BIOCONTROL OF WEEDS

Abstract: The present invention provides a biological solution for controlling weeds of the family Chenopodiaceae in crops, but especially for the annual herbaceous weed Chenopodium album. In particular, the invention relates to a method of selectively controlling the weed Chenopodium album in crops, which comprises applying a herbicidally effective amount of the fungal pathogen Ascochyta caulina to the said weed plants, the seeds or seedlings thereof or the crop area. The invention further relates to strains of Ascochyta caulina that can be suitably used within the context of the present invention as mycoherbicides for controlling Chenopodium album in crops and to compositions comprising the same.

Description: BIOCONTROL OF WEEDS The present invention is in the field of biocontrol of weeds. In particular, the invention relates to novel microorganisms that are capable of controlling weed plants and to herbicidal compositions containing the same. The invention further relates to a novel herbicidal composition that contains a combination of a biocontrol agent according to the invention and one or more herbicides suitable for selectively controlling weeds in crops of cultivated plants, typically in crops of cereals. maize, rice, rape, sugar beet, sugar cane, plantations and soybeans. The invention also relates to a process for controlling weeds in crops of cultivated plants and to the use of said novel biocontrol agents and of compositions containing same. The biological approach to weed control aims towards offering alternative solutions for specific problems that cannot be satisfactorily dealt with by chemical control methods. One reason for the failure of the chemical approach may be that a specific chemical solution can only be offered for a specific crop while in other crops no such solution is available. Biocontrol of weeds has a long tradition, the first reports going back to the year 1865. Previously, the prevailing attention as control agents is given to insects owing to the good knowledge of their systematics, life history and plant association. the high degree of host specificity and the conspicuous damage they cause. Only recently other pathogens including viruses, bacteria, fungi and nematodes are increasingly being considered as an option for controlling weeds. Amongst the fungi, representatives of the Deuteromycetes or Fungi Imperfecti are most frequently used as mycoherbicides. For example, Morris eft at (1989) report the potential use of the leaf spot fungus Pheaeoramularia sp for the biological control of crofton weed (Ageratina adenophora) in South Africa. The potential of Colletrotrichum gloeosporioides to be employed in the biological control of Clidemia hirta in Hawaiian forests is reported by Trujillo et al (1986). Other fungal pathogens so far being examined for their potential to be used as a mycoherbicide in biocontrol of weeds include for example, Altemaria eichhomiae and Cercospora piaropi (for control of Eichhornia crassipes; water hyacinth); Attemada cassiae (for control of Cassia obtusifolia, sicklepod); Phytophthora palmivora (for control of Morrenia odorata; milkweed vine, strangler vine). The predominance of the Fungi Imperfecti is to be explained by their capability of being relatively easily cultured and induced to sporulate. They are thus potentially amenable to mass-production in large scale fermentations, one of the major prerequisites for a fungal pathogen to become employed as a biocontrol agent. Mycoherbicides which are commercially available to date include DeVine [Abbott Laboratories] and College9 [Ecogen Inc]. The active ingredient of DeVineç is Phytophthora palmivora, which is a pathogen against strangle vine (Morrenia odorata), a weed of the family Asclepiadaceael. College0 makes use of Colletotichum gloeospodoides which is a pathogen against Northem jointvetch (Aschynomene virginica), a leguminous weed. The present invention provides a biological solution for controlling weeds of the family Chenopodiaceae in crops, but especially for the annual herbaceous weed Chenopodium album, for which no such solution has been offered previously. Chenopodium album, commonly called 'white goose-foot', 'lambs quarters' or 'fett hen' on a worldwide basis is rated within the first five most common weeds. Chenopodium album is an annual herbaceous plant which can be found on arable fields and rural sites in almos all inhabitant areas of the word. It is distributed equally well in both the northern and southem hemispheres and can be found in Russia, North America, Europe, India, South Africa, Australia and South America. One of the major problems with this weed lies in its persistence and its relatively strong competitive ability. The seeds of Chenopodium album are able to remain viable for a period of up to 40 years. These characteristics rank C album the top of the list of noxious weeds. The present invention provides a novel biocontrol solution for the weed Chenopodium album employing the fungal pathogen Ascochyta caulina. Ascochyta caulina (P Karst) vd Aa & v Kest is a plant-pathogenic fungus that causes necrotic spots on leaves and stems of Chenopodium L and Atriplex L species. The fungus reproduces asexually by production of pycnidiospores in pycnidia. Prior to the present invention there is only one report on the use of Ascochyta caulina for controlling Chenopodium album [Eggers T and Thun K (1988)]. However, from the results provided in the said report it is to be concluded that the use of Ascochyta caulina is not an option for controlling Chenopodium album in crops, as the performance of this pathogen is reported to be poor. Within the scope of the present invention it is now surprisingly found that strains of Ascochyta caulina can be isolated which have the potential to control Chenopodium album in crops under field conditions. Strains of Ascochyta caulina that can be suitably used within the context of the present invention as mycoherbicides for controlling Chenopodium album in crops are those that are pathogenic to the weed plant only without negatively affecting the crop plant to be protected. Thus, in one embodiment of the invention strains of Ascochyta caulina are provided which are capable of controlling Chenopodium album in crops under field conditions. Preferred are Ascochyta caulina strains that are capable of developing appressoria-like infection structures upon infection of their host plants. The present invention further relates to a method of selectively controlling weeds of the family Chenopodiaceae , but especially of Chenopodium album, in crops, which comprises applying a herbicidally effective amount of one or more species of Ascochyta spp, but especially of the fungal pathogen Ascochyta caulina to the said weed plants, the seeds or seedlings thereof or the crop area. A further embodiment of the invention are herbicidal compositions for controlling the weeds of the family Chenopodiaceae , but especially of Chenopodium album, in crops comprising one or more species of Ascochyta spp, but preferably the fungal pathogen Ascochyta caulina, in a herbicidally-effective amount together with a suitable carrier and/or other compounds including both fertilizers or micronutrient donors or other preparations that influence plant growth of the crop plant to be protected; or selective herbicides, insecticides, fungicides, bactericides, nematicides, molluscides or mixtures of several of these preparations. Within the scope of the present invention, a herbicidally-effective amount is to be understood as an amount that is sufficient to keep the growth of the weed plant at a level that is no longer detrimental to the growth and yield of the crop plant to be protected, but does not produce disease symptoms on the crop plant itself. Preferred within the scope of the invention is a growth reduction of Chenopodium album of between 40% and 90% as compared to the untreated control, preferably of between 50% and 95%, more preferably of between 70% and 99%. Crop areas will be understood as meaning the areas already under cultivation with the cultivated plants or seeds thereof, as well as the areas intended for cropping with said cultivated plants. Surprisingly, it has now been found that a combination of a biocontrol agent according to the invention with a herbicide suitable for selectively controlling weeds in crops of cultivated plants, typically in crops of cereals, maize, rice, rape, sugar beet, sugar cane, plantations and soybeans, in a ratio varying within specific limits, exerts a synergistic effect that is able to control the majority of weeds occurring in crops of cultivated plants preemergence as well as postemergence, without substantial injury to the cultivated plants. Hence, especially preferred within the scope of the invention are synergistic herbicidal compositions comprising one or more species of Ascochyta spp, but preferably the fungal pathogen Ascochyta caulina, and one or more herbicides suitable for selectively controlling weeds, but especially weeds of the family Chenopodiaceae, in crops of cultivated plants in a synergistically-effective amount. A synergistic effect is always obtained when the herbicidal action of the combination of the biocontrol agent according to the invention and a herbicidal compound is greater than the sum of the action of the individual herbicides. In the present case, it is entirely surprising that the combination of the biocontrol agent according to the invention and a herbicidal compound exceeds the expected additive action against the weeds to be controlled and thus in particular enhances the activity range of both components in two respects: On the one hand, the rates of application of the biocontrol agent according to the invention and a herbicidal compound are reduced while the effectiveness of said compounds is retained. On the other hand, the novel herbicidal combination also achieves a high degree of weed control where the herbicidal compound is not or has become no longer agriculturally effective at low rates