WO2007139382A2 - Method for controlling a fungal foliar pathogens with an extract from yucca - Google Patents

Abstract

The invention relates to control of fungal foliar diseases in plants, preferably Rosaceae, such as apple scab (Venturia inaequalis), pear scab (Venturia pirina), brown spot (Stemphylium vesicarium) and powdery mildew (Podosphaera leucotricha) by treatment of plant with an extract of Yucca species, preferably Y. schidigera or Y. filamentosa. The treatment is effective in both prevention and treatment of the fungal infection.

Description

Title: New composition for control of foliar diseases

FIELD OF THE INVENTION

The invention relates to the field of plant pathogen control, particularly in Rosaceae, more particularly in fruit crops, such as apple, and particularly to the control of fungal foliar diseases, such as apple scab (Venturia inaequalis) and powdery mildew (Podosphaera leucotrichά).

BACKGROUND OF THE INVENTION

Apple scab is of major economic importance in the areas where apples are grown. If not controlled, the disease can cause extensive losses (70 percent or greater) where humid, cool weather occurs during the spring months. Losses result directly from fruit or pedicel infections, or indirectly from repeated defoliation which can reduce tree growth and yield.

Apple scab (see Fig. 1) can be observed on leaves, petioles, blossoms, sepals, fruit, pedicels, and less frequently, on young shoots and bud scales. The first lesions are often found on the lower surfaces of leaves as they emerge and are exposed to infection in the spring. Later, as the leaves unfold, both surfaces are exposed and can become infected. Young lesions are velvety brown to olive green and have feathery, indistinct margins. With time, the margins become distinct, but they may be obscured if several lesions coalesce. As an infected leaf ages, the tissues adjacent to the lesion thicken, and the leaf surface becomes deformed. Young leaves may become curled, dwarfed, and distorted when infections are numerous. The lesions may remain on the upper and lower leaf surface for the entire growing season; occasionally, the underlying cells turn brown and die, so that brown lesions are visible on both surfaces. The number of lesions per leaf may range from one or two to more than a hundred. The term "sheet scab" is often used to refer to leaves with their entire surfaces covered with scab. Young leaves with sheet scab often shrivel and fall from the tree. Infections of petioles and pedicels result in premature abscission of leaves and fruit, respectively. In late summer or early fall, lesions may appear whitish due to the growth of a secondary fungus on the lesion surface.

Lesions on young fruit appear similar to those on leaves, but as the infected fruit enlarge, the lesions become brown and corky. Infections early in the season can cause fruit to develop unevenly as uninfected portions continue to grow. Cracks then appear in the skin and flesh, or the fruit may become deformed. The entire fruit surface is susceptible to infection, but infections early in the season are generally clustered around the calyx end. Fruit infections that occur in late summer or early fall may not be visible until the fruit are in storage. This symptom is called "pin-point" scab, with rough circular black lesions ranging from 0.004 to 0.16 inch (0.1 - 4 mm) in diameter. Although research in New York has shown that the scab fungus can overwinter in trees as conidia on bud scales, the pathogen generally overwinters in leaves and fruit on the orchard floor. Ascospores are the major source of primary inoculum and are produced within pseudothecia that develop in leaves during the winter months. In a typical year in most locations, the first mature ascospores are capable of causing infections at about the time of bud break or soon thereafter. Ascospores continue to mature and are discharged over a period of five to nine weeks, with peak discharge during the pink to petal fall phenological stages. The length of time required for infection to occur depends on the number of hours of continuous wetness on the leaves and the temperature during the wet period. Young leaves remain susceptible for five to eight days, but their lower surfaces may become infected in late summer. For fruit, the duration of the wet period required for infection increases with the age of the fruit, which remains susceptible until harvest. Once the fungus is established in the leaf or fruit, conidia form on the surface of the lesion and become the source of secondary inoculum for the remainder of the season. Conidia are disseminated to developing leaves and fruit by splashing rain and wind. Several secondary cycles of conidial infection may occur during the growing season depending upon the frequency of infection periods and the susceptibility of host tissue.

Management of apple scab is multifaceted, with resistant cultivars, sanitation, and chemicals all being used to some degree depending on the orchard system being used and the goals of the grower.

Most of the major apple cultivars are susceptible to the fungus, although this varies somewhat. More than 25 scab-resistant cultivars have been released, included Prima, Priscilla, Jonafree, Redfree, Liberty, Freedom, Goldrush, and Pristine. Most are adapted to the more northern apple-growing areas of the U.S. All scab-resistant cultivars vary in their susceptibility to other early-season diseases; and all are susceptible to the summer diseases. Some recently released apple cultivars that have not been bred specifically for resistance to scab show varying levels of scab susceptibility, also.

Prevention of pseudothecial formation in overwintering apple leaves would probably eliminate scab as a serious threat to apple production.

Unfortunately, complete elimination of pseudothecia is not possible under orchard conditions with current methods.

Apple scab is controlled primarily with fungicide sprays. A variety of fungicide sprays with differing modes of action are available. When and how they are used depends upon their mode of action. Protectant fungicides prevent the spores from germinating or penetrating leaf tissue. To be effective, they must be applied to the surface of susceptible tissue before infection occurs. Occurrence of infection can, amongst others, be predicted with an accurate weather forecast. Protectant fungicides are applied routinely at 7 to 10 day intervals or according to anticipated infection periods.

Post infection fungicides control the scab fungus inside leaves and fruit. These chemicals can penetrate plant tissues to eliminate or inhibit lesion development. The ability of these fungicides to stop infections is limited to a few hours, or up to few days (depending upon the specific fungicide), and their effect often varies according to temperatures during the first 24 to 48 hours after infection. Some fungicides can inhibit the fungus even later into the incubation period (the time between infection and the appearance of symptoms). Eradication of scab lesions after they appear does not usually occur, but can be achieved with the proper rate and timing of certain fungicides. The selection of fungicides for management of scab is based on several factors, including the entire spectrum of other diseases that must be managed at that time, the potential for resistance in the scab fungus to the selected chemical, the history of the disease in a particular orchard, the final market for the fruit, and other social and economic factors. Good horticultural practices, such as proper site selection, tree spacing and annual pruning, facilitates better chemical control by improving spray coverage and reducing the length of wet periods. Chemical fungicides used in the treatment or prevention of apple scab include maneb, mancozeb, captan, pyrimethanil and tolylfluanide. Powdery mildew is another important disease of apple and pear trees in the world. It is caused by the fungus Podosphaera leucotricha. This fungus frequently infects new vegetative growth, causing reduced vigour, leaf malformation, and reduced viability of buds. Early infection of apple fruitlets results in a web-like russeting on the mature fruit. Infections causing fruit russet can occur from about 3 weeks before bloom to 3 weeks after bloom.

Powdery mildew may also reduce the vigour of trees and reduce return bloom (flowering next season). Young trees and vigorously growing shoots are the most susceptible.

The fungus overwinters (hibernates/survives the winter) as mycelium in buds which were infected the previous season. Infected buds are not as plump as healthy buds, and they often don't seal properly, resulting in a "feathered" appearance. Such buds are more sensitive to cold temperatures. Severe winter temperatures can reduce mildew pressure by killing infected buds, which are more susceptible to winter injury than healthy buds. However, even at lower temperatures some of the mildew survives in infected buds and inoculum is always available. The fungus also produces small black structures called cleistothecia, but the spores from cleistothecia do not play a major role in new infections.

New infections occur in the spring when infected buds open and the dormant fungus resumes growth on the developing tissues. These primary infections result in a white, powdery mass of spores which are easily spread by wind and splashing rain. Secondary infections result when these spores infect young leaves. Powdery mildew is favoured by moderate temperatures (10- 25⁰C) and high relative humidity. Late summer infection of buds results in establishment of mycelium inside the buds, that survives the winter in a dormant condition.

Cultivars such as Jonathan, Idared, Rome, Cox's Orange Pippin, Jonagold, Elstar, Mclntosh, Granny Smith, Gala and Braeburn are susceptible whereas Red and Golden DeliGious are more resistant. Pruning infected buds during the dormant season has not proven to be an effective cultural practice and is not regularly done. However, it is recommended to remove infected shoots as they appear early in the spring. Chemical control with fungicides is necessary when growing susceptible varieties.

Early spring applications of a fungicide beginning no later than tight cluster are necessary to prevent secondary spread of the mildew in apples. Neglecting control early in the year will result in poor control during the season. Repetition of applications is necessary weekly or every two weeks depending on climatic conditions. Fungicide applications should be continued until the buds are forming in July to reduce the amount of infected buds in which the fungus overwinters. To prevent fruit infection and subsequent russeting, sprays may be applied of fungicides containing of triadimenol, myclobutanil, flusilazole, kresoxim-methyl, trifloxystrobin, or sulphur. Also lime sulphur is effective for the control of powdery mildew. A good mildew control program in the current year reduces the amount of overwintering inoculum and the need for subsequent fungicide applications. Nevertheless, nowadays a tendency to reduce chemical fungicides, especially for food products, has become apparent. Also, there is a tendency to abolish the use of chemical pesticides and herbicides altogether in agriculture and horticulture, which has developed in the industry of organic growing. Organic growers prefer natural compositions, if any, for disease control on their crops. However, currently the most widely used fungicides in organic growing for control of apple scab are elemental sulphur (such as Kumulus S™ and Thiovit Jet™), lime sulphur and copper compounds (such as Kiocide™, Phyton 27™, Cuprofix Disperse™ and Funguran-OH 50 WP™). In many countries several of these fungicides can no longer be used, because of general safety and environmental risks (e.g. use of copper as a fungicide in the EU is to be phased out from 2006).

There is thus still need for a natural fungicide, which is effective for the control of apple scab and powdery mildew, and which is environment- friendly and non-toxic for humans and/or animals.

SUMMARY OF THE INVENTION

The present inventors now have discovered that extracts of Yucca species, especially Y. schidigera and Y. filamentosa are able to control foliar pathogen infection, especially fungal infections, e.g. of Venturia inaequalis and Podosphaera leucotricha. It is expected that similar results will be obtained from the use of extracts from other Yucca species.

The present invention, in a first aspect, relates to a method to control a fungal foliar pathogen in plants, preferably plants of the family Rosaceae, comprising treating said plants with a composition comprising an extract from Yucca as an antifungal agent. Preferably in such a composition the Yucca extract is unrefined. Also, preferably, the Yucca extract is the sole antifungal agent in said formulation.

