GB2331520A - Enzyme composition for protection of plants from fungal disease - Google Patents

Abstract

An enzyme composition is provided comprising: (i) at least three enzymes selected from a carboxymethyl cellulase, an α-mannanase, a xylanase, a chitinase, a #-glucanase, a cutinase and a pectinase, wherein at least one of the enzymes is a carboxymethyl cellulase, an α-mannanase, or a xylanase, and (ii) a diluent. A method of treating a plant having a fungal disease, or protecting a plant from a fungal disease is also provided. The method comprises applying to the plant an enzyme composition comprising: (i) a carboxymethyl cellulase, an α-mannanase, or a xylanase, and (ii) a diluent. The composition and method are useful alternatives to agrochemical fungicides, because they are environmentally safe, and possess a similar or improved activity against fungal pathogens.

Description

k Protection of Plants from Fungal Disease b Treatment with Enzymes c y

2331520 The present invention relates to an enzyme composition for application to a plant and a method for protecting or treating a plant by applying such a composition. More specifically, the invention relates to such a composition having fungicidal activity.

Over the years, diseases in plants caused by fungi have resulted in considerable losses in both the yield and the quality of farmed produce. Therefore, improvements in techniques for protecting plants, such as crops, from fungi art constantly being sought. Traditionally, the most effective way of preventing or ZIP treating fungal attack, has been to apply appropriate chemicals to those parts of the plant which tend to be or are affected.

However, fungal pathogens have increasingly developed resistance to chemical I fungicides. Moreover, there is growing concern amongst consumers about the potential harmful side effects of chemical fungicides, to people, to animals, to the food chain and to the environment generally. Consequently, new effective, environmentally safe strategies for resisting fungal pathogens are needed.

The cultivation of micro-organisms to produce desired end-products has been employed to produce biologically active enzymes. As a result enzymes have become more available for conunercial use. In general they are natural products which have a low impact on the environment in comparison to chemical products and they have found wide-scale use in animal feeds, washing powders and detergents, and beer and wine making.

It is already known to apply an enzyme composition to a crop, to protect it from fungal pathogens. For example, WO 911063 12 discloses a method for protecting a harvested crop from such pathogens, in which a composition comprising an endoenzyme is applied to the crop. The endoenzyme catalyses the production of an oligosaccharide, which in ' turn stimulates the crop to synthesise anti-pathogenic agents. The mode of action of the endoenzyme is thus to stimulate the immune system of the plant. Several endoenz mes are mentioned, including 0-glucanase 1 y 0 0 1 and chitinase. WO 91/063 13 3 describes a similar use to that of WO 91/06312, for the control of fungal pathogens in growing crops.

It is an object of the invention to provide an enzyme composition which, on application, controls fungal pathogens which may otherwise damage a plant. A further object of the invention is to provide an environmentally safe alternative to currently available fungicides. Another object is to provide an enzyme composition which has an improved fungicidal effect against certain fungal pathogens, in comparison with currently available fungicides.

Accordingly, the first aspect of the present invention provides an: enzyme composition, comprising: at least three enzymes selected from a carboxymethyl cellulase, an ct-mannanase, a xylanase, a chitinase, a Pglucanase, a cutinase and 0 a pectinase, wherein at least one of the enzymes is a carboxymethyl cellulase> an ot-mannanase, or a xylanase, and (ii) a diluent.

(i) A second aspect of the invention provides a method of treating a plant having a fungal disease, or protecting a plant from a fungal disease, comprising applying to the plant an enzyme composition comprising: (i) (ii) a diluent.

a carboxymethyl cellulase, an cc-mannanase, or axylanase, and A third aspect of the invention provides the use of a carboxymethyl cellulase, an ot-mannanase or a xylanase as a plant fungicide.

In this invention, a carboxymethyl cellulase means a cellulase which acts on a carboxymethyl cellulose substrate.

In vitro studies by the inventors have shown that the enzymes and enzyme mixtures provided by the present invention have a fungicidal effect in the absence of a plant. Thus, fungal -pathogens can be controlled by direct action of the enzyme, without relying on stimulating the immune system of the plant. As a result of this surprising finding, the possibility has been created to protect and treat plants, using enzymes which have not been used before for this purpose. Thus, the invention opens the way for the use of enzyme fungicides which are both effective and environmentally safe as alternatives to chemical fungicides.