of application. The consequence is a substantial increase in the selectivity for the cultivated plants that is necessary and desirable in the event of unintentional overapplication of herbicide. In addition, the novel composition permits greater flexibility with respect to subsequent crops while retaining the excellent control of weeds in crops of cultivated plants. Most importantly, the synergistic effect to be observed is often most prominent two to four weeks after application of the herbicidal composition according to the invention, that is at the time most critical for crop development. Examples of herbicides that can be suitably used within the scope of the present invention in combination with one or more species of Ascochyta spp, but preferably the fungal pathogen Ascochyta caulina include, but are not limited to: 2- [ [ [ [4,6-dimethyl- pyrimidin-2-yl-amino] carbonyl] amino] sulfonyl]-benzoic acid oxetan-3-yl-ester; methyl [[2-chloro-4-fluoro-5- [(tetrahydro-3-oxo-1 H,3H- [1,3,4]thiadiazolo [3,4-a] pyridazin-1- ylidene)amino] phenyl] thio] acetate; (S)-2-chloro-N-(2-ethyl-6-methyí-phenyl)-N-(2- methoxy-1 -methyl-ethyl)-acetamide; 5-cyclopropylisoxazol4-yl 2 mesyl4trifluoromethylphenyl ketone; 4'-fluoro-N-isopropyl-2-(5-trofluoromethyl- 1 ,3,4-thiadiazol2-yloxy)acetanilide; methyl 2- [4,6-dimethoxypyrimidin-2-ylcarbamoyl)sulfamoyl] 6- (trifluoromethyl) nicotinate, monosodium salt; 1-(2-ethylsulfonylimidazol [1,2-a] pyridin 3-ylsufonyl)-3-(4 ,6-dimethoxypyrimidin-2-yl)urea; [3-(4,6-dimethoxypyrimidin-2-yl)- 1 -(2- ethoxyphenoxysulfo-nyl).urea; 2 ,4-dichloro-o-methylphenoxyacetic acid [2,4-D], 4chlorophenoxyacetic acid [MCPA]; 5- [2-chloro-4-(trifluoromethyl)phenoxy] -2nitrobenzoic acid [acifluorifen]; sodium 5-(2-chloro-4-trifluoromethylphenoxy)-2-nitrobenzoate [acifluorfensodium]; 2-chloro-N-(ethoxymethyl)-N-(2-ethyl-6-methylphenyl)- acetamide [acetochlor]; N-methoxymethyl-2',6'-diethyl-2-chloroacetanilide [alachlor]; N- [ [[ [(4,6-dimethoxy-2-pyrimidinyl)amino] carbonyl] amino] sulfonyl] -N-methylmethanesulfonamide [amidosulfuron]; 2-chloro-4-ethylamino-6-isopropylamino-1,3,5- triazine [atrazine]; 2-chloro4-ethylamino6-tertiary-butylamino-1 ,3 5-triazine [terbuthylazine]; 3-(1 -methylethyl)-(1 H)-2, 1 ,3-benzothiadiazin-4(4(3H)-one 2,2-dioxide [bentazone]; methyl 5-(2,4-dichlorophenoxy)-2-nitrobenzoate [bifenox]; 3,5-dibromo-4hydroxybenzaldehyde O-(2,4-dinitrophenol)oxime [bromfenoxim]; 3,5-dibromo-4hydroxybenzonitrile [bromoxynil]; 5-amino-4-chloro-2-phenyl-3(2H)-pyridazinone [chloridazone]; ethyl 2- [ [ [[(4-chloro-6-methoxy-2-pyrimidinyl)amino] carbonyl amino] sulfonyl] benzoate [chlorimuron]; 3-(3-chloro4-methylphenyl)-1 ,1-dimethylurea [chlortoluron]; 2-chloro-N-(4-methoxy-6-methyl- 1,3, ,3,5-triazin-2-yl-aminocarbonyl)- benzenesulfonamide [chlorsulfuron]; 2-(4-chloro-6-ethylamino- 1,3, 5-triazin-2-yl- amino)2-methylpropionitrile [cyanazine]; ethyl [3- [ [(phenylamino)carbonyl] oxy] - phenyl]carbamate [desmedipham]; 3,6-dichloro-2-methoxybenzoic acid [dicamba]; 1 ,2-dimethyl-3,5-diphenyl- 1 H-pyrazolium [difenzoquat]; N-(2,4-difluorophenyl)-2- [3 (trifluoromethyl)phenoxy ] -3-pyridinecarboxamide [diflufenican]; 2-chloro-N-(2,6dimethylphenyl)-N-(2-methoxyethyl)acetamide [dimethachlor]; (RS)-2-chloro-N-(2 4- dimethyl-3-thienyl)-N-(2-methoxy-1 -methylethyl)acetamide [dimethenamide]; (i)-2 ethoxy-2 , 3-dihydro-3, 3-dimethyl-benzofuranyl methanesulfonate [ethofumesate]; 3chloro-5-[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl] amino]sulfonyl]-1- methyl H-pyrazole-4-carboxylic acid [halosulfuron]; N-(2 ,4-difluorophenyl)-5-methyl- [ 1,2,4] triazolo 5-a] pyrimidine-2-sulfonamide [flumetsulam]; [(4-amino-3,5 dichloro-6-fluoro-2-pyridinyl)oxy] acetic acid [fluroxypyr]; (i)-2- [4, 5-d ihydro-4-methyl-4- (1 -methylethyl)-5-oxo- H-imidazol-2-yl] -4(and 5)-methylbenzoic acid (3:2) [imazamethabenz]; 2-(4-isopropyl4-methyl-ioxo-imidazolin-2-yl)-3-quinolinic [imazaquin]; (+)-2-[4,5-dihydro-4-methyl-4(1-methylethyl)-5-oxo-1H-imidazole-2-yl]-5ethyl-3-pyridinecarboxylic acid [imazaethapyr]; 4-hydroxy-3, 5-diiodobenzonitrile tioxynil]; 3-(4-isopropylphenyl)- 1,1 -dimethylurea [isoproturon]; (i)-2-ethoxy-1 -methyl-2oxoethyl 5- [2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoate [lactofen]; 3 cyclohexxyl6, 7-dihydro-l H-cyclopentapyrimidine-2,4(3H,5H)-dione [lenacil]; 4-amino 3-methyl-6-phenyl- 1,2 ,4-triazin-5(4H)-one [metamitron]; 3-(2-benzothiazolyl)-1,3dimethylurea [methabenzthiazuron]; 2-chloro-2'-ethyl-6'-methyl-N-(2-methoxy- 1 - methylethyl)acetanilide [metolachlor]; N-(2 ,6-dichloro-3-methylphenyl)-5,7-di methoxy- [1,2,4] triazolo [1,5-a] pyrimidine-2-sulfonamide [metosulam]; 4-amino-6-tertiary-butyl 3-methylthio- 1 2,4-triazin-5(4H)-one [metribuzin]; metsulfuron; methyl 2-[3-(4-methoxy6-methyl-1,3,5-triazin-2-yl)ureidosulfonyl]-benzoate [metsulfuron-methyl], 2-[[[[(4,6- dimethoxy-2-pyrimidinyl)amino] carbonyl] amino] sulfonyl] -N,N-dimethyl-3-pyridinecarboxamide [nicosulfuron]; N-(1-ethylpropyl)-3,4-dimethyl-2,6-dinitrobenzenamine [pendimethalin]; 3- [(methoxycarbonyl)amino] phenyl(3-methylphenyl)carbamate [phenmedipham]; 2-[[[[[(4,6-bis(difluromethoxy)-2-pyrimidinyl)amino]carbonyl]- amino] sulfonyl] benzoic acid lprimisulfuron]; 1-(4-methoxy-6-methyl-triazin-2-yl)-3- [2 (3,3,3-trifluoropropyi)-phenylsulfonyl]-urea [prosulfuron]; O-(6-chloro-3-phenyl-4- pyridazinyl) S-octyl carbonothioate [pyridate]; 7-chloro-3-methyl-8-quinolinecarboxylic acid [quinmerac]; N- [ [ (4,6-dimethoxy-2-pyrimidinyl)amino] carbonyl] -3-(ethylsulfonyl)2-pyridinesulfonamide [rimsulfuron]; 2-chloro-4,6-bis-(ethylamino)-1 3, 5-triazine [simazine]; Schloro-N-(1, 1 -dimethylethyl)-M-ethyl-1 ,3,5-triazine-2,4-diamine [terbuthyl- azine]; N-( 1,1 -dimethylethyl)-!'I'-ethyl(methylthio)- 1,3, 5-triazine-2,4-diamine [terbutryn]; 3- [ [ [ [(4-methoxy-6-methyl-1 ,3,5-triazin-2-yl)amino] carbonyl] amino]- sulfonyl] -2-thiophenecarboxylic acid [thifensulfuron]; S-(2,3,3-trichloro-2-propenyl) bis( I -methylethyl)carbamothioate (triallate]; 2-(2-chloroethoxy)-N- [ ((4-methoxy-6 methyl ,3,5-triazin-2-yl)amino] carbonyl) benzenesulfonamide (triasulfuron]; 2- [[[[(4 methoxy-6-methyl-1 ,3 , 5-triazin-2-yl(methyl)carbonyl] amino ] sulfonyl] benzoic acid [tribenuron]; 2,6-dinitro-N, N-dipropyl4-trifluoromethylaniline [trifluralin]. In general, within the synergistic compositions according to the invention one or more of herbicides can be used selected from the group consisting of diphenyl ether herbicides, anilide herbicides, chloracetamide herbicides, imidazolinone herbicides thiolcarbamte herbicides, pyridazine herbicides, triazine-type herbicides and sulfonylurea herbicides. Especially preferred to be used in synergistic compositions according to the invention are triazine-type, pyridazine herbicides and sulfonylurea herbicides. In this case, the synergistic effects that can be obtained are such that the dosage of the pathogen, one of the two effective ingredients, and also the dosage of the chemical herbicide can be reduced. The rate of application can vary over a wide range and will depend on the nature of the soil, the type of application (pre- or postemergence), seed dressing, application to the seed furrow; no tillage application etc.), the cultivated plant, the biocontrol strain employed, the weed to be controlled, the respective prevailing climatic conditions; and on other factors govemed by the type and time of application and the target crop. The herbicidal combination can be usually be applied in a rate of application of between 108 to 101A spores/ha, preferably between 1010 and 10Ú spores/ha of biocontrol agent and 0.01 to 2 kg/ha, preferably 0.02 to 1 kg/ha herbicide. The biocontrol strains according to the invention can be applied to the weed plants to be controlled either directly or in form of compositions comprising a suitable carrier. The biocontrol strains can be in form of vegetative cells or, preferably, in form of spores, which may be ascospores or conidiospores (pycnidiospores). Contact between the pathogen and the weed to be controlled can be achieved by applying the biocontrol strains of the instant invention or a composition comprising same to an environment in which the weed plants may grow. Application can be made to soil prior or shortly after the weed plants have emerged. Alternatively, the biocontrol strains according to the invention or a composition comprising same can be applied directly to the weed plant after the plant has emerged. If application is made to the plant, one may choose between leaf application and application to the flowering plant. The strains will be applied in an effective amount, preferably together with a suitable carrier. That is, in an amount sufficient to control or inhibit the growth of the weed plants. The rate of application may vary according to the crop to be protected, the efficacy of the biocontrol strain employed, the severity of the weed infestation and the environmental conditions. Generally, the rate of application will be in a range of about 105 spores to about 1012 spores/l, specifically of about 106 spores/l to about 1011 spores/l, more specifically of about 107 spores to about 1010 spores/l. The spore densities preferably to be achieved, expressed as spores/cm2, are in a range of about 10 to about 108 spores/cmê, preferably of about 105 to about 107 spores/cmê and more preferably of about 105 to about 106 spores/cmê. The biocontrol strains of the present invention are normally applied in the form of compositions and can be applied to the crop area or plant to be controlled, simultaneously or in succession, with other compounds. These compounds can be both fertilizers or micronutrient donors or other preparations that influence plant growth of the crop plant to be protected. They can also be selective herbicides, insecticides, fungicides, bactericides, nematicides, molluscides or mixtures of several of these preparations, if desired, together with further agriculturally acceptable carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation. Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers. Further supplements that can be advantageously used within the compositions according to the invention are compound that have a nutritional value for the biocontrol strain according to the invention. Compounds that may be suitably used in this respect are, for example, those present in Czapek Dox broth and/or Yeast extract. Target crops to be protected within the scope of the present invention comprise e.g., the following species of plants: cereals (wheat, barley, maize, rye, oats, rice, sorghum and related crops), beet (sugar beet and fodder beet), pomes, drupes and soft fruit (apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries and blackbenies), leguminous plants (beans, lentils, peas, soybeans), oil plants (rape, mustard, poppy, olives, sunflowers, coconut, castor oil plants, cocoa beans, groundnuts), cucumber plants (cucumber, marrows, melons), fiber plants (cotton, flax, hemp, jute), citrus fruit (oranges, lemons, grapefruit, mandarins), vegetables (lettuce, asparagus, cabbages, carrots, onions, tomatoes, potatoes, paprika), lauraceae (avocados, cinnamon, camphor), or plants such as maize, tobacco, nuts, coffee, sugar cane, tea, vines, hops, bananas and natural rubber plants, as well as omamentals (composites). The biocontrol strains according to the invention can be obtained from nature by isolating them in pure form from Chenopodium album plants that are naturally infected. Strains that can be suitably used within the scope of the present invention can be identified by testing the pathogenicity of the isolated strains against Chenopodium album and their safety towards the crop(s) to be protected within a specifically adapted testing program. By employing the previously mentioned isolation and screening procedures, species of Ascochyta spp, but especially Ascochyta caulina strains can be identified that are capable of controlling weeds of the family Chenopodiaceae, but especially Chenopodium album in crops under field conditions and can thus be suitably used as mycoherbicides. One of the embodiments of the present invention are, therefore, strains of Ascochyta caulina which are capable of controlling Chenopodium album in crops, that is, which are pathogenic to the Chenopodium album plant to be controlled without negatively affecting the crop plant to be protected. In a preferred embodiment, the invention relates to Ascochyta caulina strains that are capable of developing appressoria-like infection structures upon infection of their host plants; especially preferred is an Ascochyta caulina biocontrol strain which has the identification number AB 90-1 and a probe of which was deposited on January 31, 1995 at the Centraalbureau voor Schimmelcultures, P.O.Box 273, 3740 AG Baarn, The Netherlands, under accession number CBS 129.95, a depository institution recognized within the meaning of Rule 28(l)(a) under the Budapest Treaty, in accordance with the requirements of the Budapest Treaty. The invention further relates to mutants and variants of a biocontrol strain according to the invention which still has the characteristic biocontrol properties of strain AB 90-1. Mutants and variants of the biocontrol strains according to the invention can be easily prepared by a skilled artisan using standard methods very well established in the permanent art. Mutants may be derived, for example, by subjecting a biocontrol strain according to the invention to mutagenic conditions, which may be established by applying mutagenic compounds such as, for example, ethyl or methyl methansulfonate, dimethyl- or diethylsulfat; ethylenimine, methyl nitro-nitroso guanidine or, aitematively, UV- or X-ray-radiation. More sophisticated methods involving genetic engineering techniques such as oligonucleotide-mediated mutagenesis may aim at directly and purposefully manipulating the genome of the pathogen. The biocontrol strains of the invention can be used in various ways. In one embodiment of the invention vegetative stages may be used such as mycelia, which may be employed in essentially pure form or comprised in a culture extract. The vegetative stages may be used either directly or after having been specifically treated, for example by lyophilisation. In a further and preferred embodiment, spores are used, which may either be ascospores or conidiospores (pycnidiospores). The spores as well as the vegetative stages can be obtained by culturing the biocontrol strains on a suitable cultivation medium, which may be a solid medium or a liquid medium. Media that may be suitably used for cultivating the biocontrol strains according to the invention are, for example, malt agar, oat meal agar, raw rye agar, fluid rye, clear V8 agar, untreated oat seeds, barley seeds and wheat bran. The above listing of possible media is in no way to be considered limiting for carrying out the invention. The person skilled in the art will immediately recognize that other media commonly used for culturing fungal microorganisms may also be envisaged within the scope of the present invention. The production of Pycnidiospores is preferably being accomplished on an oat meal agar or a wheat bran medium. The formation of conidia may be promoted by removing grown aerial hyphae and drying the cells so obtained. Conidia can also be formed by inoculating fungal cells to a liquid medium such as, for example, a potato-dextrose medium, allowing the cells to proliferate, disrupting cells so obtained, and then drying the cells so disrupted. If the cultivation of the biocontrol strain is made on an agar medium or on a solid medium, the spores can be harvested by flooding the medium with an ample use of water or another suitable solvent such as, for example, a polysorbate. The number of spores harvested may be increased by increasing the time period of moisturizing, with a maximum to be reached after 2 to 5 hours, and/or by supporting the spore release mechanically. The spores so obtained can be stored for an elongated period of time without loss of viability. Germination rate of the spores can be stimulated by adding suitable nutrients or nutrient media such as, for example, a Czapek-Dox medium or Yeast extract. The biocontrol strains may be used in unmodified form or together with any suitable agriculturally acceptable carrier. Such carriers are adjuvants conventionally employed in the art of agricultural formulation, and are therefore formulated in known manner to emulsifiable concentrates, coatable pastes, directly sprayable or dilutable suspensions, dilute emulsions, wettable powders soluble powders, dusts, granulates, and also encapsulations, for example, in polymer substances. Like the nature of the compositions, the methods of application, such as spraying, atomizing, dusting, scattering or pouring, are chosen in accordance with the intended objective and the prevailing circumstances. Preferred methods of applying a biocontrol strain of the present invention or an agrochemical composition of the present invention are leaf application of juvenile plants and soil application, respectively. In leaf application, the preferred time for applying the biocontrol strain according to the invention or a composition comprising same is, when the weed plants have reached a growth range somewhere in-between the 2-leaf and the 8-leaf stage, preferably the 2-leaf and the 6-leaf stage and most preferably the 3leaf and 4-leaf stage. Also preferred in applying the biocontrol strain to a weed plant which is in the cotyledonary stage. One may also chose to apply the biocontrol strain or a composition comprising same to the weed plant at the start of flowering, at mid time of flowering, at the end of flowering or at the time the first seeds become mature. If directly sprayable or dilutable suspensions or dilute emulsions are employed in leaf or flowering plant application, the weed plants are preferably sprayed till run off. The number of applications and the rate of application depend mainly on the intensity of infestation by the weed plant and the environmental conditions present at the time of application. Further factors that are to be observed in evaluating the optimal conditions as to rate and way of application, spore densities, choice of formulation, etc are, for example, the efficacy of the biocontrol strain employed, that is its pathogenic potential towards the weed plant to be controlled and further its viability and vitality. In soil application, soil moisture content, soil type, and survival of spores in soil are further factors that may be taken into consideration. The formulations, compositions or preparations containing the biocontrol strains according to the invention and, where appropriate, a solid or liquid adjuvant, are prepared in known manner, for example by homogeneously mixing and/or grinding the biocontrol strains with extenders, for example solvents, solid carriers and, where appropriate, surface-active compounds (surfactants). Suitable solvents for compositions include aromatic hydrocarbons, preferably the fractions having 8 to 12 carbon atoms, for example, xylene mixtures or substituted naphthalenes, phthalates such as dibutyl phthalate or dioctyl phthalate, aliphatic hydrocarbons such as cyclohexane or paraffins, alcohols and glycols and their ethers and esters, such as ethanol, ethylene glycol monomethyl or monethyl ether, ketones such as cyclohexanone, strongly polar solvents such as N-methyl-2-pyrrolidone, dimethyl sulfoxide or dimethyl formamide, as well as epoxidized vegetable oils such as epoxidized coconut oil or soybean oil; or water. The solid carriers used e.g. for dusts and dispersible powders, are normally natural mineral fillers such as calcite, talcum, kaolin, montmorillonite or attapulgite. In order to improve the physical properties it is also possible to add highly dispersed silicic acid or highly dispersed absorbent polymers. Suitable granulated adsorptive carriers are porous types, for example pumice, broken brick, sepiolite or bentonite; and suitable nonsorbent carriers are materials such as calcite or sand. In