Should said composition contain nutrients, said nutrient is preferably not yucca extract. Should said composition contain sticking agents, said sticking agent is preferably not yucca extract. Should said composition contain additional compounds effective in controlling pathological organisms on a plant, said additional compounds preferably do not include the compound according to formula (I) as defined in claim 1 of WO 96/41528. In a preferred embodiment of a method of the invention, said extract is from Yucca schidigera or Yucca filamentosa.

In another preferred embodiment said fungal foliar pathogen is selected from the group consisting of apple scab ( Venturia inaequalis), pear scab (Venturia pirina), leaf spot (Blumeriella jaapϊ), rose black spot (Diplocarpon rosae/ Marssonina rosae), brown spot (Stemphylium υesicarium), powdery mildew (Podosphaera leucotricha/ Sphaerotheca pannosa), begonia mildew (Microsphaera begoniae), strawberry powdery mildew (Sphaerotheca macularis), sooty blotch (Gloeodes pomigena), fly speck (Zygophalia jamaicensis), peach leaf curl (Taphrina deformans), brown rot/spur canker (Monilia fructigena, M. laxά), pear rust (Gymnosporangium sabinae/G. fuscum), canker (Nectria galligenά), rose black spot (Diplocarpon rosae), rust on roses (Phragmidium tuberculatum /Phragmidiurn spp.), and rot of strawberry (Phytophthora fragariae, P. cactorum).

In an even more preferred embodiment said fungal foliar pathogen is selected from the group consisting of apple scab, pear scab, brown spot and powdery mildew.

In yet another preferred embodiment, said extract is applied by spraying.

In another preferred embodiment, control of the fungal foliar pathogen comprises prevention of infection and/or fungal foliar disease (preventive treatment).

In an alternative preferred embodiment, control of the fungal foliar pathogen comprises treatment of fungal foliar disease and/or elimination of infection (curative treatment). In another aspect, the invention provides the use of an extract from Yucca for the control of fungal foliar disease in plants, preferably in plants of the family Rosaceae, preferably said fungal foliar disease is a pathogen selected from the group consisting of apple scab (Venturia inaequalis), pear scab (Venturia pirina), leaf spot (Blumeriella jaapi), black spot (Marssonina rosae), brown spot (Stemphylium vesicarium), powdery mildew (Podosphaera leucotricha/ Sphaerotheca pannosa), begonia mildew (Microsphaera begoniae), strawberry powdery mildew (Sphaerotheca macularis), sooty blotch (Gloeodes pomigena), fly speck (Zygophalia jamaicensis), peach leaf curl (Taphrina deformans), brown rot/spur canker (Monilia fructigena, M. laoca), pear rust

(Gymnosporangium sabinae/G. fuscum), canker (Nectria galligena), rose black spot (Diplocarpon rosae), rust on roses (Phragmidium tuberculatum / Phragmidium spp.), and rot (Phytophthora fragariae, P, cactorum), more preferably from the group consisting of apple scab, pear scab, brown spot and powdery mildew.

LEGENDS TO THE FIGURES

Figure 1 Scab symptoms caused by Venturia inaequalis on leaves (a) and fruit (b). Figure 2a-c. Apple scab severity scores (0= no scab symptoms, 7=

More than 75% scabbed leaf surface) on apple seedlings following treatments with water (control), 0.27% sulphur (standard fungicide) and various Yucca extracts at various concentrations following protective (Fig. 2a) and curative treatments 1 day (Fig.2b) and 2 days (Fig.2c) after inoculation. Figure 3a-b. Apple scab severity scores (0= no scab symptoms, 7= more than 75% scabbed leaf surface) on apple seedlings following treatments with water (control), 0.27% sulphur (standard fungicide) and Yucca schidigera extract at 0.5% (w/w) applied protectively (Fig.3a) and curatively (Fig.3b).

Figure 4. Apple powdery mildew (Podosphaera leucotricha) causing fruit russeting and mildew of primary leaves. DETAILED DESCRIPTION OF THE INVENTION

The term "Yucca extract" or its equivalent "an extract from Yucca" as used herein refers both to a Yucca-derived substance (a powder, a liquid, a chemical) as well as to a composition comprising the Yucca-derived substance and suitable for agronomical application which is intended to mitigate or control a fungal foliar pathogen as referred to herein, including for instance apple scab and pear scab. (i.e. for controlling the fungal pathogens). When not expressly mentioned, the skilled person will understand from the context of the description whether the Yucca-derived substance (the anti-fungal agent) or the (anti-fungal) composition is referred to. The Yucca-derived substance has been found to exhibit anti-fungal activities and is hence referred to herein as the "antifungal agent". The term "antifungal agent" includes not only a killing (fungicidal) effect but also an effect that prevents fungal infections to establish in the plant. For purposes of this invention, a composition for mitigating or controlling a fungal foliar pathogen may in addition to the yucca extract also include other compounds which can improve the performance of the composition. A yucca extract as referred to herein is preferably applied to the plant in the form of a composition comprising Yucca extract as the antifungal agent and an agronomically acceptable carrier.

As detailed above, apple scab and powdery mildew are diseases with tremendous economical impacts for the apple growers. This is especially the case for apple scab control by organic farmers, who have few or no alternative chemical pesticides, especially now that some of the agents that are commonly used are tending to be phased out because of environmental and general toxicity risks.

An alternative is being sought in the form of extracts from plants, since plant extracts have been described to be possible candidates for treating fungal diseases and since these would comply with the requirements for organic growing. The antifungal activity of several plant extracts has been claimed in the cosmetic industry and by alternative medicine. Searching the internet for these compounds yields thousands of hits, where the compounds are presented as effective, often without any proper experimental support for those claims. However, several extracts from plants to which antifungal effects have been attributed have recently been tested in careful experiments. Plant extracts among others, included extracts from: rapeseed oil, Equiseturn arvense, Malva sylvestris, Sambucus nigra, Saponaria officinalis, Tanacetum υulgare, Thymes vulgaris, Urtica dioica and Viscum album, to all of which antifungal effects have been attributed. The present inventors now found that especially extracts of Yucca were able to effectively control fungal foliar pathogens, such as apple scab.

There is, as yet, no indication what the active ingredient(s) of the extracts would be. Yucca has a high level of vitamins, especially vitamin A and B complexes. Further, it contains a high percentage of saponins, which are compounds to which all kinds of activities have been ascribed, such as antiinflammatory and insecticidal activities. It also contains resveratrol, which acts as an anti-oxidant. In horticulture, Yucca extracts have been applied as detergent for addition to sprays, but also as a fertiliser. In the (animal) food industry Yucca is used as a nutritional enhancer to improve digestion (e.g.

Kaya, S. et al, J. Animal Vet. Adv. 5(l):57-59, 2006; Nazeer, M.S. et al., Int. J. Poultry Sci. 1(6):174-178, 2002).

A saponin from Yucca schidigera has been described for use in a preparation for control of various plant pathogens in WO 96/41528. However, there the saponin is used as an additional component (i.e. not as active ingredient) for increasing the emulsifiability of solutions and is used in addition or concomitantly to another pesticide. It is clear from the specification, that the saponin is used as an emulsifier, and according to the author saponins from a variety of sources, like quillaja, agava, tobacco, liquorice, soybean, ginseng and asparagus would be equally applicable. Additionally, this patent application does not show experimental evidence that saponin enhances the pesticidal effect of the active component. In fact, there is no indication or suggestion that saponins, let alone Yucca extract, would exhibit any anti-fungal activity itself. Therefore, the prior art does not teach that Yucca extract can be used as an antifungal agent in compositions for treating fungal foliar pathogens.

Yucca extracts, suitable for use in aspects of the present invention, may be obtained by conventional extraction procedures or by drying and grinding (thereby obtaining powder) of leaves, roots and/or stems of Yucca. As can be seen from the experimental part described below, both treatment with an extract which is enriched in saponins (Saponin) and a non-enriched extract (Norponin®) outperform the current control strategies.

The Yucca extract used in aspects of the present invention may for instance be prepared by mixing powder of a suitable Yucca source with a suitable carrier for application. Suitable carries include both solid and liquid carriers. Water is a suitable liquid carrier. As a carrier any agronomically acceptable carrier can be used, i.e. any carrier to aid the dispersion of the active composition without impairing the active composition's effectiveness and which by itself has no significant detrimental effect on the soil, equipment, desirable plants, or the agronomic environment, may be used. A very suitable agronomically acceptable carrier is water. Other suitable carriers are solid carriers such as in the form of granules as described in more detail herein below.

Commercially available extracts can be used, such as Norponin® BS liquid (Nor-Natur ApS, Denmark), Yucca powder (Nor-Natur ApS, Denmark), Yucca-extract (Nor-Natur ApS, Denmark), Yucca pulver (from Yucca filamentosa, Natur-Drogeriet A/S Denmark), Saponin (saponin extract from Yucca, 80%, Deruned BV, The Netherlands), DK-35 Powder® or Yucca Ag- Aide™ (Desert King, San Diego, USA). Also, fresh extracts can be made by either milling or pulverising (parts of) the plants and optionally drying, or by extracting the plants or plant powder with cold or hot aqueous solutions or organic solvents.

The compositions comprising the Yucca extract may be in the form of liquids, powders, or solids and may be delivered in the form of for instance a solution, an emulsion, a suspension, a powder, a foam, a paste, a granulate or an aerosol. If in powder form, it can be dissolved in water or any suitable solvent and preferably filtered through a polyester mesh.

Suitable compositions may comprise of from 0.01 to 95 wt.% of Yucca extract (as a dry powder), based on the weight of the composition, in particular from 0.1 to 90 wt.% of the composition. Preferably a composition comprises more than 40, more preferably more than 50, even more preferably more than 60, yet more preferably more than 70 wt.% of Yucca extract as active substance. The concentration of active ingredient in a deliverable or ready for use formulation may vary in a wide range and may be as low 0.001 wt.% and as high as 99 wt%. A typical composition comprises Yucca extract in a concentration of from 0.1 to 10 wt%, preferably 0.5-5 wt%.