1 The compositions according to the present invention contain at least three enzymes, at least one of which is a carboxymethyl cellulase, an amannanase, or a xylanase. Compositions which contain at least one of these enzymes have a particularly useful fungicidal effect. The compositions may also contain a chitinase, a P-glucanase a cutinase and/or a pectinase. Preferred enzyme compositions include:

i. carboxymethyl cellulaselchitinaselp-glucanase ii. carboxymethyl cellulase/chitinase/p-glucanase/(x-mannanase iii. carboxyMethyl cell ulase/chitinase/p - glucanas clcc-mannanas e/cutinase iv. xylanase/cutinase/pectinase V. xylanase/cutinase/pectinase/p-glucanase vi. xylanase/cutinaselpectinase/p-glucanase/chitinase Preferred carboxymethyl cellulases include Multifect CL (available from Genencor International) and Sumizyme C (available from Shin Nihon). Preferred oc-mannanases include YL15 (available from Amano). Preferred xylanases are those available from Genencor International. Preferred chitinases include those available from Biochemis. Preferred 0-glucanases include those available from Biochemis. Preferred pectinases are those available from Genencor International.

The compositions of the invention additionally contain a diluent. The diluent may be any substance which facilitates the storage of the enzymes, or their application to plants. It can also be any substance which assists the enzyines, in entering the plant, or the pathogen, e.g. by promoting greater contact of the applied composition with the plant. This allows the fungicidal effect to be realised more,quickly and efficiently. Preferably one diluent is used, but if required, two or more diluents can be present in the compositions.

The diluent should be compatible with the enzymes in the composition and should not have a damaging affect on the plant to which the composition is applied. Preferred diluents include water, acetone and a mixture of water and acetone.

The enzyme compositions are most effective when the enzyme sample concentration is between 1 and 10 5 jig/cm 3 and a range of 10 - 100 pg/cm 3 is particularly preferred. If one of the enzymes is present as a liquid broth, then there is often no need to add a diluent, as sufficient diluent may already be present.

The compositions of the invention may additionally contain one or more additives. Preferred additives include surfactants, buffers, humectants, stabilisers and preservatives. Examples of preferred stabilisers include sorbitol, sodium benzoate, potassium sorbate and sodium chloride.

When the additive is a surfactant, it should be compatible with the enzymes used, and should not adversely affect the plant to which the composition is applied. The surfactant ensures effective coverage and wetting of the follage when the g composition is applied to a plant. Any surfactant which fulfils these criteria can be used. Exemplary surfactants include Tween 200), Tween 800, Triton-X-100@ and Silwet(g. The surfactant is preferably present in a concentration of 0.00 1 to 0.1 % by weight based on the weight of the diluent. A concentration of 100 gg/cm 3 is particularly preferable.

In the methods according to the present invention, an enzyme composition which comprises a carboxymethyl cellulase, an (x-mannanase, or a xylanase is applied to a plant. The compositions may also contain a further enzyme, e.g. a carboxymethyl cellulase, an cc-mannanase, a xylanase, a chitinase, a P-glucanase and/or a pectinase and preferred enzymes and enzyme mixtures for the compositions used in the method include:

i. carboxymethyl cellulase ii. cc-mannanase iii. xylanase iv. carboxymethyl cellulase/chitinaseffl-glucanase V> carboxymethyl cellulase/chitinase/o-glucanaselct-mannanase vi. carboxymethyl cellulase/chitinase/o-glucanase/(x-mannanaselcutinase vii. xylanase/cutinase viii. xylanase/cutinase/pectinase ix. xylanase/cutinase/pectinase/p-glucanase xylanase/cutinaselpectinase/oglucanaselchitinase The preferred enzymes, diluents, additives and concentrations of constituents are the same as those described above in regard to the compositions of the invention.

In the methods of the invention, the preferred plants are cereals, fruit, vegetables, nuts, etc. The method may be carried out on growing plants, or plants which have 1 been harvested. For example, it may be used to treat or protect growing plants, or 1 1 1:

1 to protect harvested plants from decay, thus increasing their post-harvest life. In particular, the methods can be applied to cereals, (e.g. wheat, barley, oats, rice, maize, rye, triticale) potatoes, tomatoes, brassicas (e.g. oilseed rape), strawberries, lemons, grapevines, tree crops (e.g. apples, peaches, coffee), soybean, peanuts, bananas, and avocados.

The pathogens which are especially effectively controlled are those in the classes Oomycetes, Ascomycetes, Basidiomycetes, and Fung i Imperfecti. Exemplary fungi in the class Oomycetes include Phytophthora infestans, Pythium ultimum and Plasmospora viticola. Other pathogens which can be controlled by the methods of the present invention include Erysiphe grarninis, Leptosphaeria odorum, Puccinia recondita, Pyricularia oryzae, Botrytis cinerea, Rhizoctonia solani, Pseudocercosporella herpotrichoides, Peronospora parasitica, Venturia inaequalis and Penicillium digitatum.