addition, a great number of pregranulated materials of inorganic or organic nature can be used, e.g. especially dolomite or pulverized plant residues. Suitable surface-active compounds are nonionic, cationic and/or anionic surfactants having good emulsifying, dispersing and wetting properties. The term "surfactants" will also be understood as comprising mixtures of surfactants. Suitable anionic surfactants can be both water-soluble soaps and water-soluble synthetic surface-active compounds. Suitable soaps are the alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts of higher fatty acids (chains of 10 to 22 carbon atoms), for example the sodium or potassium salts of oleic or stearic acid, or of natural fatty acid mixtures which can be obtained- for example from coconut oil or tallow oil. The fatty acid methyltaurin salts may be used. More frequently, however, so-called synthetic surfactants are used, especially fatty sulfonates, fatty sulfates, sulfonated benzimidazole derivatives or alkylarylsulfonates. The fatty sulfonates or sulfates are usually in the form of alkyli metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts and have a 8 to 22 carbon alkyl radical which also includes the alkyl moiety of alkyl radicals, for example, the sodium or calcium salt of lignonsulfonic acid, of dodecylsulfate or of a mixture of fatty alcohol sulfates obtained from natural fatty acids. These compounds also comprise the salts of sulfuric acid esters and sulfonic acids of fatty alcohol/ethylene oxide adducts. The sulfonated benzimidazole derivatives preferably contain 2 sulfonic acid groups and one fatty acid radical containing 8 to 22 carbon atoms. Examples of alkylarylsulfonates are the sodium, calcium or triethanolamine salts of dodecylbenzenesulfonic acid, dibutylnaphthalene-sulfonic acid, or of anaphthalenesulfonic acid/formaldehyde condensation product. Also suitable are corresponding phosphates, e.g. salts are preferably in the form of halides, methylsulfates or ethylsulfates, e.g. stearyltrimethylammonium chloride or benzyldi(2-chloroethyl) ethylammonium bromide or salts of the phosphoric acid ester of an adduct of p-nonyl- phenol with 4 to 14 moles of ethylene oxide. Non-ionic surfactants are preferably polyglycol ether derivatives of aliphatic or cycloaliphatic alcohols, or saturated or unsaturated fatty acids and alkylphenols, said derivatives containing 3 to 30 glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms in the alkyl moiety of the alkylphenols. Further suitable non-ionic surfactants are the water-soluble adducts of polyethylene oxide with polypropylene glycol, ethylenediamine propylene glycol and alkylpolypropylene glycol containing 1 to 10 carbon atoms in the alkyl chain, which adducts contain 20 to 250 ethylene glycol ether groups and 10 to 100 propylene glycol ether groups. These compounds usually contain 1 to 5 ethylene glycol units per propylene glycol unit. Representative examples of non-ionic surfactants are nonylphenolpolyethoxyethanols, castor oil polyglycol ethers, polypropylene/polyethylene oxide adducts, tributylphenoxypolyethoxyethanol, polyethylene glycol and octylphenoxyethoxyethanol. Fatty acid esters of polyoxyethylene sorbitan and polyoxyethylene sorbitan trioleate are also suitable non-ionic surfactants. Cationic surfactants are preferably quatemary ammonium salts which have, as N-substituent, at least one C8-Czalkyl radical and, as further substituents, lower unsubstituted or halogenated alkyl, benzyl or lower hydroxyalkyl radicals. The salts are preferably in the form of halides, methylsulfates or ethylsulfates, e.g. stearyltrimethylammonium chloride or benzyldi(2-chloroethyl)ethylammonium bromide. The surfactants customarily employed in the art of formulation are described, for example, in "McCutcheon's Detergents and Emulsifiers Annual, "MC Publishing Corp. Ringwood, New Jersey, 1979, and Sisely and Wood, "Encyclopedia of Surface Active Agents, "Chemical Publishing Co., Inc. New York, 1980. Preferred within the scope of the invention are surfactants selected from the group consisting of polysorbitan based surfactants Tween 20, Tween 80; Merck, Germany and Lamers & Indemans, the Netherlands, respectively], silicone based surfactants [Sylgard 309, Silwet L-77; Union Carbide, NY, USA and Dow Coming Europe, Belgium, respectively] and lecithin based surfactants [Natipide (a soy bean lecithin surfactant); A.Natterman & Cie GmbH, Germany), in concentrations of between 0.1% and 0.001%, dependent on the surfactant used. Especially preferred are silicone based surfactants such as, for example, Slygard 309 and Silwet L-77. The agrochemical compositions usually contain from about 0.1 to about 99%, preferably about 0.1 to about 95%, and most preferably from about 3 to about 90% of the active ingredient, from about 1 to about 99.9%, preferably from about 1 to about 99%, and most preferably from about 5 to about 95% of a solid or liquid adjuvant, and from about 0 to about 25%, preferably about 0.1 to about 25%, and most preferably from about 0.1 to about 20% of a surfactant. EXAMPLES The invention will be further described by reference to the following detailed examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Various modifications of the specific details provided in the examples will be apparent to those skilled in the art. Such modifications are intended to fall within the scope of the invention. Media Oat meal agar is made by suspending 60 g oat meal [Quaker Oats BV, The Netherlands] in 1 I demineralized hot water. The suspension is heated to 100"C and subsequently strained over a mesh wire with holes of 2 by 2 mm. Finally, 15 g agar [Oxoid Agar Bacteriological, Unipath LTD, England] is added to 1 I oat meal suspension. The oat meal agar is sterilized at 1200C for 30 min. Czapek Dox broth [g] [Difco, USA] Saccharose 30.0 Sodium nitrate 3.0 magnesium sulfate 0.5 potassium chloride 0.5 ferric(ll) sulfate 0.01 di-potassium hydrogenphosphate 1.0 Yeast Extract [Merck, Germany Wheat Bran [Zonnatura B.V., The Netherlands] Formulation Examples 1. Wettable powders a) b) c) Ascochyta caulina pycnidiospores 25% 50% 75% sodium lignosufonate 5% 5% sodium laurylsulfate 3% - 5% sodium diisobutylnaphthalenesulfonate - 6% 10% octylphenol polyethylene glycol ether - 2% (7-8 moles of ethylene oxid) highly dispersed silicid acid 5% 10% 10% kaolin 62% 27% The pycnidiospores are thoroughly mixed with the adjuvants and the mixture is thoroughly ground in a suitable mill, affording wettable powders which can be diluted with water to give suspensions of the desired concentrations. 2. Emulsifiable concentrate Ascochyta caulina pycnidiospores 10% octylphenol polyethylene glycol ether (4-5 moles ethylene oxide) 3% clacium dodecylbenzensulfonate 3% castor oil polyglycol ether (36 moles of ethylene oxide) 4% cyclohexanone 30% xylene mixture 50% Emulsions of any required concentration can be obtained from this concentrate by dilution with water. 3. Dusts a) b) Ascochyta caulina pycnidiospores 5% 8% talcum 95% kaolin - 92% Ready for use dusts are obtained by mixing the active ingredient with the carriers and grinding the mixture in a suitable mill. 4. Extruder Granulate Ascochyta caulina pycnidiospores 10% sodium lignosulfonate 2% carboxymethylcellulose 1% kaolin 87% The active ingredient or combination is mixed and ground with the adjuvants and the mixture is subsequently moistened with water. The mixture is extruded, granulated and the dried in a stream of air. 5. Coated Granule Ascochyta caulina pycnidiospores 3% polyethylene glycol (mol wt 200) 3% kaolin 94% The active ingredient or combination is uniformly applied in a mixer to the kaolin moistened with polyethylene glycol. Non-dusty coated granulates are obtained in this manner. 6. Suspension Concentrate Ascochyta caulina pycnidiospores 40% ethylene glycol 10% nonylphenol polyethylene glycol ether (15 moles of ethylene oxide) 6% sodium lignosulfonate 10% carboxymethylcellulose 1% 37% aqueous formaldehyde solution 0.2% silicone oil in the form of a 75% aqueous solution 0.8% water 32% The active ingredient or combination is intimately mixed with the adjuvants giving a suspension concentrate from which suspensions of any desired concentration can be obtained by dilution with water. Example 1 Isolation and Selection of Biocontrol Strains 1.1 Isolation Naturally infected Chenopodium album plants are collected. Ascochyta caulina is isolated from infected leaves exhibiting medium to severe disease symptoms. Prior to the fungus isolation small pieces of diseased tissue are cut out and surface sterilized by submerging them in a 3% (v/v) sodium hypochlorite solution for about 30 s. The isolate is then axenically transferred to oat meal agar slants in test tubes. The tubes are kept at room temperature (18"C -24 C) till some mycelium appears, and subsequently stored at 5"C in the dark. 1.2 Selection Selection of strains is accomplished by testing the pathogenicity and host specificity of the isolated strains in a specifically adapted test program. Three isolates are evaluated in the test program, (1) an isolate from a leaf of a plant obtained from an arable field (isolate 90-1); (2) an isolate from a stem of a plant obtained from a ruderal site (isolate 90-2); and (3) and isolate from a leaf of a plant from another arable field (isolate 91-2) 1.2.1 Soon application and wetness period When plants of a batch had reached a particular development stage, they are sprayed with a spore suspension till run off 4 h before the climate chambers went dark. A De Vilbiss atomizer is employed. Sprayed plants are placed in high-humidity chambers for a specified wetness period. Subsequently, they are returned to the climate chamber in which they are originally grown. Conditions in the high-humidity chambers are a nearsaturated atmosphere ( > 95% RH) with a day-night regime of 14 h light (average PAR 210 .limol/m2/s; Philips TLD 50W/84) at 18"C and 10 h darkness at 12"C. The surface of sprayed plants dried slowly but not completely during exposure to high humidity. When sprayed plants had been retumed to the climate chamber at 7585% RH, the surface of the plants dried within 30 minutes. 