Suitable carriers or diluents or solvents for providing liquid formulations of the active substance include aromatic hydrocarbons such as xylene, toluene or alkylnaphtalene, chlorinated aromatic or chlorinated aliphatic hydrocarbons such as chlorobenzene, chloroethylene or methylenechloride, aliphatic hydrocarbons such as cyclohexane or paraffin, for instance mineral oil fractions, alcohols such as butanol or glycol and ethers and esters thereof, ketones such as aceton, methylethylketon, methylisobutylketon or cyclohexanon or strongly polar solvents such as dime thy lformamide, dimethylsulfoxide and water.

Volatile gas-like diluents or carriers, i.e. substances that are in the gas- phase at normal environmental temperature and pressure, may also be used and may include for instance butane, propane, nitrogen gas and carbondioxide. As solid carriers pulverized natural minerals such as kaolin clay, talc, chalk, quartz, montmorillon or diatom earth or pulverized synthetic minerals such as dispersed silica, aliminiumoxide, and silicate may be used. As solid carriers for granulate, also size reduced and fractionated natural stone materials such as calcite, marble, sepiolith and dolomite, or synthetic granulates from inorganic and organic powders such as from polymers or granulates from organic materials as coconut shells, corn cobs and tobacco stems may be used.

Typical amounts of said carrier in a composition comprising the yucca extract are between 1 and 99 % by weight of the composition, depending on the type of carrier chosen. The skilled person will appreciate that suitable amounts of carriers can be chosen to suit the particular application wherein the composition of the invention is used.

The composition may further comprise other ingredients to aid in dispersibility of the yucca extract (in particular the ingredient(s) that form(s) the active fungicidal agent) in water, to modify surface tension of the spray, or to promote adhesion of the composition to the plant leaves. These additional ingredients are collectively termed excipients and may include for instance inert carriers, diluents, surfactants, dispersants, spreaders, stickers, antifoam agents, thickeners, dyes, colorants, antifreeze compounds and emulsifiers. One skilled in the art will recognize circumstances where such excipients are typically combined with the yucca extract to be applied. Further inert ingredients useful in the present invention can be found in McCutcheon's, vol. 1, "Emulsifiers and Detergents," MC Publishing Company, Glen Rock, N,J,, U.S.A., 1996. Additional inert ingredients useful in the present invention can be found in McCutcheon's, vol. 2, "Functional Materials," MC Publishing Company, Glen Rock, New Jersey, U.S.A., 1996.

Any additives that can normally be present in plant sprays, such as wetting agents (like Zipper™) or co-solvents (such as DMSO), may be used in the compositions used in aspects of the invention.

Biological efficacy of anti-fungal agents is influenced by many factors, particularly the residence time of the anti-fungal agent on the treated surface, which is often a plant leaf or seed surface. A major factor influencing the residence time is the degree to which the anti-fungal agentresists wash-off by rain, that is, rainfastness. With liquid formulations, rainfastness may be improved by including ingredients in the formulation or adding such ingredients to the spray tank (tank mixing) that, during drying, provide a water-resistant bond between the anti-fungal agent and the substrate. For example, emulsified oil or water insoluble polymers prepared in emulsion have been used to improve liquid formulation rainfastness. One widely used material is a formulation comprising a carboxylated synthetic latex emulsion polymer, a primary aliphatic oxyalkylated alcohol, and water. This material has been shown to improve the efficacy of a variety of anti-fungal agents. Surfactants that may be employed in compositions of this invention include for instance one or more of various non-ionic, anionic, and amphoteric surfactants. Examples of non-ionic surfactants which are useful include polyalkylene glycol ethers and condensation products of alkyl phenols, aliphatic alcohols, aliphatic amines, and fatty acids with ethylene oxide, propylene oxide or their mixtures such as the ethoxylated alkyl phenols or ethoxylated aryl and polyaryl phenols and carboxylic esters solubilized with a polyol or polyoxyethylene. Anionic surfactants include salts of alkyl aryl sulphonic acids, sulphated polyglycol ethers, salts of sulfosuccinic acid esters with hydrophobes such as 2-ethylhexanol, salts of phosp hated polyglycol ethers, alkyl sarcosine salts, alkyl isethionate salts, and derivatives of taurine.

As dispersants, substances such as lignosulphonates and methylcellulose may be used. The compositions described herein comprising the Yucca extract are preferably applied as spray, to which extent the powderous extracts are for instance dissolved or dispersed in a suitable carrier and the liquid extracts are optionally diluted with a suitable solvent. The amount of spraying solution to be applied is about 1 to 50 litres per hectare, most preferably about 5-10 1/ha. The compositions described herein may for instance be applied to plant foliage as aqueous sprays by methods commonly employed, such as conventional hand-held (low-litre) spray bottles, high-litre hydraulic or pressurized sprayers, air-blast, and aerial sprays. Powders can be delivered by hand or by dispensers that are pushed or towed. Solid formulations can be delivered by hand or by machine.

The Yucca extracts of the invention can be applied for prevention and/or treatment of fungal foliar pathogens in a wide variety of plants, including crop plants and ornamental plants. Examples of ornamental plants include maple, elm, acer, juniper, spruce, magnolia, lavender, begonia, birch, oak, horsechestnut, and pine. Examples of crop plants include cereals, such as wheat, barley, corn, sorghum, and soy; asparagus; alfalfa; Solarium species, such as tomato and potato; sugar beet; and a large number of fruit plants, in particular species of the Rosaceae family. The plants in aspects of the present invention are preferably plants of the family Rosaceae. More preferably, the Rosaceae plant is a plant of the genus Prunus (plum and cherry), Malus (apple), Pyrus (pear), Fragaria (strawberry), Rosa (roses) and/or Rubus (e.g. raspberry). Even more preferably, the plant is a Malus spp. and/or Pyrus spp.. The various aspects of the present invention do not pertain to grape vine (Vitis υiniferά), and in preferred embodiments of aspects of the invention also not to other Vitis species. These species are not referred to when reference is made to a crop plant or ornamental plant in the context of the present invention.

It is believed that the extracts can be applied for a broad range of fungal foliar pathogens, which are able to infect these plants, such as, but not limited to: apple scab (Venturia inaequalis), pear scab (Venturia pirina), leaf spot (Blumeriella jaapi), rose black spot (Diplocarpon rosae/ Marssonina rosae), brown spot (Stemphylium υesicarium), powdery mildew (Podosphaera leucotricha/ Sphaerotheca pannosa), begonia mildew (Microsphaera begoniae), strawberry powdery mildew (Sphaerotheca macularis), sooty blotch (Gloeodes pomigena), fly speck (Zygophalia jamaicensis), peach leaf curl (Taphrina deformans), brown rot/spur canker (Monilia fructigena, M. laoca), pear rust (Gymnosporangium sabinae/G. fuscum), canker (Nectria galligenά), rose black spot (Diplocarpon rosae), rust on roses (Phragmidium tuberculatum / Phragmidium spp.), and rot of strawberry (Phytophthora fragariae, P. cactorum).

The effects of treatment of apple scab and powdery mildew with Yucca extracts have been demonstrated in the examples given below, which are meant to illustrate the invention and should not be regarded as limiting the invention in any way.

EXAMPLES

Example 1. Screening of yucca materials for Ventuvia inaequalis (Vi) control in apple (LIFE) on seedlings.

Summary

Among several potential materials for apple scab control an extract of Yucca schidigera (Norponin® BS Liquid, NorNatur ApS, Denmark) was identified under controlled conditions (project: DARCOF II-1.14: StopScab; Bengtsson et α/., 2005 IOBC /wprs Bulletin Vol. 29 (1): 123-127; and REPCO Project No. 501452). This extract has been further studied for its mode of action on the conidial infection of Venturia inaequalis . In the REPCO project other extracts made from Yucca have also been tested in plant assays and the conidium germination test and all were found very effective to control apple scab. These materials have been shown to have protective as well as curative action against the apple scab pathogen. Based on the results of LIFE the Yucca extract Norponin® BS Liquid was selected for testing under orchard conditions at DIAS (Danish Institute of Agricultural Sciences, Research Centre Aarslev, Denmark) in 2005. Material and Methods

Plant material

Seed oϊMalus x domestica 'Golden Delicious', were obtained from Eichenberg & Co, Miltenberg, Germany. For stratification, seeds are primed under running tap water for 24 hours, surface sterilised for 1 min. in 70% ethanol, 5 min. in 2-3% sodium hypochlorite and rinsed three times in tap water. Seeds are placed in moist sand and incubated in a refrigerator (ca.4°C) for 3-6 weeks. Germinated seeds are sown in small pots (5.5 cm) with a peat based substrate mixed with sand (3:1) and grown in a growth chamber (15-16⁰C, 12 hours light / 12 hours darkness) under plastic cover until use. Plants with 4-6 leaves are used, eight replicates per treatment.

Fungal inoculum Conidia of a single spore isolate of Venturia inaequalis (MB 363B, isolated in 1998 from 'Jonagold', Aarslev, Denmark) are produced by the "bottle wick method" modified from Williams (1976) Proceedings of Apple and Pear Scab Workshop, Kansas City, Missouri, July 1976. New York State Agricultural Experiment Station, Geneva. Special Report 28: 16-18. 300-ml flat glass bottles are mounted with a gauze wick (1 layer) along one side of the bottle and 20 ml Potato Dextrose Broth is poured in. The bottles are autoclaved and cooled before use. 250μl of a starter-spore suspension of Vi is pipetted into each bottle. The bottles are placed on their flat side with the gauze wick submerged in the medium and incubated for 4 days at 18°C in the dark. The bottles are turned on their narrow side so that the gauze dips into the medium and incubated for additional ten days at 18°C in the dark. The liquid medium is carefully poured off. The bottom of the bottle is rinsed with sterile water. Fifteen ml of water is added per bottle and agitated vigorously until a majority of conidia are dislodged from the gauze. The suspensions are strained through new gauze and centrifuged at 10000 rpm at 4°C for 30 min. The supernatant is eliminated and the residue containing conidia is re-suspended in sterile water. The conidial concentration is adjusted to 1.5 x 10⁵ conidia per ml. Inoculum is stored in Falcon tubes at -18⁰C until use.