The methods of the present invention are particularly effective for the following plant/pathogen combinations:

0 i.

wheatlLeptosphaeria nodorum ii. wheatlPuccinia recondita iii. ricelRhizoctonia solani iv. ricelPyricularia oryzae V. brassicas (especially oilseed rape)lPeronosporaparasitica vi. potatoesIPhytophthora infestans vii. tomatoesIPhytophthora infestans viii. strawberries/Botrytis cinerea ix. lemonslPenicillium digitatum X. grapevineslPlasmospora viticola xi. grapevinesIBotrytis cinerea The preferred enzyme compositions for treating or protecting these plants from their respective pathogens include the following.

0 i. wheatILeptosphaeria nodorum - carboxymethyl cellulase ii. wheatILeptosphaeria nodorum - oc-mannanase iii. wheatILeptosphaeria nodorum - carboxymethyl cellulase + chitinase + glucanase + cc-mannanase + cutinase iv. wheatILeptosphaeria nodorum - xylanase + cutinase k (I'-wheat/Puccinia recondita - carboxyrnethyl cellulase + chitinase + Pglucanase + (x-mannanase + cutinase vi. wheatlPuccinia recondita xylanase + cutinase vii. wheatIPuccinia recondita - xylanase + cutinase + chitinase + P-glucanase + pectinase ricelRhizoctonia solani or Pyricularia oryzae - carboxymethyl cellulase + chitinase + P-glucanase + ot-mannanase + cutinase ix. ricelRhizoctonia solani or Pyricularia oryzae - xylanase + cutinase X. brassicas (especially oilseed rape) - Peronospora parasitica/cc-mannanase xi. brassicas (especially oilseed rape) Peronospora parasiticalxylanase + cutinase xii. brassicas (especially oilseed rape) - Peronovpora parasiticalxylanase + cutinase + pectinase xiii. potatoes or tomatoesIPhytophthora infiestans - carboxymethyl cellulase xiv. potatoes or tomatoesIPhytophthora infiestans - (x-mannanase xv. potatoes or tomatoesIPhytophthora infestans - carboxymethyl cellulase + chitinase + P-glucanase + (x-mannanase + cutinase xvi. grapevineslPlasmospora viticola - carboxymethyl cellulase xvii. grapevineslPlasmospora viticola - cc-mannanase xviii. grapevineslPlasmospora viticola - carboxymethyl cellulase + chitinase + Pglucanase + a-mannanase + cutinase xix. grapevinesIPlasmospora viticola xylanase + cutinase + pectinase xx. grapevineslPlasmospora viticola xylanase + cutinase + pectinase + P- glucanase + chitinase viii.

The compositions are preferably applied to the plants by spraying, but any method can be used which ensures adequate coverage of the plant with the composition. The amount of the composition required for treatment or protection of the plant will depend on the particular type of plant involved, and its condition. This can be readily determined by a person skilled in the art in each case. The compositions are preferably applied in a single dose, but several smaller doses can be used if necessary. If required, several repeat doses can be applied throughout the season to effect control.

The present invention is further explained by way of the following Examples- In the in vivo tests, solid enzyme samples were all diluted with water to a concentration of 10 mg/cm 3 and Tween 200 was added to a concentration of 100 Lg/CM3 ID The liquid samples remained undiluted, but Tween 20 was added to the broth to a concentration of 100 ig/cm 3. In the in vitro tests, solid enzyme samples were added to liquid agar, to a concentration of I mg/cm 3. The liquid samples were added to liquid agar to give a 33 % by volume concentration in the final medium.

For comparison, a standard wide-spectrum chemical fungicide, Dithane (mancozeb available from Rohm and Haas) was tested in vivo and in vitro, at a concentration of 100 Lg/CM3 in water, and liquid agar respectively. The mancozeb sample for the I in vivo test also contained Tween 200 at a concentration of 100 Lg/cm'. A control was also tested in each case, consisting of a 100 Lglcm 3 solution of Twe,en 20 & in water.

The enzymes and enzyme mixtures which were prepared for testing are shown below in Table 1.

Table 1

Sample number Enzymes present ..................................................................... --.

CP 1 carboxymethyl cellulase (Multifect CL, Genencor) ....................................................

CP 2 ct-mannanase (YLIS, Amano) ... -_------.....

CP 3 carboxyinethyl cellulase (Multifect CL, Genencor), 150 mI + chitinase (Biochemis), 2 g + 01glucanase (Biochemis), 1 g ................... Z.... j........................

e celluIase (Multifect CL, Genencor), 150 m.1 + chitinase (Biochemis), 2 g + P-glucanase (Biochemis), 1 g + a-mannanase (YL 15, Amano), 2 g ......... 1..............

carbo xym ethy 1 cell ul ase ( Mult ifec t CL, Genencor), 150 mI + chitinase (Biochemis), 2 g + P-glucanase (Biochemis), 1 g + cc-mannanase (YL 15, Amano), 2 g + cutinase (Genencor), 2 g ................ 1....... 1........... 1. 1..

carboxymethyl cellulase (Multifect CL, Genencor), 150 nil + chitinase (Biochemis), 2 g + P-glucanase (Biochemis), 1.2 g + cc-mannanase (YL 15, Amano), 2 g + 1 1 cutinase (Genencor), 2 g 1 i 1 CP 7 carboxymethyl ce ' Itulase (Sumizyme C, Shin Nihon), 2 g + chitinase (Biochemis), 2 g + P-glucanase (Biochemis), 1.2 g + cc-mannanase (YL 15, Amano), 2 g + cutinase (Genencor), 2 g ......................................................