1.2.2 Disease assessment and data analysis Plants are separated in leaves and stems. The proportion of necrotic leaf area on each individual leaf of a plant is estimated by means of standard diagrams (Fig. 1). The area of each individual leaf is measured by means of a leaf area meter (LI-COR, USA; Model 3100). The necrotic area of each leaf is calculated by multiplying the leaf area with the estimated proportion of necrosis. The proportion of necrotic leaf area of a plant is calculated by dividing the summarized necrotic leaf area of a plant by the summarized leaf area of that plant. The proportion of necrotic stem area on a stem of a plant is directly estimated by means of standard diagrams. A mono-molecular model [equation 1; e.g. Campbell and Madden, 1990] and a nonmonotonic, critically damped model (equation 2; Gilligan, 1990) are used to describe necrosis dynamics, y = 1 - et°4) (1) Y = (b+c*(t a))* eire (2) In both models, y is the proportion of necrotic area per plant at time t, r is a rate parameter, t is time after spore application in days, and a is the incubation period (intercept of the curve with the x-axis). Parameters b and c in equation 2 are shape parameters. The models are fitted to the data by means of non-linear regression procedures of Genstat 5 [Payne et al., 1987]. Treatment effects on angular-transformed proportions are determined by means of analysis of variance (A NOVA). 1.2.3 Effect of source population and isolate on disease development C album plants of three populations (arable field 1, arable filed 2, and ruderal site) are grown at densities of 4 plants/pot. When the plants had reached the 6-leaf stage, they are sprayed with a suspension of 0 or 5 x 106 spores/ml. Spore suspensions of three isolates (90-1, 91-1 or 91-2) are applied. Sprayed plants are placed in the high-humidity chamber for a wetness period of 24 h and subsequently retumed to the climate chamber at 75-85% RH. The experiment consisted of 12 treatments (3 plant populations * 4 suspensions). A treatment consists of 2 pots with 4 plants each. The pots are placed in a complete randomized design. Leaf necrosis on individual plants is assessed one week after inoculation. At spraying time, one ml spore suspension of each isolate is plated on 1% (w/v) water agar in a Petri dish ( 9cm). The Petri dishes are placed in the high-humidity chamber for 24 h. Percentages of spore germination are determined by observing 300 spores per isolate. A spore is considered germinated when the length of the germination tube exceeded the width of the spore. One of the strains performing well in the experimental test systems described above with the identification number AB 90-1 was selected and further tested for host specificity. 1.2.4 Host-sDecificitv Plants of 22 taxons, mostly Chenopodiaceae, are grown at densities of 4 plants/pot. When the plants had reached the 4 to 6-leaf stage, they are sprayed with spore suspensions of 0 or 106 spores/ml of isolate 90-1. Nutrients are added to the suspensions as described for experiment 3. Sprayed plants are placed in high-humidity chambers for a wetness period of 24 h and subsequently returned to the climate chamber at 75-85% RH. The experiment consisted of 44 treatments. A treatment consisted of 2 pots with 4 plants each. Leaf necrosis on the individual plants of a pot is assessed one week after spore application. The remaining plants are monitored for disease development for another two weeks. 1.2.5 Strain identification The strain AB 90-1 was identified as Ascochyta caulina (P. Karst.) v.d. Aa & v. Kest. based on criteria published by Van der Aa and Van Kesteren (1979). 1.3 SDore Production Pycnidiospores are produced either on oat meal agar or on a wheat bran medium, which may preferably be mixed with sand. Spore production is initiated by axenically transferring some fungal mycelium from stock cultures stored at 5"C to oat meal agar in plastic Petri dishes ( 9 cm). The Petri dishes are placed in a climate cabinet at 20"C in continuous light [75 limol/m2/s; Philips TL 13W/83]. The growth conditions are considered optimal for production of spores of A caulina. After an incubation period of ca 3 weeks, cultures on the oat meal agar are flooded by 10 ml sterile demineralized water per Petri dish. After three hours, supematants with suspended spores are poured into a flask. Spore density of the collected suspension is determined by means of a hemocytometer under a light microscope. The suspension is diluted to 106 spores/ml and used a seed inoculum. 1.3.1 Production of spores on oat meal agar In experiments 1.1.3.1 and 1.3.1.2, production of spores on oat meal agar in plastic Petri dishes ( 9 cm; height 1.3 cm), and germination of harvested spores are studied. 1.3.1.1 Twenty-five Petri dishes with 40 ml oat meal agar each, are inoculated by spreading 1 ml seed inoculum over the surface of each plate using a Drigalski spatula. The Petri dishes are placed in the climate cabinet (continuous light and 20"C). Five Petri dishes are randomly taken from the climate chamber at different intervals within a period of a month. The cultures in the Petri dishes are flooded by 10 ml distilled water with 0.05 % Tween 80 (v/v) surfactant per Petri dish. The Petri dishes are regularly shaken. Supematants with suspended spores are poured into test tubes after 3 h. Volume and spore density of the collected spore suspensions are determined to calculate the number of spores produced per Petri dish. Samples of 1 ml are taken from each spore suspension, and spread over the surfaces of a 1 % (w/v) water agar plates in Petri dishes ( 9 cm). The Petri dishes are placed in the climate cabinet (continuous light and 20"C) for 20 h. Proportions of germinated spores are determined by observing 200 spores per Petri dish under a light microscope. A spore is considered germinated when the germination tube is longer than the width (circa 5 Cun) of the spore. 1.3.1.2 Experiment 1.3.1.2 is a replication of experiment 1.3.1.1, but a second experimental factor is added to the experiment. Each collected spore suspension is divided into two equal parts. 3.5 g Czapek-Dox broth/l spore suspension and 0.4 g yeast extract spore suspension (media of Difco, USA and Merck, Germany, respectively) are added to one part, whereas the other part is used as the addition-free control. Germination of spores of suspensions with and without added nutrients is determined as described for experiment 1.3.1.1. 1.3.2 Production of spores on wheat bran In experiment 1.3.2, spore production on a wheat bran medium in glass Petri dishes ( 20 cm; height 4 cm; content 1 I) is studied. Wheat bran medium is prepared by wetting a mixture of 30 g wheat bran (Zonnatura B.V., The Netherlands) and 90 g dry quartz sand with 60 ml distilled water in a Petri dish. The medium is sterilized at 1200C for 30 min. Five Petri dishes with wheat bran medium are inoculated with 10 ml seed inoculum per Petri dish. The Petri dishes are thoroughly shaken, placed in the climate cabinet (continuous light and 20"C), and subsequently shaken every two to three days to prevent agglomeration of medium by fungal growth. Six g of wheat bran medium inclusive fungal biomass are taken out of each Petri dish at different intervals within a period of a month. The samples are divided into two parts, 5 g is used to determine spore production and 1 g is used to determine the moisture content. The samples of 5 g are transferred to flasks, and flooded by 20 ml distilled water with 0.05 % (v/v) Tween 80 per flask. The flasks is regularly shaken. Supematants with suspended spores are poured into test tubes after 3 h. Volume and spore density of collected suspensions are determined. The samples of 1 g are dried at 70"C for 2 days. Numbers of spores produced per gram dry medium (including fungal dry matter) are calculated. 1.4 Effect of storaae of sDores on germination rates In experiment 4, Petri dishes with wheat bran medium are inoculated and incubated as described for experiment 3. After 11 days, the contents of the Petri dishes are placed between two layers of blotting paper in the laboratory at 18-24"C and 30-60 % RH for three to four days. Subsequently the cultures (inclusive medium) are transferred to glass jars, and stored at 5"C in the dark. The cultures in cold storage had a moisture content of 2 to 4 %. Samples of 5 g are taken from the cultures in cold storage at different intervals over a period o 385 days. Spores are harvested from the samples, and the number of spores harvested per gram dry medium is determined as described for experiment 3. Spore germination is determined as described for experiment 2. 1.5 Data analysis Spore production is analyzed with a Gompertz model for sigmoidal growth (e.g. Campbell and Madden, 1990): y=c*exp(-exp(-b*(t-m))) (1) where y is the number of spores produced per unit medium at time t, c is the maximum spore production per unit medium, b is a shape parameter, t is the day number after inoculation of the medium (age of the culture), and m is a location parameter. Spore germination is analyzed by means of analysis of variance and described by an exponential model: 1.6 Spore Formulation Addition of nutrients to suspensions of pycnidiospores of A. caulina stimulates germination of the pycnidiospores and infection of the plant Therefore, the addition of nutrients to the mycoherbicide is a standard formulation of the mycoherbicide. Highest level of performance of the mycoherbide can be attained by addition of Czapek Dox broth and Yeast extract. The use of a surfactant in spore formulation is recommended. Two surfactants are successfully being used in the application experiments described below: Tween 80 (Merck, Germany) and Sylgard (Dow Coming, Belgium). When Tween 80 is used, the spray volume is preferably 0.15 liter per square meter. When Sylgard is used, the spray volume is preferably 0.04 liter per square meter. 