Preparation of Yucca extracts

Yucca materials were obtained from several sources: Norponin® BS Liquid (Nor-Natur ApS, Denmark), Yucca powder (Nor-Natur ApS, Denmark), Yucca extract (Nor-Natur ApS, Denmark), Yucca pulver (Yucca filamentosa; Natur- Drogeriet AJS, Denmark), Saponin (Deruned, NL). Concentrated solutions are diluted on a v/v basis. Water extracts are made of powdered materials on a w/v basis by adding water, mixing and filtering through polyester mesh. Standard concentrations of 0.5, 1, 2.5 or 5% are used in seedling assay, 0.1-10% are used in conidia germination test.

Seedling assay

Yucca solutions are applied 1 day before inoculation (preventive treatment) or 1 or 2 days after (curative treatment) pathogen inoculation. Water and elemental sulphur (0.27% w/v) are used as standard control treatments in all tests. Preventive treatments: Yucca solutions are applied to the 4-5 youngest leaves with a hand sprayer to run off. Incubation at 15-16°C under a polyethylene cover to ensure humid conditions for 24 hours with no additional light. Inoculation: A conidial suspension of the fungus is applied with a hand sprayer to the 4-5 youngest leaves with a hand sprayer to run off. Plants are incubated for an additional 48 h at a temperature of 15-16⁰C under humid conditions (polyethylene bags) with no additional light. Hereafter the light is turned on, holes are made in the plastic bags and the plants are grown at 15-16°C, under 16-h photoperiod for 2 weeks. Curative treatments: Yucca solutions are applied 1 or 2 days after inoculation, otherwise as above. Symptom assessment: Symptoms normally begin to appear after 7-8 days after inoculation and the assessment is performed 14 days after inoculation. Symptoms are assessed using a grading scale derived from Croxall et al. (1952) Plant Pathology 1: 39-41; and Parisi et al. (1993) Phytopathology 93: 533-537: 0 = No visible symptoms; 1 =: 0% < percentage of scabbed leaf surface (sis) < 1%; 2 = 1%< sis <5 %; 3 = 5%< sis <10%; 4 = 10< sis <25%; 5 = 25%< sis <50%; 6 = 50%< sis <75%; 7 = sis > 75%. Disease severity is determined from the median obtained for the scores of all susceptible leaves on the day of inoculation. Effect of treatments on diseases severity is analysed statistically using the GLM procedure in SAS, version 8.2 (Statistical Analysis System,

SAS Institute, Cary, NC). The least significant differences (LSD) test is used to detect significant differences among means (P<0.05).

Conidium germination test Different concentrations (e.g. 0.1%, 0.5%, 1%, 2.5%, 5% or 10%) of the Yucca extracts are tested for effect on conidial germination of Vi. On single-well glass slides 35 μl of conidial inoculum of Vi is mixed with 35 μl of test solution and incubated at 18°C in a moist chamber. After 24 and 48 hours, the percentage of germinated conidia is assessed using a light microscope and compared to a water control, which is set to 100% germination. The tests are repeated three times per test solution.

Results

Treating apple seedling with the Yucca extract Norponin® BS Liquid (Nor- Natur ApS, Denmark) in concentrations 1, 2.5 and 5% have repeatedly shown significant control of apple scab in the plant assays. While the standard treatment with sulphur only acts preventively, this Yucca extract also acts curatively applied one or two days after inoculation. In the conidium germination test this yucca extract inhibited strongly the conidia germination at concentrations 0.5, 1, 2.5, 5%. In a histopathological study of its mode of action it was shown that this Yucca extract primarily acted by inhibiting conidial germination and germ tube elongation. In addition, on surviving conidia the yucca extract also had a significant inhibitory effect on several pre- and post penetration stages of the infection process.

Treatment with Norponin® BS Liquid (Nor-Natur ApS, Denmark) and other Yucca extracts have all shown promising apple scab control in the seedling assay as protective treatments at concentrations of 1 and 2.5 % (Fig. 2a) and also as curative treatments 1 day after inoculation (Fig. 2b) and 2 days after inoculation (Fig. 2c). A concentration of 0.5% Yucca extracts has also shown effect both as protective treatment (Fig. 3a) as well as curative treatment (1 day after inoculation) (Fig. 3b). Similarly, in the conidium germination test, all yucca extracts in the tested concentrations strongly inhibited germination.

Example 2. Field experiment 2004 (PPO)

Material

Orchard and equipment A field experiment was carried out in 2004 in the organic orchard of PPO in Randwijk, The Netherlands, during the summer epidemics. The experiment was done on Jonagold "Jonaveld" (Malus x domestica Borkh.) on M 27 rootstock and pruned as slender spindles. The trees were planted in a single row. Three rows of Jonagold were alternated with a row of Alkmene and a row Discovery which served as pollinators. The trees were planted in 1999 at a planting distance of 3 x 1.1m. Treatments were applied with a handheld spray gun (manufacturer EMPASS, Veenendaal, The Netherlands) with a 1.2 mm ceramic hollow cone nozzle at 1.1-1.2 Mpa with a volume of 1000 litre ha-1. Spray volumes were calibrated depending on the number of trees per hectare. Apple scab infection periods were determined by the Mety computer-based weather recorder (Boshuizen & Verheyden, 1994) and the Rimpro warning system (Trapman, 1994) throughout the ascospore infection period. The scab warning system calculated infection periods based on the hourly detected meteorological data, the modified Mills table, the simulation of ascospore release and effect of previously used sprays. The parameters measured were temperature, relative humidity, rainfall, leaf wetness, global radiation and wind speed. Tree growth stage was recorded according to Fleckinger-growth stage scale (Anon., 1984). Observations were made only on cv. Jonagold, which is highly susceptible to apple scab.

Treatments

Experiments were done according to EPPO-Guidelines 1/5 (3). Nine treatments, of which four are presented here, were replicated 6 times in different blocks, completely randomised within blocks. Each plot consisted of 5 trees. Observations were only made on the three middle trees of each plot. The bordering trees served as a buffer for neighbouring treatments. Rows of untreated trees served also as buffer. The treatments started from beginning of bud break until 10 June 2004. The treatments that were carried out were: 1) untreated, 2) copper hydroxide (0.2 kg/ha; Funguran-OH®), 3) sulphur (4 kg/ha; Thiovit Jet®) and 4) Yucca extract (7.5 1/ha; Saponin) plus sulphur (4 kg/ha; Thiovit Jet®) as a tank mix. The treatments were sprayed preventively just before a scab infection period according the RimPro scab warning system. Treatment schedules in 2004 are shown in Table 1. All treatments received 4-5 kg/ha wettable sulphur at weekly intervals from the end of June till harvest on 27 September in 2004. Sulphur was sprayed more frequent during rainy periods.

Applied products were: Funguran-OH® 50 WP (copper hydroxide, 50%, Asepta BV, The Netherlands), Thiovit Jet® (wettable sulphur, 80%, Syngenta Crop Protection BV, The Netherlands) and Saponin (saponin extract from Yucca, 80%, Deruned BV, The Netherlands).

Table 1. Treatment schedules applied during the scab infection season, 2004 phenological stage* treatment schedule

2004 copper sulphur saponin hydroxide + sulphur

1 April C3/D P P P

7 April D2/E P P

8 April D2/E P P P

20 April E2 P P P

28 April F/F2 P P P

4 May H P P P

28 May J P P P

1 June J P P P

8 June J P P P p = preventive according RIMPRO and weather forecast; *according to Fleckinger- growth stage scale (Anon., 1984)

Measurements

Scab incidence and severity

Disease assessments were made on leaves and fruits. For leaf assessment, 50 at randomly chosen clusters with rosette leaves from the 3 middle trees per plot were recorded on 24 May 2004. Leaf scab was also examined on 30 extension shoots from the 3 middle trees on 21 and 22 June 2004. The incidence of leaves was calculated as the percentage of leaves diseased. The severity was expressed as the mean number of lesions per leaf. Percentage of diseased fruits was examined by assessing all the fruits from the 3 middle trees of each plot between 4 and 14 October 2004. Incidence of fruit was calculated as the percentage of fruit diseased. The fruit were also assessed according to the following scale: 1 = no attack, 2 = 1-3 lesions per fruit, 3 = 4-6 lesions per fruit, 4 = 7-9 lesions, 5 = 10-20 lesions, 6 = 20-50 lesions and 7 = >50 lesions per fruit. The severity of the scab infection was expressed as a severity index (Sf). The Sf is calculated as [(number of fruits in scale I x O) + (number of fruits in scale 2 x l) + (number of fruits in scale 3 x 3) + (number of fruits in scale 4 x 5) + (number of fruits in scale 5 x 7) + (number of fruits in scale 6 x 9) + (number of fruits in scale 7 x 11)]/ total number of fruits.

Flower and leaf phytotoxicity

Flower phytotoxicity measurement was done on 29 April 2004. Leaf phytotoxicity observations were made on 14 June 2004. Phytotoxicity was scored by estimating the frequency and intensity of the damage. The three middle trees within a plot were assessed according the following scale: 0 = no phytotoxicity (pt), 1 = light pt (1-10%), 3 = moderate pt (10-33%), 5 = heavy pt (>33%). For each plot the mean phytotoxicity was calculated. The amount of russeting of the fruit was measured from all the fruits from the 3 middle trees of a plot between 4 and 14 October 2004. The fruit were assessed according to the following scale: 1 = non russet (0%), 2 = light russet (1-10%), 3 = moderate russet (11-33%), 4 = heavy russet (>33%). The russeting was expressed as a russet index (Ri). The Ri was calculated as [(number of fruits in scale I x I) + (number of fruits in scale 2 x 3) + (number of fruits in scale 3 x 5) + (number of fruits in scale 4 x 1)1 total number of fruits.

Class 1

The apples without scab and less than 10% russet yield the highest profit and are classified as class 1 apples. The percentage of class 1 apples was calculated by dividing the apples without scab and less than 10% russet from the total number of fruit. Statistical analyses

All data were subjected to analysis of regression using Genstat 6 Release 6.1 statistical package (Lawes Agricultural Trust, Rothamsted Research, UK). I^mprobabilities were calculated for pair wise comparison of treatment means. The number of lesions on fruit was subjected to analysis of variance (ANOVA). Significant F-tests (P < 0.05) were followed by a Least Significant Difference (LSD)-test for pair wise comparisons of treatment means using LSDo.os values.