CP 8 . CP 9 ........... -1......... 11 CP 10 xylanase (Genencor), 15 0 ml + cutinase (Genencor), 2g . xylanase (Genencor), 75 mI + pectinase (Genencor), 75 ml + cutinase (Genencor), 2 g ...................................................................

xylanase (Genencor), 75 mI + pectinase (Genencor), 75 mI + cutinase (Genencor), 2 g + 0-glucanase (Biochernis), 1 g ............. 1................... 1 1..................

xylanase (Genencor), 75 mI + pectinase (Genencor), 75 mI + cutinase (Genencor), 2 g + P-glucanase (Biochemis), 2 g + chitinase (Biochemis), 0. 2 g In vivo teSI5 In all cases the compositions were applied to run off by high-volume spraying, using an airbrush. Each treatment was comprised of a single dose. Three plants were used for each test sample. The treated plants were laid out in a random fashion to avoid local differences in the growing enviroment or effects from proximity to other treated plants.

Example 1

Five test samples from Table 1 were tested to determine their effect on Leptosphaeria nodorum in wheat. This pathogen is responsible for wheat glume blotch. Wheat of the variety Hornet was grown to the three leaf stage in a glasshouse. Plants of identical stature and vigour were selected and treated with the test compositions. When dry (at least 3 hours after spraying) the plants were inoculated with a spore suspension, prepared from agar plates grown for 7 - 14 1 1 days at 20 'C and exposed to uv light for 12 hrs each day. The suspension was prepared by adding sterile distilled water containing 0.1 wt.% Tween 200 to each petri dish and scraping the mycelial surface to release the spores. The suspension was strained through muslin and the spore number counted using a 5 haemocytometer. The spore number was adjusted to ive 5 x 10 s ores cm-'. The 91 p spore suspension was then applied to the plants to the point of run off using a low pressure spray. The plants were then placed in polythene bags containing water, the bags were sealed to ensure a very high relative humidity and the plants were transferred to a Fisons growth cabinet. The cabinet temperature was maintained at 18 - 20 'C and the relative humidity was kept at 85 % or higher. Disease control was assessed after 14 days with each plant being scored on percentage disease on treated leaves. The results are shown in Table 2.

Table 2

Sample First plant Second plant Third plant Average % control Control 100 80 60 80 -- CP 1 -30 20 20 23 71 CP 2 10 10 10 10 88 CP 3 35 0 20 40 62 CP 5 10 10 10 10 88 CP 8 10 20 10 13 83 Mancozeb 10 20 10 All of the samples showed useful ontrol of the pathogen, and CP 2 and CP 5 were not only effective, but were actually superior to the wide- spectrum chemical fungicide, Mancozeb. Moreover, CP 8 was comparable to Mancozeb in effectiveness.

Examp-le-2 Six test samples from Table I were tested to determine their effect on Puccinia recondita in wheat. This pathogen is responsible for wheat brown rust. Wheat of the variety Avalon was grown to the three leaf stage in a glasshouse. Plants of identical stature and vigour were selected and treated with the test compositions. When dry (at least 3) hours after spraying) the plants were inoculated with a spore suspension prepared from 'stock plants infected with the pathogen which was sporulating freely. The spore suspension was prepared by cutting the leaves and placing them in a beaker containing distilled water and 0. 1 wt.% Tween 20 Gentle washing removed the spores and the concentration was determined usinc, a 1 0 haemocytometer. The spore numberwas adjusted to give 2 x 105 spores cm-3. The spore suspension was then applied to the plants to the point of run off using a low pressure spray. The plants were then transferred to a Fisons growth cabinet maintained at 18 - 24 'C where they were kept in the dark for 24 hrs following inoculation. Thereafter, the plants were grown in a 14: 10 hr daymight regime at a relative humidity of around 80 %. Disease control was assessed after 10 days with each plant being scored on percentage disease on treated leaves. The results are shown in Table 3.