1.7 Plant Production Seeds of Beta vulgaris subspecies vulgaris cv Carla (sugar beet), Spinacia oleracea cv Martine (spinach), Zea mays cv Mandigo (maize), Triticum aestivum cv Arminda (wheat) and Pisum sativum cv Eminent (pea) are taken from commercial seed batches. If coatings are present on seeds, they are removed by several washings in water. Germination of seeds is induced prior to sowing. The seeds are placed on water agar (1% w/w) in Petri dishes and these are placed in an incubation chamber with a daynight regime of 14 h light (20 CLmol/mZ/s; Philips TL 8W/33) at 25"C and 10 h darkness at 15"C for two days. Germinated seeds are sown in soils in plastic pots (soil volume 600 ml and soil surface 72 cm2). The seeds are sown in square pattern, 25 seeds per pot for C album and 16 seeds per pot for the other plant species tested. The pots are placed in a greenhouse with the following growth conditions: 12-22"C, 65-90% relative humidity and a 15-17 h day light period. Daily incoming photosynthetic active radiation is ca 20 to 30 mol/m2/day. A sterilized sandy soil, a mixture of coarse sand and silver sand (1:1; w/w), is used in all soil application experiments. The water content of the soil is kept between pre-set levels. In spray application experiments, germinated sees are planted in peat soil, which consists of a mixture of 10 volumes of peat (Trio bv, The Netherlands, Triomf no 17) an 1 volume of coarse sand. Example 2 Performance of Biocontrol Strains under Greenhouse Conditions 2.1 Pre-Emeraence Acolication + Soil Applicabon: The performance of A caulina in soil application of spores is tested in plot experiments in the greenhouse. Soil application is done by spraying a suspensions of pycnidiospores of A. caulina on the soil surface or by mixing of a granulate of pycnidia of A. caulina and a barley medium through the top layer of the soil. Granulate application and subsequent mixing through the soil is done by hand. Essentially two application methods are used. With method 1, spores are applied prior to sowing. Pots are partly filled with 500 ml soil and topped with a layer of 100 ml soil (layer thickness 1 cm) through which a spore suspension or granulate is mixed. Germinated seeds are sown in the soil at specific depth, C album at a depth of 0.5 cm and the other plants species at a depth of 1.5 cm. With method 2, spores are applied after sowing. Geminated seeds are sown in pots with 600 ml of soil. Consequently, 5 ml spore suspension are sprayed on the soil of a pot with a commonly used spray equipment. Spore density in/on soil is calculated from the number of spores applied. 2.1.1 Dlsease assessment and data analvsis The number of emerged, diseased and dead plants per pot is regularly counted over a period of about one month. The numbers of diseased and dead plants per pot are summarized which results in the number affected plants per pot. Numbers are converted into proportions. Proportions of emerged plants per pot are related to number of seeds per pot and proportions of affected and dead plants per pot are related to the number of emerged plants per pot. A mono-molecular regression model [equation 1; Cambell and Madden, 1990) is used to describe disease incidence with time and mortality with time. In equation 1, y is a proportion of plants at time t, r is the rate parameter (effective contacts per plant per day), t is time after sowing and a is the intercept of the regression line with the x-axis. y o A logit-log regression model equation 2; Zadoks and Schein, 1979] is used to estimate spore densities that caused disease symptoms on 50% of the plants [ED50] and mortality of 50% of the plants [LD5o]. In equation 2, y is a proportion of plants, a is the intercepts of the regression line with the y-axis, x is the spore density and b is the slope of the regression line. EDso and LD50 are calculated according to equations 3 and 4.: Logit(y) = a + b*Log(x) (2) ED50 = 10(-aED/bED) (3) LDso = 1 O{4LÓ (4) Treatment effect on proportions are determined using a generalized linear regression model with a logistic link function [McCullagh and Nelder, 1989]. Treatment effects on bED and bLo log-transformed EDso, LDso and dry matter weight per plant are studied by analysis of variance. Regression analyses and analyses of variance are done by means of GENSTAT 5 software [Payne et al, 1987]. 2.1.2 Host SDecificitv Germinated seeds of C album and cultivars of B vulgaris subspecies vulgads, S oleracea, Z mays, T aestivum, and P sativum are sown in sand through which spore suspensions are mixed. Spore densities are 0 or 106 spores/cmê soil surface area. Each treatment consists of two pots with each 25 (C album) or 16 (other species) seeds. The pots are placed in a complete randomized design. The soil moisture content is 15% at sowing and fluctuated between 12% and 18%. Disease development is monitored till the plants have four to six leaves. Seventeen days after sowing some diseased plants are harvested to make re-isolations from diseased plant tissue. Small pieces of diseased tissue are surface-sterilized with sodium hypochlorite (3% vN) for 30 s and placed on oat meal agar as described previously in Example 1. Isolated colonies are compared with those of the original A caulina isolate. 2.1.3 Effect of spore densitv and appilcation method on disease incidence (a) Germinated seeds of C album are sown in sand through which spore suspensions are mixed as described previously. Spore densities ranged from 0 to 1.5 x 106 spores/cm2 soil surface area. The experimental unit is a treated pot with 25 sees. Each treatment consisted of five pots which are randomly assigned to five blocks. The experiment has a one-factor (spore density) randomized block design with five replicates. Each replicate is a block with seven pots, one pot of each spore density. (b) In a second experiment, spore suspensions are sprayed over sand (application method 2) in which germinated seeds of C album are already sown at a depth of 0.5 cm. In total there are five spore density treatments ranging from 0 to 1.6 x 106 sporesicm soil surface area. The experimental design is identical with that of experiment (a). In both experiments the soil moisture content is 15% at sowing and fluctuated between 12% and 18%. Emergence, disease incidence and mortality are determined for both experiments every three to five days till 32 (a) or 30 (b) after sowing. The proportions of emerged, affected and dead plants on the final day of observation are analyzed statistically. The monomolecular model is fitted to data of each treatment. The logit-log model is fitted to data of each block of the final days of observation. The ED50 and LD5o are calculated. 2.1.4 Effect of solid moisture content on disease incidence Germinated seeds of c album are sown in sand through which spore suspensions are mixed as described previously. Spore densities ranged from 0 to 1.4 x 106 spores/cm2 soil surface area. Soil moisture contents are either 10%, 15% or 18% at sowing and average (and min-max) soil moisture contents over a period of three weeks are 9.4% (8-10%), 14.4% (13-15%) or 17.1% (16-18%), respectively. The soil moisture range (circa pF 2.0 - pF 2.8) allows plant growth to be undisturbed by water stress. The experiment has a two-factor (soil moisture content and spore density) randomized block design with five replicates. The logit-log model is fitted to data of each block of the final day of observation (28 days after sowing). The proportion of emerged plants, ED5o, bED, LD5o, bLo on this day are analyzed statistically. 2.2 Application of mixtures comprising herbicidal compounds Observations on spore germination in mixtures of herbicides and pycnidiospores of A. caullna are made to estimate the potential of joint application of A. caulina and a herbicide. The herbicides tested are rimsulfuron (Titus, Du Pont de Nemours), triflusulfuron methyl (Safari, Du Pont de Nemours), prosulfuron (Peak, Ciba Geigy), metribuzin (Sencor, Bayer) and asulam (Asulox, Rhone Poulenc). The chemicals are solved in distilled water on the days experiments are started. Concentrations of surfactants and Asulox are expressed as volume percentages, concentrations of Titus, Safari, Peak and Sencor as weight percentages. 2.2.1 Experiments to assess compatibility of A. caulina spores and herbicides On the day of testing, spores of A. caulina are harvested from cultures on wheat bran medium according to a standard procedure (see item 1.3.2). The spores are suspended in distilled water. Nutrients are added to the spore suspensions (0.35 g Czapek Dox broth per I and 0.04 g yeast extract per I, which is 10% of the recommended dose). Herbicides to be tested on compatibility are solved in distilled water at different concentrations. Herbicides and spore suspensions are mixed in 100-ml flasks. Each chemical solution (20 ml) is mixed with an equal volume of spore suspension. Spore density in the mixtures is 5x104 spores per ml. Two 80-R1 droplets are sampled out of each spore/chemical mixture circa 15 minutes after it is prepared. The samples are placed on glass slides developed for observations on spore germination. Such a slide had a small cavity on the upper side. The cavity had a concave shape (the diameter is 15 mm and the depth in centre is 2 mm). The droplets are placed in the cavities. Droplets spread over the bottom of the cavity in dependence of the surface tension of the droplets. It is assumed that this difference in spreading did not effect spore germination. The slides with the droplets are placed in an incubator at 20 "C and > 99% relative humidity for one day. The droplets do not dry during incubation. After incubation, spore germination is assessed using a transmission light microscope. The percentage of germinated spores is determined by observing 200 spores per sample. The mean length of germination tubes is determined by measuring lengths of germination tubes per sample. The herbicides are tested in five experiments. In each experiment, a treatment consisting of a chemical-free spore suspension is included as a reference. It is used to assess fungitoxic effects, and to assign a compatability rating. A herbicide at a specific concentration is considered incompatible with A. caulina when either the estimated percentage of germinated spores is less than 70%, or when the estimated mean length of germination tubes is significantly smaller than the mean length of germination tubes of the herbicide-free control treatment. 