sterilized needle from the nematode suspension to be used as a nematode specimen. Each of Compound B2 (50mg) and Compound Bl (50 mg) was dissolved in methanol to prepare standard solutions (2,500 μg/ml). Each of the standard solutions was diluted with methanol to prepare 5 sample solutions having 5 different concentrations (concentration: 0.04 μg/ml, 0.2 μg/ml, 1.0 μg/ml, 5.0 μg/ml and 25.0 μg/ml). In each Syracuse watchglass, stelirized water (5.0 ml) was poured, and 50 1 out of the stelirized water was replaced by each sample solution prepared as above. Into the sample solution of each Syracuse watchglass, 30 nematode specimens fished from the nematode suspension were dipped. These Syracuse watchglasses were left standing in a thermostat kept at about 25°C for about 24 hours. Then, mortality, morbidity and paralysis of nematodes were determined by stimulation method under microscopy. Those nematodes whose heads intermittently moved without stimulation by a sterilized needle were judged to be paralyzed. Those nematodes which moved only after the stimulation given by the needle were judged to be morbid. Those nematodes which showed no movement and no reaction against the stimulation by the needle were judged to be dead. The mortality, morbidity or paralysis rate was obtained by dividing the total number of dead, morbid or paralyzed nematodes by 30. The results of the testing are as shown in Table 2.

Table 2

Concentration of Active Ins iredient

0.04 0.2 1.0 5.0 25.0

⁽uzl ml)

Example 12 (Compoi ind Bl .

Paralysis 3.3 26.7 83.3 73.3 86.7

Morbidity 0 0 16.7 26.7 13.3

Mortality 0 0 0 0 0

Total 100 100 100 100 100

Example 13 (Compound B2)

Paralysis 6.7 93.3 83.3 63.3 70

Morbidity 0 0 16.7 36.7 30

Mortality 0 0 0 0 0

Total 6.7 93.3 100 100 100

As seen from Table 2, it was confirmed that Compound B2 showed the nematocidal activity similar to Compound Bl . Examples 14 and Comparative Example 7

In order to measure nematocidal activity of a mixture of Compound Bl and Compound B2, the following experiment was carried out.

A nematode suspension used was prepared as in the same manner with Exampes

12. A standard sample solution used in this experiment had a methanol concentration of 83.5

%, and 22 μg/ml of Compound Bl and 275 μg/ml of Compund B2. The standard sample solution was sequentially diluted with sterilized water to prepare sample solutions of 5, 10, 50, 100, 500, 1 ,000, 2.500 and 5,000 fold dilutions. Into each sample solution (5 ml) poured in a Syracuse watchglass, 30 nematode specimens fished from the nematodes suspension were dipped. These Syracuse watchglasses were left standing in a thermostat kept at 25°C for 24 hours. Then, mortality and morbidity rates were investigated in a manner similar to Example 12. The results of the testing are as shown in Table 3.

Table 3

Dilution .Fold .

10 50 100 500 1000 2000 5000

Example 14 (Mixture of Compound B l and B2)

Morbidity 100 93.3 3.3 0 0 0 0 0

Mortality 0 6.7 80 53.3 60 23.3 0 0

Total 100 100 83.3 53.3 60 23.3 0 0

Comparative Example 7 (Methanol)

Morbidity 100 6.7 0 0 0

Mortality 0 10 0 0 0 -

Total 100 16.7 0 0 0

As seen from Table 3, it was confirmed that a mixture of Compound Bl and B2 showed good nematocidal activity.

Claims

1. A nematocidal composition for treatment or prevention of nematodes infections in trees, which comprises a compound of the general formula:

(I) wherein R when taken individually is hydrogen; R¹ when taken individually is hydrogen or hydroxy; R and R¹ when taken together represent a double bond; R² is a C₃.₈ cycloalkyl; and R³ is hydrogen or methyl, and an inert carrier.

2. A nematocidal composition according to claim 1 , wherein R and R¹ together represent a double bond; R² is cyclohexyl and R³ is hydrogen.

3. A nematocidal composition according to claim 1 , wherein R is hydrogen, R^l is hydroxy; R² is cyclohexyl and R³ is hydrogen.

4. A nematocidal composition according to claim 2 or 3, wherein the inert carrier is water, methanol, acetone, hexane, acetonitrile or mixtures thereof.

5. A nematocidal composition according to claim 2 or 3, comprising 0.001 to 70 weight percent of the compound of formula (I) and 99.999 to 30 weight percent of the inert carrier.

6. A nematocidal composition according to claim 1 , wherein the nematodes infection is caused by pine wood nematode Bursaphelenchus xylophilus.

7. A method for treatment or prevention of nematodes infections in trees, which comprises treating trees to be treated with the nematocidal composition of claim 1.

8. A method according to claim 7, wherein the treatment of the trees is carried out by an injection method or infusion method.