GB2250438A - Control of parasitic nemotodes - Google Patents

Abstract

The compound L-2-amino-4-(guanidinooxy)butyric acid (L-canavanine), <IMAGE> or an acid addition salt thereof is useful in controlling diseases caused by parasitic nematodes in plants or mammals. Seeds may be dressed, coated or impregnated with the compound.

Description

CONTROL OF PARASITIC NEMATODES (D) Field of the invention This invention relates to the control of diseases caused by parasitic nematodes in plants and animals.

Prior art Since the early 1940's many chemical anti-nematode compounds have been available. These have often displayed undesirable toxic effects, for example the fumigant dibromochloropropane was withdrawn from the market in 1977, as It was thought to cause sterility in workers. During the 1960's fumigant type nematicides were largely superseded by granular systemic nematicides. These have been in use since then, a representative compound being oxamyl. These compounds are mainly organocarbamates or organophosphate derivatives, and because of their toxicity have to be used in a strictly controlled manner. Accordingly it would be of benefit to have anti-nematode agents that are environmentally favourable, i.e. being non-toxic themselves and in their degradation products to non-target organisms.

Additional prior art is referred to in a separate section after "Summary of the invention", without which its context would not be clear.

Summarv of the invention The present invention provides the use of the compound L-2-amino-4-(guanidinooxy)butyric acid, (L-canavanine).

or an acid addition salt thereof, for use in controlling diseases caused by parasitic worms in plants, including crops, and in mammals. The invention includes also seeds, dressed, coated or impregnated with L-canavanine or a said salt thereof.

Additional prior art L-canavanine (referred to hereinafter simply as canavanine, for brevity) is a naturally occurring arg i n i ne analogue. Its extraction from jack beans - Canavalia ensiformis was first described by Kitagawa (J. Biochem. (Tokyo), 1929, 11, 265). It is found extensively in leguminous plants (see Turner et Phytochem., 1977, 6, 863). It has been shown that canavanine can be acted upon in some organisms by arginyl-transfer RNA synthetase, whereby it can be incorporated into plant and animal proteins in place of arginine. This property may be the basis for some of its biological effects. It has been described as a potent growth inhibitor of many organisms, (Pilcher et al., Proc. Soc. Exp. Biol.

Med., 1955, 88, 79) and recently as an anti-tumour agent (Thomas et al., Cancer Res., 1986, 46, 2898-2903). Its effects on certain insects have been reviewed by G. A. Rosenthal, J. Chem. Ecology 12, 1145-1156 (1986) and differ considerably between species. Although the metabolism of the tobacco hornworm Manduca sexta is severely affected by canavanine, the larvae of the bruchid beetle Carvedes brasiliensis, the curculionid weevil Sternechus tuberculatus and the tobacco budworm Heliothis virescens are unaffected by canavani ne.

It has been stated that -canavanine is extremely toxic to mammalian cells (Tschiersch, Pharmazie, 1962, 17 621). This has severely prejudiced the notion of its use as an applied chemical agent in the environment.

Brief description of the drawinq The Figure shows the dose response in vitro toxicity of canavani ne.

Description of the Dreferred embodiments Canavanine is believed to break down rapidly in soil, thus minimising any possible uptake by mammals and the possibilities of its appearing in water supplies. Fears about its toxicity to mammals now appear to have been over-stated.

Canavanine displays its properties against a wide range of worms affecting plants or crops, e.g. root-knot nematodes, cyst nematodes and virus-transmitting nematodes. Of particular note is its activity against the crop damaging nematodes of the following genera: Meloidoavne, Globodera, Lonaidorus, Heterodera, Radopholus, Pratvlenchus, Hirschmanniella, Scutellonema, Helicotylenchus, Tvlenchus, Rotylenchus, Ditvlenchus and Xiphinema.With regard to nematodes which infest mammals, canavanine is active against a wide range of helminthic nematodes, especially those of the following genera: Haemonchus, Teladorsaaia, Nematodfrus, Trichostronavius, Dictvocaulus and Cooperia, particularly the species Haemonchus contortus and Teladorsagla circumcincta (previously classified as Ostertaqia circumcincta).

Canavanine can be isolated as described by Kitagawa (above) or synthesized as described by Yamada et al., Agr. Biol. Chem., 1973, 31, 2201.

Canavanine and its salts are water-soluble and can therefore be applied without a surfactant or dispersing agent. For foliar spraying, it is suggested that normally the plants by sprayed with a solution containing 0.1 to 1 g./litre until the spray runs off.

The above description of canavanine applies also to its acid addition salts, which can be any which are compatible with the intended use, e.g. agriculturally or veterinarily acceptable if the use is on plants or non-human animals respectively. Such salts can be made in the conventional way from the free base. Conveniently, canavanine is administered as its free base or Its sulphate.

As shown in Example 1 below, canavanine is highly nematotoxic, but when applied at too low a pH can be phytotoxic to plants.

There is a possible correlation between phytotoxicity and charge on the guanidoxy group. The guanidoxy group is positively charged at a pH of around 5.6, whereas at higher pHs, at which no phytotoxicity was evident, It Is only partially charged, becoming uncharged. The preferred pH is thus above 5.6, most especially above 6.0.

The following Examples illustrate the invention. Unless otherwise stated, canavanine was administered as a free base. The units "ppm" are used to signify mg. of test compound per litre of water.

Example 1 Gall Test In the gall test, tomato seedlings, stimulated to produce fine adventitious roots by removing the main root system, were planted in tubes containing 25g of fine, sieved, dry sand, 350 Meloidoqvne iavanica (J2) and canavanine, in solution in water. The effect of canavanine on the ability of the nematodes to gall the plant roots was studied over a 10-12 day perIod. A phosphate buffer control was included in the test. There were 10 replicates of each treatment.