Results In 2004 nine scab infection periods were recorded during the 12 weeks from the mid of March until the mid of June. The infection periods were severe on 20 March and 8, 9, 10, 29 April; moderate on 19 March, and low on 7 April and 4 and 7 May 2004. The treatment Saponin plus sulphur gave the best scab control. Saponin plus sulphur had significantly lower scab infestation than Funguran-OH® on the leaves of the extension shoots and on fruits (Table 2).

Table 2: Effect of treatments on incidence and severity of apple scab in 2004.

Cluster leaf Leaf of extension shoot Fruit

Treatment Incidence Severity Incidence Severity Incidence Severity schedules (%) (number of (%) (number of (%) (index) lesions) lesions)

Untreated 21.9 h 113.5 g 85.6 e > 1000.0 f 96.6 e 6.9 f

Funguran-OH® 2.2 bed 9.8 cd 25.4 b 206.8 be 39.9 b 0.9 ab

Thiovit Jet® (TJ) 5.2 ef 18.5 def 47.1 cd 478.3 de 73.5 cd 2.3 d

Saponin + TJ 0.7 ab 1.7 ab 13.5 a 85.2 a 14.8 a 0.2 a

F-test <0.001 <0.001 <0J 001 <0.001 <0.001 <0.001

The treatments with the highest efficacy against scab also gave the highest phytotoxicity on flowers. This did not result in a lower yield per plot (Table 3). Fruit treated with Funguran-OH®, and Saponin plus sulphur showed significantly more russeting than untreated fruit. Russeting caused by Saponin plus sulphur was not significantly different from that of Funguran- OH®.

Table 3: Effect of treatments on flower and leaf phytotoxicity, fruit russet index, yield and class 1 in 2004.

Flower Leaf Fruit Yield

Treatment Phytotoxicity Phytotoxicity Russet Fruit Class 1* schedules

(0-5) (0-5) (1-4) kg plot-1 (%)

Untreated 0.0 a 0.0 a 2.00 a 23.9 a 3.3 a

Funguran-OH® 0.2 a 0.0 a 3.13 cd 34.8 be 44.1 ef

Thiovit Jet® (TJ) 0.0 a 0.0 a 2.26 a 31.6 b 23.6 be

Saponin + TJ 0.7 ab 0.0 a 3.45 d 33.0 be 53.0 f

F-test O.001 - <0. 001 <0.001 <0.001

Class 1 = percentage fruit with russeting less than 10% without scab

Example 3. Field experiments 2006 (PPO)

Material and methods

Short description of the experiment

Natural compounds were sprayed according the RimPro scab warning system from start of bud break until the mid of June. Severity and incidence was measured on the leaves and the fruit. Phytotoxicitiy and russet was assessed. The natural compounds were compared with the standard biological fungicides copper hydroxide and sulfur.

Orchard and equipment A field experiment was carried out in 2006 in the organic orchard of PPO in Randwijk, The Netherlands, and in Tuil, the Netherlands during the summer epidemics. The experiment was done on Jonagold "Jonaveld" (Malus x domestica Borkh.) on M 27 rootstock in Randwijk and on Jonagold "Jonagored" on M 9 rootstock at the grower in Tuil and pruned as slender spindles. The trees were planted in a single row. Three rows of Jonagold were alternated with a row of Alkmene and a row Discovery, which served as pollinators in Randwijk. The trees were planted in 1999 at a planting distance of 3 x 1.1m. Treatments were applied with a modified air assisted "Urgent" cross flow sprayer (manufacturer Homeco Holland), with 2 x 5 nozzles Albuz yellow (height 0.50m - 2.00m) at a pressure of 8 bar (1000 1/ha). At the grower Jonagold rows were pollinated by Elise. The trees were planted in 1992-93 at a planting distance of 3.25 x Im. Treatments were applied with a handheld spray gun (Zeewolde sprayer) with a 1.2 mm ceramic hollow cone nozzle at a pressure of 8 bar with a volume of 1000 litre ha-1.

Apple scab infection periods were determined by the Mety computer-based weather recorder in Randwijk and Herwijnen (8.5 km from the grower in Tuil) and the Rimpro warning system throughout the ascospore infection period. The scab warning system calculated infection periods based on the hourly detected meteorological data, the modified Mills table, the simulation of ascospore release and effect of previously used sprays. The parameters measured were temperature, relative humidity, rainfall, leaf wetness, global radiation and wind speed. Tree growth stage was recorded according to

Fleckinger-growth stage scale. Observations were made only on cv. Jonagold, which is highly susceptible to apple scab.

Treatments Experiments were done according to EPPO-Guidelines 1/5 (3). Fifteen treatments were replicated five times in different blocks, completely randomized within blocks in 2006 in Randwijk. At the grower seven treatments were replicated four times in different blocks, completely randomized within blocks. Each plot consisted of 5 trees. Observations were only made on the three middle trees of each plot. The bordering trees served as a buffer for neighbouring treatments. Rows of untreated trees served also as buffer.

Field experiment 2006 The treatments were applied from 6 April until 29 May 2006 at PPO. The treatments that were carried out in Randwijk were:

1) Untreated,

2) Copper hydroxide (Funguran-OH; 0.5 kg/ha),

3) Sulfur (Thiovit Jet; 4 kg/ha), 4) Yucca extract 2.5 1/ha,

5) Yucca extract 5 1/ha,

6) Yucca extract 7.5 1/ha,

7) Yucca extract 2.5 1/ha + sulfur (4 kg/ha),

8) Yucca extract 5 1/ha + sulfur (4 kg/ha), 9) Yucca extract 7.5 1/ha + sulfur (4 kg/ha),

10) Potassium bicarbonate (85 % (Armicarb; KHCO₃; 2.5 kg/ha)),

11) Potassium bicarbonate (85 % (Armicarb; KHCO3; 5 kg/ha)),

12) Potassium bicarbonate (85 % (Armicarb; KHCO₃; 10 kg/ha)),

13) Potassium bicarbonate (85 % (Armicarb; KHCO₃; 2.5 kg/ha)) and sulfur (4 kg/ha),

14) Potassium bicarbonate (85 % (Armicarb; KHCO₃; 5 kg/ha)) and sulfur (4 kg/ha) and

15) Potassium bicarbonate (85 % (Armicarb; KHCO₃; 10 kg/ha)) and sulfur (4 kg/ha). The treatments were applied from 14 April until 19 June 2006 at the grower. The treatments that were carried out in Tuil were:

1) Untreated,

2) Copper hydroxide (Funguran-OH; 0.5 kg/ha),

3) Sulfur (Thiovit Jet; 4 kg/ha), 4) Yucca extract 7.5 1/ha, 5) Yucca extract 7.5 1/ha + sulfur (4 kg/ha),

6) Potassium bicarbonate (85 % (Armicarb; KHCO3; 5 kg/ha)),

7) Potassium bicarbonate (85 % (Armicarb; KHCO3; 5 kg/ha)) and sulfur (4 kg/ha). The potassium bicarbonate was applied in a more or less weekly schedule at Randwijk and sprayed as a tankmix with sulfur at the grower in Tuil. Sulfur was sprayed preventively just before a scab infection period according the RimPro scab warning system. Yucca extract was sprayed together with sulfur as a tankmix. The other treatments were sprayed when the RimPro scab warning system together with the weather forecast expected an infection period.

Applied products were: Funguran-OH 50 WP (copper hydroxide, 50%, Asepta BV, The Netherlands), Thiovit Jet (wettable sulfur, 80%, Syngenta Crop Protection BV, The Netherlands), Armicarb IOOF (potassium bicarbonate, 85%, Helena Chemical Company, United States of America) and Saponin (saponin extract from Yucca, 80%, Deruned BV, The Netherlands) Treatment schedules in 2006 are shown in Table 4 and 5. All treatments recieved 4-5 kg/ha wettable sulphur weekly intervals from end of June till harvest on 26 September in 2006. The grower also sprayed sulfur after the treatments. Sulfur was sprayed more frequent during rainy periods. Table 4. Treatment schedules applied during the scab infection season at PPO in Randwijk, 2006

Phenolo Treatment schedules 2006 gical 2 3 4 5 6 7 8 9 10 11 12 13 14 15 stage*

6 C w w w w w w

April

14 C2 P P P P P P P P pw PW pw pw pw pw April

19 D2-E P P P P P P P P pw pw pw pw pw pw

April

26 E-E2 p p p p p p p p pw pw pw pw pw pw

April

1 May E2 p p p p p p p p pw pw pw pw pw pw

15 G-H w w w w w w May

17 H-I P P P P P P P P P P P P P P

May

23 H-I w w w w w w

May

29 I P P P P P P P P pw pw pw pw pw pw

May w=weekly, p=preventive according RIMPRO and weather forecast; *according to Fleckinger- growth stage scale Table 5. Treatment schedules applied during the scab infection season at the grower in Tuil, 2006

Pheno Treatment schedules

2006 logical 2 3 4 5 6 7 stage*

*

3 April* - P P P P P P

12 - P P P P P P

April*

14 D P P P P P P

April

26 E2 P P P P P P

April

2 May E2-F P P P P P P

17 May H-I P P P P P P

24 May I P P P P P P

2 June I-J P P P P P P

19 June J P P P P P P p=preventive according RIMPRO and weather forecast; * only sulfur was sprayed by the grower, **according to Fleckinger-growth stage scale

Measurements

Disease assessments were made on leaves and fruits. For leaf assessment, 50 at randomly chosen spurleaf clusters from the 3 middle trees per plot were recorded on 31 May at PPO and on the 2nd of June 2006 at the grower. Leaf scab was also examined on 30 extension shoots from the 3 middle trees on 17 and 18 July at PPO and on 17 July 2006 at the grower. The incidence of leaves was calculated as the percentage of leaves diseased. The severity was expressed as the mean number of lesions per leaf. The fruit was harvested at 26 and 27 September at PPO and at 4 October at the grower. Percentage of diseased fruits was assessed by assessing all the fruits from the 3 middle trees of a plot between 5 and 10 October 2006 at PPO and on 12 and 13 October at the grower. Incidence of fruit was calculated as the percentage of fruit diseased. The fruit were also assessed according to the following scale: 1 = no attack, 2 = 1-3 lesions per fruit, 3 = 4-6 lesions per fruit, 4 = 7-9 lesions, 5 = 10 lesions, 6 = >10 lesions per fruit. The severity of the scab infection was expressed as a severity index (Sf). The Sf= [(number of fruits in scale I x O) + (number of fruits in scale 2 x l) + (number of fruits in scale 3 x 3) + (number of fruits in scale 4 x 5) + (number of fruits in scale 5 x 7) + (number of fruits in scale 6 x 9)]/ total number of fruits. No flower phytotoxicity was found in 2006. Leaf phytotoxicity observations were made on 19 June at the grower and on 17 and 18 July 2006 at PPO. Phytotoxicity was scored by estimating the frequency and intensity of the damage. The three middle trees within a plot were assessed according the following scale: 0 = No phytotoxicity (pt), 1 = Light pt (l_:10%), 3 = Moderate pt (10-33%), 5 = Heavy pt (>33%). For each plot the mean phytotoxicity was calculated. The amount of russeting of the fruit was measured from all the fruits from the 3 middle trees of a plot between 5 and 10 October 2006 at PPO and on 12 and 13 October at the grower. The fruit were assessed according to the following scale: 1 = non russet (0%), 2 = light russet (1-10%), 3 = moderate russet (11-33%), 4 = heavy russet (>33%). The russeting was expressed as a russet index (Ri). The Ri = [(number of fruits in scale I x I) + (number of fruits in scale 2 x 3) + (number of fruits in scale 3 x 5) + (number of fruits in scale 4 x 1)1 total number of fruits.