2.2.2 Plantmatenal Seeds of Chenopodium album are collected from mature plants on an arable field near Wageningen, the Netherlands. They are stored in the dark at 50 C until use. Plants to be used in experiments are grown in a climate chamber on a peat soil in plastic pots. Several seeds per pot are sown on a depth of circa 0.5 cm. The peat soil is a mixture of 10 volumes of commercial peat (Trio B.V., the Netherlands; Triomf no. 17) and 1 volume of coarse sand. The soil volume of each pot is 600 ml. The pots are placed in a climate chamber with a day-night regime of 14 h light at 18 C, 75% RH, and 10 h darkness at 12" C, 85% RH. Light is obtained from Philips TLD 50W184 lamps. The average photosynthetic active radiation (PAR) at the soil level is 240 11 mol.m-2.s-1. The soil is watered twice a week with a Steiner nutrient solution. After emergence, plants density is thinned to 4 plants per pot. 2.2.3 Mixed application of A. caulina and rimsulfuron Two experiments are carried out to investigate a joint control effect of A. caulina and rimsulfuron. They are numbered experiment 1 and 2. Each experiment consisted of 8 treatments: 1. Application of A. caulina spores, 2. Application of rimsulfuron, 3. Application of a mixture of A. caulina and rimsulfuron, 4. No application (untreated), 5. Application of A. caulina spores + exposure to high humidity during first 24 h after treatment, 6. Application of rimsulfuron + exposure to high humidity during first 24 h after treatment, 7. Application of a mixture of A. caulina and rimsulfuron + exposure to high humidity during first 24 h after treatment, 8. No application, exposure to high humidity during first 24 h after treatment. The C. album plants are treated when they had two to four leaves, circa 10 days after emergence. Four pots with 4 plants each are randomly assigned to each treatment. After the applications are made, the pots are either placed back in the climate chamber where the are originally grown, or placed in a climate chamber with a relative humidity > 95% and a day-night regime of 14 h light at 18"C and 10 h darkness at 12 "C for a period of 24 h and then placed in the climate chamber where they are originally grown. The pots are placed in a randomised block design. Applications are made in a spray cabin. The cabin is 4 by 1.5 by 2 m (length x width x height). It contained a track sprayer with 2 Birchmeier 1.2 mm flat-fan nozzles (perforated pike 0.6 mm). The sprayer is used at a pressure of 2.5 bar and a rate of 2 km/h. Spray deposition is 400 Vha. Plants are sprayed per treatment in one run of the track. The distance between the nozzles and the plants at spraying is circa 75 cm. The density of A. caulina spores in the applied suspensions is 5x109 spores per I. The concentration of rimsulfuron in the applied solutions/suspensions is 0.0031 g per I (0.00031% w/w). Titus contains 25% rimsulfuron. Expressed as doses per ha, 2X1012 A. caulina spores and 1.25 g rimsulfuron are applied per ha. Sylgard 309 is used as surfactant. It is added to the treatment suspensions/solutions to a concentrations of 0.05% (v/v). Nutrients are not added to the suspensions/solutions.The doses of A. caulina and rimsulfuron are selected based on dose response curves of A. caulina and Titus. The doses are that high that mortality of C. album plants is not expected when the control agents are applied separately. They are circa a factor 10 lower than the doses that are recommended for weed control in arable crops. Example 3 Performance of Biocontrol Strains under Field Conditions 3.1 Post-Emeraence Anolication The performance of the biocontrol strain according to the invention is tested under field conditions. Experimental fields are made in maize and sugar beet crops. 3.1.1 Spray Application to 4leaf staae Dlants. The experimental fields exist of plots which have lengths of ca 6 meter and widths of ca 3 meter. C album is sown in the crop rows at densities twice as high as the crop density. C album seedlings emerge ca 1 week after the crop seedlings have emerged. When C album has reached a 4-leaf stage, the mycoherbicide is applied. Plant are sprayed till run off with a suspension of spores of A caulina, which is formulated as described in Example 1.6. Spore densities in the suspension have been determined to be ca 10'0 spores/l. The spray volume amounts to ca 0.15 I/m2. Spray application is done by standard spray equipment. Spray pressure is 3 bar, spray volume as mentioned in Example 5, depended on the surfactant used. Spray application of formulated A caulina spores is preferably done when the environmental conditions are favorable for infection. If the environmental conditions are sub-optimal, extra treatments of the C album seedlings may become necessary. 3.1.2 Application to flowering Dlants. Application of spores to flowering plants is done on experimental fields in maize and sugar beet crops. The experimental design is identical to that already described in Example 3.1.1. Plant are sprayed at four different growth stages, which are: (1) at the start of flowering (begin of July); (2) at mid time flowering (half July); (3) at the end of flowering (end of July); and (4) at the time that the first seeds are mature (August). 3.2.4 Mixed application of A. caulina and Rimisulfuron and Prosulfuron. resDectivelv Four experiments were carried out in a maize crop grown on a lot of AB-DLO experimental farm 'Droevendaal' near Wageningen. Soil type of the experimental site was a loamy fine sand (%-organic material 2-3%, pH-KCI 5-6). In 1994, sugar beets had been grown on the experimental site. The maize crop was grown according to common practice in the region. Maize seeds of cultivar 'Brazil' were sown in a row structure (mutual distance between rows was a multiple of 75 cm) using a precision sowing machine. Circa 110.000 seeds per ha were sown. The date of sowing was 2 May 1995. On 6 May, pre-emergence of the maize, the top layer of the soil was harrowed as part of the weed control strategy on the experimental site. No other weed control measures were carried out in the experiments except for the experimental treatments. 3.2.4.1 Experiments. treatments and expenmental designs Three experiments (three field trials) were laid out in the maize crop. The experiments should be analysed as separate experiments because of the variation in weed infestation. The experiments are numbered 1, 2, 3 or in chronological order of application dates. Each experiment consisted of several plots (8 to 40). The plot seize was 4 m by 3 m. The treated area of a plot was 6 m2 (width 2 m, length 3 m). The plots of each experiment were arranged in randomised block designs with either 5 replicates (expts 2) or 4 replicates (expts 1 and 3). Eighteen treatments were carried out. They consisted of application of a formulated control agent (A. caulina, rimsulfuron or prosulfuron) at specific growth stages of the crop. They differed in rates, compositions and timings. The treatments are summarised in table 10. 3.2.4.2 Active ingredients and formulation The active ingredients that were applied were obtained in the following way. A. caulina (isolate AB 90-1) was grown on a wheat bran medium. Spore production reached its maximum within 14 days after inoculation of the medium, and was 107-108 spores per gram medium. The medium with the fungal spores were dried and stored at 5 "C until use. At the date of application, the cultures were taken from cold storage and prepared for application. Rimsulfuron was obtained from Du Pont de Nemours as a formulated product Titus (25% a.i.). Prosulfuron was obtained from Ciba as a formulated product A8714C (75% a.i.). 3.2.4.3 Application methods The treatments were applied either with water or with wheat bran medium as carrier. When water was the carrier, the active ingredients were solved/suspended in water with 0.1% Sylgard (the surfactant was obtained from Dow Coming in January 1993), and applied using field spray equipment. The spray equipment consisted of an air pressure device, and a spray boom (width 2 m) with 6 1.2 mm Birchmeier flat fan nozzles (perforated pike 0.6 mm). The sprayer was used at a pressure of 2.5 bar and a rate of 1.5 km/h which resulted in a deposition of 50o600 ljha. All post-emergence treatments were supplemented with nutrients (0.35% Czapek Dox broth and 0.04% Yeast extract to stimulate spore germination. The 'wheat bran' application method can be considered as a granulate application method. The granulate the inoculated wheat bran medium. It consisted of small particles covered by fungal pycnidia containing the spores. The wheat bran medium with the fungal culture on it, was broadcasted by hand over the experimental plots, and the plots were superficially raked. It is to be expected that the spores will be released from the pycnidia once the pycnidia gets wet. 3.2.4.4 Observations and measurements Weather data were obtained from a small weather station on the experimental site, and from weather stations near the experimental site. Temperature ("C), relative humidity (%), surface wetness (yes or no, as measured by a 'grid' sensor), and wind speed (m/s) at 0.5 m height were recorded on the site. Solar radiation and rain fall were recorded near the site (not available yet when this report was made). At the dates of application, the weather type was recorded (i.e. presence of clouds). Plant development stages were recorded regularly using the extended BBCH-scale (Stauss, 1994). Phytotoxicity/disease development were monitored regularly. The amount of necrosis on plants was quantified by estimating the proportion of necrotic leaf area at 2 and 4 weeks after treatment. Plant densities per plot were