Table 3

Sample First plant Second plant Third plant Average % control Control 90 70 80 80 -- CP 2 20 50 10 27 64 CP 3 20 30 10 20 75 CP 5 20 10 10 H 8n J CP 7 10 10 20 13 83 CP 8 10 20 10 13 H CP 11 10 20 10 1 1 H J Mancozeb 10 10 30 17 79 IA n All of the samples showed useful control of the pathogen. CP 5, CP 7, CP 8 and CP 11 were significantly superior to Mancozeb. Moreover CP 3 was comparable to Mancozeb in effectiveness and CP 2 was effective enough to provide a useful alternative to the chemical fungicie.

Exam-PLI C-1 Four test samples from Table 1 were tested to determine their effect on Phytbphthora infestans in tomatoes. This pathogen is responsible for late blight in 1 tomatoes and potatoes. Tomato seedlings of the variety First in the Field were grown to the two leaf stage in a glasshouse. Plants of identical stature and vigour were selected and treated with the test compositions. When dry (at least 3 hours after spraying) the plants were inoculated with a spore suspension prepared from agar plates that were 17 18 days old and sporulating regularly. The spore suspension was prepared by adding cold, sterile distilled water to the plates and agitating with a glass rod to release the sporangia. The liquid was decanted into a

C=.

large glass container and left at room temperature for 1 hr to allow for eneystment.

Z1 When encystment was 90 % complete, the spore number was adjusted to give 2 x 10 5 spores cm-3. The spore suspension was then applied to the plants to the point of run off using a low pressure spray. The plants were then placed in a metal tray containing water, covered with clear polythene to maintain the relative humidity at around 100 % and incubated in a growth room for 6 days at a temperature of 15 17 'C in a 12:12 day:night regime. Disease control was assessed after 6 days with each plant being scored on percentage disease on treated leaves. The results are shown in Table 4.

Table 4

Sample First plant Second plant Third plant Average % control Control 80 70 60 70 CP 1 10 5 0 5 93 CP 2 0 5 0 1.7 97 CP 4 20 10 20 17 66 CP 7 10 0 10 7 90 Mancozeb 0 0 0 0 100 The chemical fungicide proved very effective in this test giving complete control, but the compositions of the invention provide very effective alternatives. CP 1, CP 7 and in particular CP 2 showed control at 90 % or better which is more than satisfactory from a commercial viewpoint. CP 4 also showed a useful effect.

Examplc-4 Five test samples from Table -1 were tested to determine their effect on Peronospora parasitica in oilseed rape. This pathogen is responsible for downy mildew in brassicas. Oilseed rape plants were grown to the cotyledon stage. Plants of identical stature and vigour were selected and treated with the test compositions. When dry (at least 3 hours after spraying) the plants were inoculated with a spore suspension prepared from infected leaves containing sporulating mycelium. The spore suspension was prepared by agitating the excised leaves in distilled water containing 0. 1 wt.% Tween 200, The suspension was strained through muslin and the spore number was adjusted to give 2 x 105 Spores CM-3. The spore suspension was then applied to the plants to the point of run off using a low pressure spray. The plants were then placed in a metal tray containing a layer of water to maintain high relative humidity. Dise.ase control was assessed after I I days with each plant being scored on percentage disease on the treated cotyledons. The results are shown in Table 5.

11 Table 5

Sample First plant Second plant Third Average % control plant Control 100 80 60 - so --- CP 2 20 10 20 17 80 CP 5 30 40 20 30 61 CP 8 20 30 10 20 75 CP 9 0 10 0 3 96 CP 10 20 o 30 27 66 j Mancozeb 10 is 10 12 85 1 1 All of the samples showed good effect against the pathogen. CP 9 was significantly superior to Mancozeb and represents a particularly exciting alternative in controlling this pathogen. CP 2 was comparable to Mancozeb in effectiveness and the degree of control by CP 5, CP 8 and CP 10 was also useful.

In vitro test5 In all cases, agar plates were inoculated with the enzyme samples used in the invention. Discs of growing myeelium were placed at the centre of the plate. They were incubated at 22 'C in the dark for 8 days. Growth inffibition was measured by comparing the radial growth of the myeelium in comparison to untreated plates.

Example 5

Samples CP 4 - CP 6 and CP 8 - CP 11 were tested on the pathogen Rhizoctonia solani. The results are shown in Table 6.

Table 6

1 Sample number % control CP 4 79 CP 5 91 CP 6 81 CP 8 100 CP 9 100 CP 10 82 CP 11 7 3 Mancozeb 100 1 j.

Mancozeb provided total control in this test, but this is to be expected of an agrochernical fungicide in an in vitro test. However, CP 8 and CP 9 were just as effective and the other samples also displayed a useful degree of control, all being higher than 70 % and thus effective alternative fungicides.

Exampl?, 6 Samples CP 1, CP 5, CP 6, CP 9 and CP 10 were tested on the pathogen Pseudocercosporella herpotrichoides. The results are shown in Table 7.