The apples without scab and less than 10% russet yield in the highest profit and are classified as class 1 apples. The percentage of class 1 apples was calculated by dividing the apples without scab and less than 10% russet from the total number of fruit.

Statistical analyses

All data were subjected to analysis of regression using GenStat Release 8.11 statistical package (Lawes Agricultural Trust, Rothamsted Research, UK). T- probabilities were calculated for pair wise comparison of treatment means. Significant F-tests (P < 0.05) were followed by a Least Significance Difference (LSD)-test for pair wise comparisons of treatment means using LSDO.05 values.

Results

Table 6: Effect of treatments on incidence and severity of apple scab at PPO in Randwijk in 2006

2006 Cluster leaf Leaf of extension Fruit shoot

Treatment schedules Incidence Severity Incidence Severity Incidenc Severity

(%) (number (%) (number e (%) (index) of of lesions)* lesions)*

Untreated 5.7 f 16.4 f 52.9 g - - 83.7 e 5.1 f

Copper 0.1 a 0.2 a 14.6 a - - 15.4 a 0.3 a

Sulfur 1.5 cde 3 cd 25.4 cd - - 47.4 be 2.0 bed e

Yucca extract 2.5 1/ha 2.3 e 4.6 e 37.3 f - - 61.4 be 2.8 de d

Yucca extract 5 1/ha 2.2 e 5.2 e 32.4 def - - 61.5 cd 3.4 def e

Yucca extract 7.5 1/ha 1.7 de 3.4 de 32.8 def - - 55.4 be 2.6 cde

Yucca extract 2.51/ha + sulfur 1.2 bcde 2.4 be 22.3 be - - 41.8 b 1.3 be de

Yucca extract 5 1/ha + sulfur 0.6 abc 1.6 ab 22.2 be - - 38.7 b 1.1 ab cd

Yucca extract 7.5 1/ha + sulfur 0.4 ab 0.8 ab 16.5 ab - - 44.0 be 1.9 bed

Potassium bicarbonate 2.5 2.1 e 4.8 e 36.3 ef - - 59.2 cd 3.4 def kg/ha e

Potassium bicarbonate 5 kg/ha 0.7 abed 1.6 ab 33.7 ef - - 72.5 de 4.2 ef cd

Potassium bicarbonate 10 0.5 abc 1 ab 29.8 cdef - - 49.7 be 2.1 bed kg/ha c

Potassium bicarbonate 2.5 0.7 abed 1.4 ab 29.7 cdef - - 49.5 be 2.1 bed kg/ha and sulfur cd

Potassium bicarbonate 5 kg/ha 0.5 abc 1 ab 31.9 def - - 41.9 b 1.0 ab and sulfur c Potassium bicarbonate 10 0.3 ab 0.6 ab 28.1 cde - - 46.7 be 1.2 abc kg/ha and sulfur

F-test O.001 <0.001 ^^ ; <0.001 <0.001

*Number of spots per 200 leaves

At PPO the scab incidence was low at the time of the cluster leaf observation. But in July the incidence of the untreated leaves of the extension shoot was over 50%. More than 80% of the untreated fruit was infected by scab at the harvest (Table 6). Both Yucca extract and potassium bicarbonate gave significant control of scab on leaves and fruit except for the middle concentration of Yucca extract and the lowest two concentrations potassium bicarbonate on the fruit. There was a tendency that the highest concentration of Yucca extract and potassium bicarbonate gave more control than the lower concentrations. Combining Yucca extract with sulfur gave a lower scab incidence but this was not always significant. On fruit the efficacy of sulfur alone was not significant different from the combinations of Yucca extract and sulfur. The severity on fruit after treatment of the middle concentration of Yucca with sulfur was not significant different from copper. Also the combination of potassium bicarbonate with sulfur gave no significant better scab control than without the combination with sulfur and the treatment sulfur alone (Table 6). Copper gave the best control of scab on leaves and fruit. The percentage of class 1 fruit was lower (35%) compared with some other treatments but this was not significant different. The treatments Yucca (5 1/ha) and potassium bicarbonate (5 kg/ha) both with sulfur gave the highest percentage of class 1 fruit with more than 50% fruit of class 1. No flower toxicity was found. Copper and sulfur caused significant leaf damage compared with the other treatments but the damage was low. On the other hand copper caused severe russet on the fruit and was significant different from the other treatments. No effect on yield was found (Table 7). Table 7: Effect of treatments on flower and leaf phytotoxicity, fruit russet, yield and class 1 in 2006

2006 Flower Leaf Fruit Yield

Treatment schedules Phytotoxicit Phytotoxic Russet Fruit Class 1* y ity

(0-5) (0-5) (1-9) kg plot- (%)

Untreated 0.0 a 0.0 a 1.7 abed 13.1 a 15.2 a

Copper 0.0 a 0.4 b 4.2 e 23.3 a 35.0 bcdef

Sulfur 0.0 a 0.4 b 1.7 abed 24.7 a 45.8 def

Yucca extract 2.5 1/ha 0.0 a 0.0 a 2.1 bed 25.4 a 31.9 abcde

Yucca extract 5 1/ha 0.0 a 0.0 a 2.2 d 23.5 a 29.4 abc

Yucca extract 7.5 1/ha 0.0 a 0.0 a 1.9 abed 29.3 a 38.1 bcdef

Yucca extract 2.5 1/ha + sulfur 0.0 a 0.0 a 2.2 cd 27.8 a 44.3 cdef

Yucca extract 5 1/ha + sulfur 0.0 a 0.0 a 2.1 cd 23.3 a 50.8 ef

Yucca extract 7.5 1/ha + sulfur 0.0 a 0.0 a 2.1 bed 21.0 a 44.7 bcdef

Potassium bicarbonate 2.5 kg/ha 0.0 a 0.0 a 1.6 abc ~23JT a 35.8 abed

__

Potassium bicarbonate 5 kg/ha 0.0 a 0.0 a 1.6 ab a 25.0 ab

__

Potassium bicarbonate 10 kg/ha 0.0 a 0.0 a 1.9 abed a 41.3 bcdef

__

Potassium bicarbonate 2.5 kg/ha and 0.0 a 0.0 a 1.5 a a 46.9 cdef sulfur

Potassium bicarbonate 5 kg/ha and 0.0 a 0.0 a 1.5 a ^"UT^" a 53.6 f sulfur

Potassium bicarbonate 10 kg/ha and 0.0 a 0.0 a 2.1 bed 23.9 a 43.0 cdef sulfur

F-test - 0.008 <0. 001 0.078 0.008

Class 1 = percentage fruit with russet less than 10% without scab

At the grower little scab was found at the time of the cluster leaf observation. Therefore no statistical analysis was done of the cluster leaf. Scab incidence was over 50% in the untreated plots when the leaves of the extension shoots were measured and almost 40% of the fruit was infected after harvest (Table 8). No flower and leaf phytotoxicity was found at the grower in none of the treatments (Table 9). Table 8: Effect of treatments on incidence and severity of apple scab at the grower in Tuil in 2006

2006 Cluster leaf Leaf of extension Fruit shoot

Treatment schedules Incidenc Severity Incidenc Severity Incidenc Severit e (%) (number e (%) (number e (%) y of of (index) lesions)* lesions)*

Untreated 0.2 - 0.5 51.1 d 394.5 c 39.8 e 1.0 d

Copper 0.2 - 0.5 5.8 a 18.3 a 7.5 ab 0.2 b

Sulfur 0.1 - 0.3 20.3 c 110.8 b 8.5 be 0.2 ab

Yucca extract 7.5 1/ha 0.0 - 0.0 20.8 c 98.5 b 11.6 cd 0.2 b

Yucca extract 7.5 1/ha + sulfur 0.0 - 0.0 6.1 a 25.0 a 5.1 ab 0.1 a

Potassium bicarbonate 5 kg/ha 0.1 - 0.3 12.6 be 48.0 ab 15.4 d 0.5 c

Potassium bicarbonate 5 kg/ha + 0.0 - 0.0 7.6 ab 34.5 a 3.3 a 0.1 a sulfur

F-test - - <0.001 <0.001 <0.001 <0.001

*Number of spots per 200 leaves

The treatments with copper, Yucca extract + sulfur and potassium bicarbonate + sulfur gave the best scab control on both leaves and fruit. There was no significant difference between these treatments except for the severity on fruit. Copper gave a significant higher severity than Yucca extract + sulfur and potassium bicarbonate + sulfur. On leaves sulfur alone was significant less effective than copper. On fruit sulfur was not significant different from copper. Yucca extract alone was significant effective against scab but the combination with sulfur was significant better on both leaves as fruit. Potassium bicarbonate also showed significant control compared with the untreated plots, the combination with sulfur was only on fruit significant better than potassium bicarbonate alone. The combination was also significant better than sulfur alone (Table 8). Copper showed good control of scab but also increased the amount of russet of the fruit compared with the other treatments. Therefore the percentage of class fruit was the lowest of all treatments including the untreated plots. The combination of potassium bicarbonate with sulfur gave the highest percentage of class 1 fruit but this was not significant different from the treatment with sulfur. No effect on yield was found (Table

9).