Initially, canavanine (200 ppm) was tested in solutions of different pH to determine the optimum pH for its activity. A dose response study was then performed at the optimum pH.

Table 1 shows the ability of canavanine (200 ppm) at pH 5.6, 6.4 and 7.2 to control root galling of M. javanica. Good control was observed at pH 6.4 and 7.2. At pH 5.6 almost no root growth was observed.

The gall test was then repeated at pH 6.4 over a range of canavanine concentrations. Table 2 shows that canavanine was effective at 200ppm. Its effect wore off upon dilution until a second effective dosage range was observed at 6.25 ppm and 3.125 ppm. This effect is also shown in the Figure. In contrast to the higher doses that were tested (100 and 200 ppm) no phytotoxic effects were observed at the lower (3.125 and 6.25 ppm concentrations).

Table 1 Effect of pH on activity of canavanine at 200 ppm Mean No. galls/root pH Treatment Control (Buffer) pH 5.6 0+ 10.0 pH 6.4 0 (100%) 14.8 pH 7.2 0.1 (99%) 9.0 + very small root system compared to buffer control, pH 5.6.

( ) = % reduction in galls compared to control.

Table 2 Gall Test. (M. Javanica/Tomato) dose-response study at pH 6.4 Treatment Mean no. galls/root Control 13.6 200 ppm 0.25 (98%)+ 100 ppm 3.35 (76%) 50 ppm 7.24 (47) 25 ppm 7.62 (45%) 12.5 ppm 8.64 (37) 6.25 ppm 4.88 (64%) 3.125 ppm 3.69 (73%) +( ) = % # reduction in galls compared to control.

Example 2 Hatch Test The hatch test examines the effect of the test chemicals on the egg hatch of Globodera rostochiensis, the Potato Cyst Nematode (PCN).

Ten PCN cysts were put in a tube with 0.25 ml of the test compound solution (conc. 50 ppm and 100 ppm) and 0.75 ml of potato root diffusate. Root diffusate normally stimulates the juveniles to hatch from eggs in the cysts. There were 5 replicates of each treatment. Twice each week the liquid was removed and the number of hatched live and dead juveniles counted. The diffusate/chemical mixture was replenished after each nematode count. The tubes were stored at 19"C between counts.

Table 3 shows the number of hatched larvae, dead or alive. The same data are also expressed as per cent effect. Application of canavanine greatly decreases the number of juveniles hatching from cysts.

Table 3 Potato cyst nematode hatch test Treatments Hatched larvae after 23 days Alive Dead Control 2071 60 Canavani ne 200 ppm 432 (79%)+ 172 100 ppm 654 (68%) 155 50 ppm 986 (52%) 93 25 ppm 1219 (41irk 146 12.5 ppm 1140 (45%) 71 6.25 ppm 1532 (26%) 71 3.125 ppm 1249 (40%) 39 + ( ) = % reduction from control value.

Example 3 In Vitro Nematotoxicity Test Groups of ten active adult Globodera rostochiensis were hand-picked into individual watchglasses containing distilled water. At a given time the batches of nematodes were transferred into 1 ml of a solution of test compound having varying concentrations of the test compound, or for the control into 1 ml of distilled water. There were three replicates of each treatment. At various time -intervals up to 72 hours the number of nematodes which were Immobilised were recorded. They were considered as immobile if they failed to move when stimulated by prodding with a bristle. All tests were carried out at 35"C.

Table 4 shows the in vitro nematotoxicity of canavanine over a range of concentrations. Results are shown as the fraction of nematodes immobilised expressed as a percentage of the mobile nematodes of the control.

Table 4 ifl vitro toxicity (G. rostochiensis) Test Compound Conc (ppm) 2.5 5 10 100 % immobility Canavanine 36 hrs. 38+ 62 62 62 72 hrs. 83 92 92 67 + X immobility expressed as a fraction of control.

A similar experiment was performed over a 24 hour period on Longidorus elongatus. Canavanine was administered in its free base and sulphate forms.

Table 5 shows that after 24 hours all L. elonaatus were still mobile in 200 ppm free base while 90X were immobile in the sulphate salt. Lowering the concentration of test compound enhanced the effect of the free base but reduced the effect of the sulphate salt. Results are shown in the same way as for Table 4.

Table 5 In vitro toxicity (L. elonqatus) Test Compound Conc ppm 10 200 % immobility Canavanine - free base 50+ 0 Canavanine - sulphate salt 30 90 + % reduction in immobility expressed as a fraction of control.

The following claims define some important aspects of the invention, but do not purport to include every conceivable aspect for which protection might be sought in subsequent continuing and foreign patent applications, and should not be construed as detracting from the generality of the inventive concepts hereinbefore described.

Claims (7)

1. The use of the compound L-2-amino-4-(guanidinooxy)butyric acid, (L-canavanine),

or an acid addition salt thereof in controlling diseases caused by parasitic nematodes in plants or mammals.

2. The use according to Claim 1 wherein the compound is applied at a pH of above 5.6.

3. The use according to Claim 1, wherein the compound is applied to plants or crops in a foliar spray.

4. The use according to Claim 1, wherein the compound is applied to plants through the soil.

5. The use according to Claim 1, 2, 3 or 4 wherein the parasitic nematode attacks plants or crops and is of the genus Meloidogvne, Globodera or Longidorus.

6. The use according to Claim 1, 2 or 3 wherein the parasitic nematode Infests mammals and is of the genus Haemonchus, Teladorsagla, -Nematodirus, Trlchostrongvlus, Dictvocaulus or Cooperia.

7. Seeds, dressed, coated or impregnated with a compound specified in Claim 1 or 2.