Table 7

Sample number % control CP 1 81 CP 5 69 CP 6 83 CP 9 100 CP 10 85 Mancozeb 97 1 Mancozeb provided excellent control in this test, but it was surpassed by CP 9, This is surprising in view of the expected greater effect of an agrochemical fungicide in in vitro tests. SampIds CP 1, CP 6 and CP 10 also showed excellent control, whilst CP 5 had a useful effect.

Examplg,. 7 Samples CP 1, CP 4 - CP 6 and CP 9 - CP 11 were tested on the pathogen Botrytis cinerea. The results are shown in Table 8.

Table 8

Sample number % control CP 1 100 CP 4 77 CP 5 65 CP 6 78 1 1.

CP 9.71 CP 10 63 CP 11 67 Mancozeb 100 Mancozeb provided total control in this test, but CP I was just as effective. The other samples also displayed a useful degree of control, all being higher than 60 % and would be useful alternative fungicides.

Example 8

Samples CP 4, CP 6 - CP 8, CP 10 and CP 11 were tested on the pathogen Venturia inequalis. The results are shown in Table 9.

Table 9

Sample number % control CP 4 100 CP 6 93 CP 7 85 CP 8 62 CP 10 100 CP 11 75 Mancozeb 100 Mancozeb again provided total control as would be expected in an in vitro test, but CP 4 and CP 10 also gave total control. In addition, CP 6 was very effective, giving over 90 % control, whilst the remaining samples proved to be useful alternatives.

1 Tests on enzyme concentration The following tests were carried out on various pathogen/plant combinations in vivo to assess the effectiveness of the enzyme compositions and methods of the present invention with varying enzyme concentration. A dilution series of varying concentrations was arranged in each case. The initial test made use of the relevant sample as in Table 1. Each subsequent test used the same sample, but diluted to one tenth of the previous concentration. Where phytotoxicity was encountered, the disease control could not be determined and this is indicated by n.d. (not determined) in Tables 10 - 13.

Example-2

Five samples having the components listed in Table 1 were tested to determine their effect on Leptosphaeria nodorum in wheat. Winter wheat seedlings were incubated at 19'C under a 16:8 hr daymight regime at a relative humidity of 85 %, and inoculated with Leptosphaeria nodorum. The intensity of the infection was assessed 17 days after the treatments. Each value corresponds to the mean of 10 replicates. Untreated plants were tested for control purposes. The results are shown in Table 10.

Table 10

Compounds Concentration / dilution series % leaf area infected % Disease control 1 11- Untreated 63.5 CP2 1 13.6 78.6 0.1 13.9 78.1 0.01 25.7 59.5 0.001 45.8 27.8 CP4 1 Phytotoxicity n.d.

0.1 Phytotoxicity n.d.

0.01 16.0 74.8 0.001 23,1 63.6 CP8 1 slight phytotoxicity n.d. TI 0.1 18.0 71.6 1 0.01 16.1 74.6 0.001 42.2 33.6 CP9 1 9.7 84,7 0.1 8.6 86.4 0.01 5,9_ 90.7 0.001 20.1 68.3 CP1 1 1 Phytotoxicity ti.d 0.1 26.6 58.1 0.01 31.5 50.3 0.001 35.5 44.0 i 1 1_---. 1) - 17 Example 10

Four samples having the components listed in Table 1 were tested to determine their effect on Puccinia recondita in wheat. Winter wheat seedlings were incubated at 19'C under a 16:8 hr daymight regime at a relative humidity of 85 and inoculated with Puccinia recondita. The intensity of the infection was assessed 17 days after the treatments. Each value corresponds to the mean of 10 replicates. Untreated plants were tested for control purposes. The results are shown in Table 11.

Example 11

Four samples having the components listed in Table 1 were tested to determine their effect on Pyricularia oryzae in rice. Rice seedlings are incubated at 27C under a 16:8 hr daymight regime at a relative humidity of 95 %, and inoculated with Pyricularia oryzae. The disease index was assessed 14 days after the treatments. The disease index is the following: 0, no symptoms; 1, smaH lesions; 2, lesions with a light centre and a necrotic edge; 3, lesions with a grey centre more or less limited; 4, typical lesions with a grey centre and a diamond shape; 5, linked lightened area without necrotic, edge. Each value corresponds to the mean of 10 replicates. The results are shown in Table 12.

Example 12 Five samples having the components listed in Table 1 were tested to

determine their effect on Plasmopara viticola on grape vines. Leaf discs were incubated at 2CC under a 14:10 hr daymight regime at a relative humidity of 100 %, and inoculated with Plasmopara viticola. The intensity of the spor-ulation of Plasmopara viticola was assessed 9 days after the treatments. Each value corresponds to the mean of 10 replicates. The results are shown in Table 1 -33.

j I h Table 11

Compounds Concentration / dilution series % leaf area infected % Disease control Untreated 49.0 CP7 1 0.1 100.0 0.1 0.1 100.0 0.01 0.1 100.0 0.001 0.1 100.0 CP8 1 Slight phytotoxicity n. d.