Table 9: Effect of treatments on flower and leaf phytotoxicity, fruit russet, yield and class 1 at the grower in Tuil in 2006

2006 Flower Leaf Fruit Yield

Treatment schedules Phytotoxicit Phytotoxicit Russet Fruit Class 1* y y

(0-5) (0-5) (1-9) kg plot'¹ (%)

Untreated 0.0 a 0.0 a 1.4 a 28.1 a 59.7 b

Copper 0.0 a 0.0 a 4.4 b 25.9 a 41.1 a

Sulfur 0.0 a 0.0 a 1.5 a 27.7 a 87.3 cd

Yucca extract 7.5 1/ha 0.0 a 0.0 a 1.5 a 27.3 a 83.3 cd

Yucca extract 7.5 1/ha + sulfur 0.0 a 0.0 a 1.7 a 29.5 a 89.2 cd

Potassium bicarbonate 5 kg/ha 0.0 a 0.0 a 1.6 a 27.0 a 80.0 C

Potassium bicarbonate 5 kg/ha + sulfur 0.0 a 0.0 a 1.6 a 27.0 a 91.9 d

F-test - - <0.001 0.485 <0.001

Class 1 = percentage fruit with russeting less than 10% without scab

Example 4. Field experiment 2 2005 (DIAS)

Material

Orchard and equipment

A field experiment was carried out in 2005 at the Danish Institute of Agricultural Sciences, Research Centre Aarslev, Denmark, during 5 weeks in the main period of ascospore discharge. The experiment was done on Jonagold (Malus x domestica Borkh.) on M. 9 rootstock and pruned as slender spindles. The trees were planted in a single row with every 11^th tree as the pollinator Discovery. The trees were planted in 1999 at a planting distance of 3.5 x 1.3m. Treatments were applied with a custom-built tunnel sprayed with 8 Tee-jet 110-02 with a volume of 1000 litre ha ¹. Results were assessed on cv. Jonagold, which is highly susceptible to apple scab.

Treatments

Experiments were done according to EPPO-Guidelines 1/5 (3). Twenty three treatments were replicated 5 times in different blocks, completely randomised within blocks. Each plot consisted of 5 trees. Results were assessed on the three middle trees of each plot. The bordering trees served as a buffer for neighbouring treatments.

Treatments were carried out with 3 — 5 days interval in total 10 applications at the following dates: 3/5 - 6/5 - 9/5 - 13/5 - 18/5 - 21/5 - 24/5 - 27/5 - 31/5 - 3/6

2005. All treatments were applied with 4 kg/ha Kumulus S® (Sulphur 80%) weekly for apple scab control from green tip until trial period and again after finish of trial period until beginning of September.

The treatments that were carried out and the dose rate are listed in table 10.

Material and Methods

Scab incidence and severity

Disease assessments were made on leaves and fruits. For leaf assessment, 100 spur leaf clusters from at least 10 clusters on the 3 middle trees per plot were recorded on 7 June 2005. Leaf scab was also examined on 200 leaves on at least 20 extension shoots from the 3 middle trees on 12 July 2005. The incidence of leaves was calculated as the percentage of leaves diseased. The severity was expressed as the mean number of lesions per leaf. Percentage of diseased fruits was examined by assessing all the fruits from the 3 middle trees of a plot on the 25 October 2004. Incidence of fruit was calculated as the percentage of fruit diseased. The fruit were also assessed according to the following scale: 0 = no attack, 1 = <0,25 cm² lesions per fruit, 2 = < 1 cm² lesions per fruit and 3 = > 1 cm² lesions per fruit. The severity of the scab infection was expressed as a severity index using Townsend & Heubergers equation: p _ «^» (v-l)*100

(Fmax-l) * iV

Where n = frequency of the relevant infection class v = the infection class,

Vhiax = strongest infection class N = number of leaves assessed

Fruit phytotoxicity The amount of russeting of the fruit was measured from all the fruits from the 3 middle trees of a plot 25 October 2005. The fruit were assessed according to the following scale: 0 = no russeting 1 = 1-10 %, 2 = 11-50 %, 3 = 51-100 % and cracs. The russeting was expressed as % fruits with russeting and as a severity index.

Table 10. Effect of treatments on incidence anc L severity of leaf scab in 2005.

Treatment Dose rate Rosette leaves 3 7/6 Long shoot 12/7

% % Spur No. of scab % Leaves No. of scab leaves with spots per with scab spots per scab spur leaf

1 Untreated 36,8 a 6,4 a 62,3 a 6,5 a

2 Copper oxychloride 0,02 10,0 be 2,0 ab 24,1 e 3,6 de

3 Kumulus S® (Sulphur) 0,4 5,9 be 1,0 b 32,0 cde 3,8 cde

4 Rapeseed oil 2,0 5,9 be 1,2 ab 29,3 de 3,5 de

5 Norponin BS® liquid 2,5 2,2 C 0,2 b 12,3 f 2,7 e

6 Quiponin BS® liquid 2,5 9,4 be 1,4 ab 43,1 bed 5,3 abc

7 Quiponin BS® liquid 5,0 10,2 be 1,7 ab 42,3 bed 5,1 abc

8 Nor-Spice® Fenugreek

2,5 16,3 be 3,0 ab 48,6 ab 5,4 abc Powder

10 Nor-Spice® Liquorice

2,5 9,4 be 1,7 ab ^' 45,5 abc 5,5 abc Powder

11 Nor-Spice® Liquorice

5,0 7,7 be 1,6 ab 42,4 bed 5,1 abc Powder

12 Nor-Spice® Tang 5,0 12,2 be 2,0 ab 50,9 ab 6,0 ab

13 Green meal 5,0 19,3 abc 4,1 ab 52,6 ab 6,2 ab

16 Equisetum arvense 5,0 14,1 be 2,9 ab 48,0 ab 5,6 ab

17 Malva sylvestris 5,0 16,8 be 3,0 ab 53,3 ab 5,9 ab

18 Sambucus nigra 5,0 18,1 abc 2,9 ab 55,3 ab 6,2 ab

19 Saponaria officinalis 5,0 8,9 be 1,9 ab 51,1 ab 5,2 abc

20 Tanacetum vulgare 5,0 17,8 abc 3,1 ab 50,9 ab 6,2 ab

21 Thymes vulgaris 5,0 27,1 ab 6,4 a 50,3 ab 6,0 ab

22 Urtica dioica 5,0 10,5 be 2,3 ab 53,9 ab 5,5 abc

23 Viscum album 5,0 21,8 abc 4,9 ab 56,3 ab 5,7 ab

24 Saponin NL product 0,75 4,9 be 1,0 b 38,5 bed 4,4 bed

25 Eugenol 2,5 9,7 be 1,2 ab 52,9 ab 5,5 abc

26 Chitoplant® 0,1 20,1 abc 3,5 ab 53,0 ab 6,1 ab

Means followed by same letter do not significantly differ (P = 0.05, Student- Newman-Keuls) Results

Treatments were carried out during the main ascospore discharge, however the first treatment was executed the day after the biggest discharge. The apple scab infections have been severe in 2005. Treatment 5, Norponin BS® liquid 2,5 % had the best apple scab control on leaves of rosettes and annual shoots in 2005 (Table 10). However the treatment also showed severe phytotoxicity on leaves in July (Data not shown). Rapeseed Oil, treatment 4 and Saponin NL product, treatment 24 also had an effect on apple scab on leaves (Table 10) and caused severe phytotoxicity (data not shown).

Due to a very dry autumn there were no infections of soothy blotch on the fruits. Fruit russeting, more severe than on untreated fruits were only observed in treatment 7, Quiponin BS® liquid 5% (Table 11). Apple scab infections on fruits were very severe. Untreated fruits were 100 % infected. Only treatment 5, Norponin BS® Liquid 2,5 % had a significant lower percentages of infected fruits compared to the control. Pest severity was significant lower for treatment 5 Norponin®, than for the two control treatment Copper oxychloride and Sulphur (Table 11). Treatment 4 Rapeseed oil had a disease severity at the same level as the control treatments. Treatment 24 Saponin NL-product had a tendency towards a control of apple scab on fruits (Table 11).

Table 11. Effect of treatments on incidence and severity of fruit scab and fruit russeting in 2005.

¹^ Means followed by same letter do not significantly differ (P=0.05, Student-Newman-Keuls)

Example 5. Field experiment Stemphylium vesicarium 2006 (PPO) Material

Orchard and equipment

A pear orchard located in Randwijk, The Netherlands was used for the field trials. The orchard was naturally infected by brown spot. The trees were planted in a single row. The cultivar that was used was Conference on Quince MC rootstock. Treatments were applied with a modified air assisted "Urgent" cross flow sprayer, manufacturer Homeco Holland, with 2 x 5 nozzles Albuz lilac (height 0,50m - 2,00m) at a pressure of 5 bar (3201/ha). Spray volumes were calibrated depending on the number of trees per hectare. Hourly weather data were recorded with a METY weather stations (Bodata, Dordrecht, The

Netherlands). The parameters measured were temperature, relative humidity, rainfall, leaf wetness, global radiation and wind speed.

Treatments The experiment was done in 4 replicates with 9 treatments. The treatments in 2006 were: 1) untreated control, 2) preventive thiram, 3) treatment a, 4) treatment b, 5) treatment c, 6) treatment d, 7) treatment e, 8) Yucca extract, 9) treatment f. The preventive treatments were sprayed weekly after full bloom until the harvest on the 21st of September 2006. The applied products and their dose were: 2kg ha-1 of Liro-Thiram Granuflo 80 WG (thiram, 80%, Syngenta Crop Protection BV, The Netherlands) and 7.51 ha-1 Yucca extract (Saponin (extract from Yucca), 80%, Deruned BV, The Netherlands).

Measurements

Disease incidence both on leaves and fruit was measured. The number of spots was measured only on fruit. The plots were 7 to 13 trees depending on the replicate. Observations were made on the 3, or 7 middle trees. The bordering trees served as a buffer for neighbouring treatments. Per plot, 800 leaves chosen at random were evaluated on the 28th and 29th of August 2006. At the harvest on the 21st of September, all fruit were picked. Fruit were stored until disease assessment. All the fruit that was harvested was used for disease assessment.

Statistical analyses

The percentage of diseased leaves and fruit were subjected to analysis of regression using Genstat 8.11 statistical package (Lawes Agricultural Trust, Rothamsted Research, UK). T-probabilities were calculated for pair wise comparison of treatment means. The number of spots on fruit was subjected to analysis of variance (ANOVA). Significant F-tests (P < 0.05) were followed by a Least Significance Difference (LSD)-test for pair wise comparisons of treatment means using LSDO.05 values.

Results In table 12 the mean incidence is presented of infected leaves by brown spot in August 2006. The first symptoms on leaves and fruit were found in the beginning of July. The percentage of infected leaves of the untreated control was 2.4 %. All treatments gave significant control of brown spot on leaves except for treatment f. Most of the treatments had less than 0.4 % of infected leaves. Only the Yucca extract had a higher incidence of 0.8 %.

On fruit 9.7 % was infected by brown spot at harvest (table 12). No efficacy was found on fruit of the treatments e and f were not significant different from untreated. Thiram, treatment a until d and the yucca extract gave significant control of brown spot on fruit. Treatment a and the Yucca extract were not significant from the treatment with thiram. Treatment b until d had all a significant higher efficacy than thiram. Also the number of spots was examined. Thiram and the Yucca extract had less spots than untreated but this was not significant. Treatments a until d had significant less spots than untreated. No effect on production was found between the treatments. Table 12: Effects of treatments on production and on brown spot on leaves and fruits in 2006

Example 6. Field experiment Powdery mildew on apple 2006 (PPO) Materials and methods

Short description of the experiment

Fungicides were applied with a cross flow sprayer from beginning of May 2006 (pink bud stage) till half June 2006. The set-up of the experiment was a randomised block design with nine treatments and four replicates. Assessments were made according to the EPPO-guideline PP 1/69 (3), on leaf and fruit infections. The new fungicides were compared to standards and a control treatment.

Orchard and equipment The experiment was conducted at parcel East 2 from Applied Plant Research

(PPO-fruit). This is an integrated maintained orchard with cultivars Jonagold and as a pollinator Alkmene, planted in single row system (3 x 1.1 m). The experiment was conducted on the cultivar Jonagold.

The trial area was marked with tape and individual plots were marked with a bamboo stick with the number of the treatment. The experiment was arranged according to a randomised block design with nine treatments and four replicates. Each plot consisted of 6 trees of which the middle 4 trees were used for observations and the other 2 were buffer trees. The lay-out of the experiment is depicted below, where A to D are replicates and numbers 1 to 9 are treatment numbers.

Treatments were applied with Homeco Urgent (Dieren, the Netherlands) cross flow sprayer, with 6 nozzles on each side and air support. The sprayer was operated single sided with 5 opened hollow cone Albuz orange nozzles (height 0,50 m — 2,00 m), a pressure of 5 bars and a forward speed of 1.8 km/hour. This resulted in a spray volume of 10001/ha.

Treatments

The following fungicides were applied from pink bud stage. 1. untreated

2. Exact (50 g triadimenol/1; 0,5 1/ha) + Merpan (80 % captan; 1,5 kg/ha)

3. Thiovit Jet (80 % sulphur; 4 kg/ha) + Merpan (80 % captan; 1,5 kg/ha)

4. Merpan (80 % captan; 1,5 kg/ha)

5. Thiram Granuflo (80 % thiram; 2 kg/ha) 6. Yucca-extract; 7,51/ha)

Dose of the treatments

Treatments were applied at about weekly interval, depending on weather conditions.

Measurements

Weather conditions during spraying will be noted together with the phenology of the tree. Disease assessments were made according the EPPO -guideline. A description of the measurements is shown below. 1. Counting the number of primary infections per plot during the end of bloom (May 2006).

2. Secondary mildew infection evaluation on June 2006 on 30 well developed shoots per plot (15 on each side of the tree). This was done by evaluating the four youngest leaves including the youngest fully unrolled leaf from each shoot. Hence 5 leaves per shoot. This was done on each side of the tree, hence in total 150 leaves per plot were evaluated. The number of leaves with and without mildew was recorded.

3. If occurring phytotoxicity will be a) described in words and b) evaluated according the scale: 0 = none; 1 = light; 2 = moderate and 3 = severe. Statistics

All data were subjected to analyses of variance (ANOVA) using Genstat Release 8.11 statistical package (Lawes Agricultural Trust, Rothamsted Research, UK). Significant F-tests (P < 0.05) were followed by a Least Significant Differences (LSD)-test for pair wise comparisons of treatment means using LSDO.05 values.

Results Spray schedule and weather conditions

In the following table (13) the dates of spray applications and weather conditions are shown, together with phenological stage (Phen) according to the Fleckinger scale.

Table 13

Phytotooάcity

No phytotoxicity was observed during the experiment.

Presence of primary infections

In the trial field, primary infections were counted on 29 May 2006, representing the infection pressure and distribution of inoculum over the area. The following table (14) shows the number of primary infections per plot. The presence of primary infections of mildew shows that powdery mildew is reasonable equally spread over the experimental field. Table 14 treatment number primary infections average per treatment untreated 3 1.8

Exact 1 0.8

Thiovit Jet 2 0.8

Merpan 1 0.8

Thiram 2 2.0

Yucca-extract 0 1.3

Presence of secondary infestation at 21 June 2006 The numbers of leaves with secondary infections, counted on 21 June 2006, are shown in the following table (table 15). Note that over one third of leaves in untreated plots had mildew symptoms. All treatments reduced mildew infection with respect to the untreated control. However Merpan and Thiram were less effective than the standard treatments Exact and Thiovit Jet. Topaz was more effective than the standard Exact. All other experimental treatments were equally effective as the standards Exact and Thiovit Jet.

Table 15 treatment % infected leaves average untreated 33.5 a

Exact + Merpan 12.8 d

Thiovit Jet + 9.9 de

Merpan

Merpan 22.9 be

Thiram 23.8 b

Yucca-extract 18.6 cd

F-value < 0.001

LSDo.05 = I 5.97 Yield

All fruits were taken from the middle four trees per plot. Fruits were stored at 5 ⁰C until examination of fruits. Numbers and weight of fruits were determined per plot. Data on numbers and weight are in Appendix 1. There were no significant differences in yield expressed as numbers (P = 0.465) and weight (P = 0.725) of fruits (Appendix 2).

Discussion

Spring was extremely late, cold and relatively dry in 2006 compared to average. Therefore, primary infections were examined almost four weeks later than in 2005. The presence of primary infections of mildew shows that powdery mildew is reasonable equally spread over the experimental field. The level of infection should be characterised as moderate considered from a fruit grower's point of view. The level of infection is considered rather low for testing fungicides, however.

The disease spreads from the primary infections to newly developed leaves, which are called secondary infections. The epidemic developed quickly in May and June 2006, resulting in high level of infection of about one third of all leaves, in untreated plots. Differences in efficacy between treatments were clearly visible end of June when all seven treatment sprays were applied. It is noted that the treatment of Exact is equally effective as that of Thiovit Jet, while in general Exact in known to have a better efficacy. Speculations about a reduced efficacy of Exact can not substantiated by this experiment, however. Other types of experiments are needed for that purpose.

Conclusions

We conclude that the efficacy of Yucca-extract tended to be less than that of the standard treatments Exact and Thiovit Jet, but this was not significant.

Claims

Claims

1. Method to control a fungal foliar pathogen in plants, preferably plants of the family Rosaceae, comprising treating said plants with a composition comprising an extract from Yucca as an antifungal agent.

2. Method according to claim 1, wherein said extract is from Yucca schidigera or Yucca filamentosa.

3. Method according to claim 1 or 2, wherein said fungal foliar pathogen is selected from the group consisting of apple scab (Venturia inaequalis), pear scab (Venturia pirinά), leaf spot (Blumeriella jaapi), rose black spot (Diplocarpon rosae/ Marssonina rosae), brown spot (Stemphylium υesicarium), powdery mildew (Podosphaera leucotricha/ Sphaerotheca pannosά), begonia mildew (Microsphaera begoniae), strawberry powdery mildew (Sphaerotheca macularis), sooty blotch (Gloeodes pomigena), fly speck (Zygophalia jamaicensis), peach leaf curl (Taphrina deformans), brown rot/spur canker (Monilia fructigena, M. laxa), pear rust (Gymnosporangium sabinae/G. fuscum), canker (Nectria galligena), rose black spot (Diplocarpon rosae), rust on roses (Phragmidium tuberculatum /Phragmidium spp.), and rot of strawberry (Phytophthora fragariae, P. cactorum), preferably said fungal foliar pathogen is selected from from the group consisting of apple scab, pear scab, brown spot and powdery mildew.

4. Method according to any of the previous claims, wherein said extract is applied by spraying.

5. Method according to any of claims 1-4, wherein control of the fungal foliar pathogen comprises prevention of infection and/or fungal foliar disease.

6. Method according to any of claims 1-4, wherein control of the fungal foliar pathogen comprises treatment of fungal foliar disease and/or elimination of infection.

7. Use of an extract from Yucca for the control of fungal foliar disease in plants, preferably in plants of the family Rosaceae, preferably said fungal foliar disease is a pathogen selected from the group consisting of apple scab (Venturia inaequalis), pear scab (Venturia pirind), leaf spot (Blumeriella jaapi), rose black spot (Diplocarpon rosae/ Marssonina rosae), brown spot (Stemphylium υesicarium), powdery mildew (Podosphaera leucotricha/ Sphaerotheca pannosa), begonia mildew (Microsphaera begoniae), strawberry powdery mildew (Sphaerotheca macularis), sooty blotch (Gloeodes pomigena), fly speck (Zygophalia jamaicensis), peach leaf curl (Taphrina deformans), brown rot/spur canker (Monilia fructigena, M. laxά), pear rust

(Gymnosporangium sabinae/G. fuscum), canker (Nectria galligena), rose black spot (Diplocarpon rosae), rust on roses (Phragmidium tuberculatum /Phragmidium spp.), and rot of strawberry (Phytophthora fragariae, P. cactorum), preferably from the group consisting of apple scab, pear scab, brown spot and powdery mildew.

8. Use of an extract from Yucca as an antifungal agent.