0.1 0.4 99.2 0.01 0.4 99.2 0.001 15.1 69.1 CP9 1 0.0 100.0 0.1 0.0 100.0 0.01 0.0 100.0 0.001 14.6 70.2 CP1 1 1 Phytotoxicity n.d 0.1 15.6 68.2 0.01 14.6_ 70.2 0.001 3.0 93.9 Compounds 1 Untreated CP4 CP6 CP9 CP1 1 1 Concentration / dilution series 1 0.1 0.01 0.001 1 0.1 0.01 0.001 1 0.1 0.01 0.001 1 0.1 0.01 0.001 Table 12 % Disease index % Disease Control Leaf 2 Leaf 3 Leaf 2 Leaf 3 4.9 1.2 2.5 4.0 4.2 1.2 3,6 4.1 4.4 2.9 3.3 3.8 4.5 1.0 1.9 3.4 3.9 1 5.0 2.6 3.0 4.3 4.4 1.1 3.2 4.3 4.5 2.7 3.5 4.0 4.8 0.8 1.6 2.7 3.8 75.6 49.1 18.5 14.4 75.6 26.7 16.4 10.4 40.9 32.8 22.6 8.3 79.6 61.3 30.7 20.6 48.0 40.1 14.0 12.0 78.0 36.0 14.0 10.0 46.0 30.0 20.0 4.0 84.0 68.0 46.0 24.0 c - Table 13

Compounds Concentration 1 dilution series % leaf area infected % Disease Control Untreated 57.7 CP1 1 Phytotoxicity n.d.

0.1 2.9 95.0 0.01 6.7 88.4 0.001 12.0 78.0 CP2 1 2.7 '95.3 0.1 2.2 96.2 0.01 13.1 77.3 0.001 23.0 60.1 CP7 1 6.2 89.2 0.1 35.5 38.5 0.01 27.5 52.3 0.001 20.0 65.3 CP9 1 1.2 97.9 0.1 5.9 89.8 0.01 8.4 85.4 0.001 27.7 52.0 CP1 1 1 Phytotoxicity n.d.

0.1 5.3 90.8 0.01 14.1 75.6 0.001 19.0 67.1 1 -1, These Examples demonstrate that the enzyme compositions and methods of the present invention are effective over a wide range of concentrations. In some cases, where a large quantity of enzyme was used, some phytotoxicity was encountered. This was not serious and the enzyme compositions were effective in smaller quantities, showing significant disease control in comparison with the control samples.

From all of the above Examples it can be concluded that the compositions, methods and uses of the present invention provide useful alternatives to agrochernical fungicides which have been employed in the past. In addition to 7 being environmentally safe, in some cases they display superior effects to chemical fungicides.

\I-

Claims (1)

1. Claims:

   1 1. An enzyme composition comprising:

   (i) at least three enzymes selected from a carboxymethyl cellulase, an cc-mannanase, a xylanase, a chitinase, a P-clucanase, a cutinase and 0 a pectinase, wherein at least one of the enzymes is a carboxymethyl cellulase, an oL-mannanase, or a xylanase, and (ii) a diluent.

   i 2. An enzyme composition according to Claim 1, comprising a carboxymethyl cellulase, a chitinase and a P-glucanase.

   3. An enzyme composition according to Claim 2, additionally comprising an (x-mannanase.

   4. An enzyme composition according to Claim 3, additionally comprising a cutinase.

   5. An enzyme composition according to Claim 4, additionally comprising a further P-glucanase.

   6. An enzyme composition according to Claim 1, comprising a xylanase, a In cutinase and a pectinase.

   An enzyme composition according to Claim 6, additionally comprising a P- _ glucanase.

   8.

   An enzyme composition according to Claim 7, additionally comprising a chitinase.

   9. An enzyme composition according to Claim 9, wherein the diluent is water, and/or acetone.

   10. An enzyme composition according to any one of the preceding Claims, 1 tP additionally comprising an additive.

   ll- An enzyme composition according to Claim 10, wherein the additive is a 0 surfactant.

   12. An enzyme composition, substantially as hereinbefore described with reference to the Examples.

   13.

   A method of treating a plant having a fungal disease, or protecting a plant from a fungal disease, comprising applying to the plant an enzyme composition comprising:

   (i) a carboxymethyl cellulase, an a-mannanase, or a xylanase, a rid (ii) a diluent. t' 14. A method according to Claim 13, wherein the composition additionally comprises at least one further enzyme.

   16.

   17.

   18.

   19.

   20.

   21.

   15. A method according to Claim 14, wherein the further enzyme is a carboxymethyl cellulase, an (x-mannanase, a xylanase, a chitinase, a 0glucanase and/or a pectinase.

   A method according to Claim 15, wherein the composition comprises a carboxymethyl cellulase, a chitinase and a P-glucanase.

   A method according to Claim 16, wherein the composition additionally comprises an cc-mannanase..

   A method according to Claim 17, wherein the composition additionally comprises a cutinase.

   A method according to Claim 18, wherein the composition additionally comprises a further P-glucanase.

   A method according to Claim 15, wherein the composition comprises a 0 xylanase and a cutinase.

   A method according. to Claim 20, wherein the composition additionally comprises a pectinase.

   i i 22. A method according to Claim 2 1, wherein the composition additionally comprises a P-glucanase.

   23.

   A method according to Claim 22, wherein the composition additionally comprises a chitinase.

   24. A method according to any one of Claims 13 - 23, wherein the plant is W wheat, barley, oats, rice, maize, rye, triticale, potatoes, tomatoes, a brassica, or grapevines.

   25. A method according to any one of Claims - 24, wherein the fungus causing the disease belongs to the class Oomycetes, Ascomycetes, Basidiomycetes, or Fungi Imperfecti.

   26. A method according to Claim 25, wherein the fungus belongs to the class Oomycetes and is Phytophthora infestans, Pythium ultimum or Plasmopara Viticola.

   27. A method according to any one of Claims 135 - 24, wherein the fungus causing the disease is Erysiphe grarninis, Leptosphaeria nodorum, Puccinia recondita, Pyricularia oryzae, Botrytis cinerea, Rhizoctonia solani, Pseudocercosporella herpotrichoides, Peronospora parasitica, or Tlenturia inaequalis.

   28. A method according to Claim 13, wherein the composition comprises a 0 carboxymethyl cellulase, the plant is wheat and the fungus causing the disease is Leptosphaeria nodorum.

   29. A method according to Claim 1 -3), wherein the composition comprises an a.

   mannanase, the plant is wheat and the fungus causing the disease is Leptosphaeria nodorum.

   30. A method according to Claim 18 or Claim 20, wherein the plant is wheat and the fungus causing the disease is Leptosphaeria nodorum.

   0 1. A method according to Claim 18 or Claim 20, wherein the plant is rice and the fungus causing the disease is Rhizoctonia solani or Pyricularia oryzae.

   1 32. A method according to Claim.13, wherein the composition comprises an ctmannanase, the plant is a brassica and the fungus causing the disease is Peronospora parasitica.

   33. A method according to Claim 20 or Claim 21, wherein the plant is a brassica and the fungus causing the disease is Peronosporaparasitica.

   34. A method according to Claim 32 or Claim 33, wherein the brassica is oilseed rape.

   35. A method according to any one of Claims 18 - 20 or Claim 23), wherein the plant is wheat and the fungus causing the disease is Puccinia recondita.

   36. A method according to Claim 13, wherein the composition comprises a carboxymethyl cellulase, the plant is a tomato, or potato and the fungus causing the disease is Phytophthora infestans.

   37. A method according to Claim 13, wherein the composition comprises an ccmannanase, the plant is a tomato, or potato and the fungus causing the disease is Phytophthora infestans.

   38. A method according to Claim 19, wherein the plant is a tomato, or potato and the fungus causing the disease is Phytophthora infestans.

   39. A method according to Claim 13, wherein the composition comprises a carboxymethyl cellulase, the plant is a grapevine and the fungus causing the disease is Plasmopara viticola.

   40. A method according to Claim 13, wherein the composition comprises an ct Z mannanase, the plant is a grapevine and the ftingus causing the disease is Plasmopara viticola.

   A method according to Claim 18, wherein the plant is a grapevine and the CP fungus causing the disease is Plasmopara viticola.

   A method according to Claim 21, wherein the plant is a grapevine and the 0 fungus causing the disease is Plasmopara viticola.

   1, - 43.

   44.

   45.

   46.

   1 A method according to Claim,23, wherein the plant is a grapevine and the fungus causing the disease is Plasmopara viticola.

   A method according to any one of Claims 13 - 43, wherein the diluent water. - is A method according to any one of Claims 13 - 44, wherein the composition additionally comprises an additive.

   47.

   48.

   method according to Claim 45, wherein the additive is a surfactant.

   method according to any one of Claims 13 - 46, wherein the enzyme composition is applied to the plant by spraying.

   A method of treating a plant having a fungal disease, or protecting a plant from fungal disease, substantially as hereinbefore described with reference to the examples.

   49. Use of a carboxymethyl cellulase, an cc-mannanase or a xylanase as a plant fungicide.