determined regularly by counting plants on an area of 0.625 m2 per plot, at 0, 2, 4 and 6 weeks after treatment. Vegetation height of the maize crop, and of C. album and S. nigrum were determined regularly. Vegetation height was defined as the distance between the soil and the top of the vegetation of the species examined. For C. album, plant lengths of individual plants were also measured. Soil cover (%) by the weeds with high infestation levels were estimated per plot, at 0, 2, 4 and 6 weeks after treatment. Reduction in biomass (O/o). The C. album biomass on the treated part of a plot was compared with the C. album biomass on a nearby untreated plot. The difference between the two was expressed as a percentage, at 0, 2, 4 and 6 weeks after treatment and near the end of the growth season. Biomass dry matter weights were determined on 16 August. The aerial plant parts on an area of 1 m2 were harvested and dried. This parameter was determined for some plots only. 3.2.5 Mixed application of A. caullna and Atrazin In this experiment the maize crop was sown on April 28th. C. album was sown on the experimental field that was laid out in the crop. Emergence of C. album was circa 20 days after emergence of the crop. C. album was by far the most dominant weed species in the experimental field. Its density before treatment were applied was 22 plants ha-' . Treatments were spray application of Ascochyta caulina spores (ASCCA; 1012 spores had'), spray application of Atrazin plus Lentagran (Herbicide; 50 g atrazin ha'' and 45 g pyridate aha'', respectively), spray application of ASCCA plus Herbicide, and spray application of a control agent free solution. They were applied 10 days after emergence of C. album plants, which had 2 to 4 leaves at that time. Weather conditions after application were suitable for infection (several rain showers occurred). The herbicides were applied at a reduced rate, less than 10% of the recommended doses. Weed control assessments were done throughout the season. RESULTS Example 1 Isolation and Selection of Biocontrol Strains Effect of source population and isolate on disease development [Example 1.2.3] No significant differences are observed in the percentage of germinated spores of the three isolates tested. On average, 75% of the spores had germinated on water agar after an incubation period of 24 h. Plants sprayed with spore suspensions developed necrosis in the first week after inoculation while plants sprayed with the spore-free control fluid did not. Leaf necrosis at one week after spore application (Table 1) was influenced by isolates of A caulina (P < 0.001) but not by source population of C album (P=0.064). Table 1 Average proportion of necrotic leaf area on juvenile C album plants of three source populations, assessed one week after application of spores of three Ascochyta caulina isolates. Standard errors are given between parentheses. Population of Isolate of A. caulina C. album 91-1 91-2 90-1 mean arable field 1 0.02 (0.03) a 0.09 (0.06) b 0.37 0.11) cd 0.16 a arable field 2 0.03 (0.03) a 0.11 (0.08) b 0.37 (0.11) cd 0.17 a ruderal site 0.03 (0.03) a 0.23 (0.15) bc 0.46 (0.14) d 0.24 a mean 0.03 a 0.15 b 0.40 c 0.20 Treatment averages, and means are separated with a LSD-test (P < 0.05). Host specificity [Example 1.2.4] Disease symptoms are observed only on plants of the genera Chenopodium, Atriplex and Spinacia. Plants sprayed with the spore-free control fluid did not develop disease symptoms. Average leaf necrosis at one week after spore application is shown in Table 2. Plants that had not developed disease symptoms in the first week after spore applications remained symptom-free in the two consecutive weeks. Table 2 Average proportion of necrotic leaf area (severity) on juvenile plants of various plant taxons, assessed one week after application of spores of Asochyta caulina. Standard errors are given between parentheses. Plant taxon..) Cultivar Severity of leaf necrosis Chenopodium album 0.30 (0.11) Chenopodium ficifolium 0.35 (0.12 Chenopodium quinoa Elsevier 0.24 (0.10) Wild type 0.06 (0.05) Chenopodium glaucum 0.11 (0.1 1) Chenopodium polyspermum 0.02 (0.02) Chenopodium rubmm O 2) Attiplex prostrata 0.35 (0.12) Atriplex patula 0.27 (0.1 I) Spinacia oleracea Martine 0.02 (0.02) Amsterdams reuzenblad 0 Beta vulgalis subspecies vulgaris Carla 0 Lucy o Univers o Kyros 0 Egyptische platte ronde 0 Corispemum marschallii 0 Zea mays Brazil 0 Mandigo 0 Pisum safivum Eminent 0 Triticum aestivum 0 Brassica oleacerea spp capitata 0 1) The first 17 taxons are from genera of the plant family Chenopodiaceae 2) Chlorosis on leaves Strain 90-1 that performed best in the above test system was deposited at the Centraalbureau voor Schimmelcultures, P.O.Box 273, 3740 AG Baarn, The Netherlands, under reference number CBS 129.95. Swore Production [Example 1.3] Maximal production is 2x 108 per Petri-dish (diameter 85 mm) on oat meal agar, and 1.3 x 10'0 per Petri dish (diameter 20 cm containing 30 g wheat bran) on wheat bran medium. On the wheat bran medium the spores can be stored at 5 C for over 1 year without loss of viability. Example 2 Performance of Biocontrol Strains under Greenhouse Conditions Pre-Emergence Application - > Soil APPlication: [Example 2.1] In Tables 3, 4, and 5 the effect of spore density on disease incidence and mortality of C album is shown. [Example 2.1.3] Table 3 Rate parameters rd and rm of a mono-molecular regression model fitted to data of disease incidence and mortality of experiments (a) and (b). Se are estimates of standard errors of the parameters. R ad are percentage of variance accounted for by the fits. Example Spore density Disease incidence Mortality (spores/cm2) rd se Rêadj rm se Rêadj 7.2.3 (a) 1.5 x 106 0.124 0.013 82 0.055 0.006 60 1.3x105 0.119 0.013 86 0.027 0.004 31 1.0 x 104 0.038 0.011 72 0.0061 0.0014 18 1,9x103 0.00550.000654 0.0015 0.0005 13 7.2.3 (b) 1.6 x 106 0.545 0.024 54 0.283 0.012 86 1.7 x 105 0.121 0.012 71 0.012 0.0009 67 1.7 x 104 0.04650.003955 0.0042 0.0009 14 1.7x103 0.0073 0.000646 0.0003 0.0001 7 Table 4 Densities (EDso) of pycnidiospores of Ascochyta caulina in/on soil that caused disease symptoms on 50% of the Chenopodium album plants on the final day of observation (days after sowing). ED5o values are determined by means of a logit-log regression model. Standard errors of the mean are given in parentheses. Example Treatment Time of a b ED50 ( x observation spores/cm2) 7.2.3 (a) Spore density - 32 -6.9 (0.6) 1.9 (0.1) 3.8 (0.9) 7.2.3 (b) Spore density 30 -7.6 (0.7) 2.4 (0.2) 1.9 (0.2) 7.2.4 Soil moisture 10% 28 -10.5(0.5) 2.3 (0.1) 42.4 (4.8) content 15% 28 -8.3 (0.6) 2.0 (0.1) 18.2 (5.1) 18% 28 -10.0(1.1) 2.6 (0.3) 6.5 (0.8) Table 5 Densities (also) of pycnidiospores of Ascochyta caulina in/on soil that caused mortality of 50% of the Chenopodium album plants on the final day of observation (days after sowing). LD50 values are determined by means of a logit-log regression model. Standard errors of the mean are given in parentheses. Example Treatment Time of a b ED50 ( x 103 observation spores/cm2) 7.2.3 (a) Spore density -- 32 -7.6 (0.9) 1.4 (0.2) 4.5 (1.2) 7.2.3 (b) Spore density -- 30 -14.0 (0.6) 3.1 (0.1) 0.4 (0.1) 7.2.4 Soil moisture 10% 28 -19.9(2.1) 3.3 (0.4) 14.0 (2.7) content 15% 28 -24.0 (2.4) 4.1 (0.5) 5.8 (1.3) 18% 28 -26.4 (2.3) 4.7 (0.4) 3.8 (0.3) Anolication of mixtures comprising herbicidal compounds [Example 2.2] The herbicides tested did not have a negative effect on spore germination (table 6). The concentrations tested are in the range of recommended doses for weed control in arable crops. In both experiments a joint control effect was demonstrated (tables 7 and 8). Plant mortality occurred only when a mixture of A. caulina and rimsulfuron was applied. Based on the mortality data, the joint effect of A. caulina and rimsulfuron is synergistic. Control levels 14 days after treatment are signifiant different, both in an absolute and in a proportional sence. This is demonstrated by the analyses of variance (ANOVA) of the data on biomass dry matter weight. ANOVA of the data of experiment 1 showed a significant effect of the factor 'control agents applied' (P < O.05) and of the factor 'relative humidity after treatment' (P < 0.001) on biomass dry matter weight. Such an analysis of the data of experiment 2 showed a weak interaction between 'control agent applied' and 'relative humidty after treatment' (P=0.054). The difference in the control levels in the two experiments is mainly caused by difference in necrosis development after treatment. Application of A. caulina resulted in higher levels of necrosis in experiment 1 than in experiment 2. We explain this difference by a difference in the quality of the spores applied in the two experiments. In experiment 2, spores of a relatively old batch are used while in experiment 1 the spores came from a relatively young batch. Plant lengths at different dates after treatment are presented in tables 3 and 4 in the appendix. Analysis of these data confirm the treatment effects previously indicated in this section. Table 6. Compatibility of A. caulina spores and herbicides in a germination test. A herbicide concentration was compatible (C) when no negative effect on spore germination was observed Herbicide Concentration Compatibility rating (product) (%) 1 rimsulfuron 0.01 C (Titus) 0.001 C triflusulfuron 0.01 C methyl (Safari) 0.001 C prosulfuron 0.01 C (Peak) 0.001 C metribuzin 0.2 C (Sencor) 0.02 C asulam 12 C (Asulox) 0.1 2 C 1 (w/w) 2 (vlv) Table 7. Mortality of Chenopodium album plants and biomass dry matter weight 14 days after treatment as affected by control agent applied, and relative humidity during the first 24 h after treatment in experiment 1. Entries are treatment averages. The control agents tested were Ascochyta caulina spores (ASCCA) and rimsulfuron (Titus). The experiment was carried in a climate chamber. Control Relative Mortality Dry matter weight agent(s) humidity (% dead plants) gram-plant¯ during first 24 h Untreated moderate 1 o 0.63 a 2 ASCCA moderate 0 0.49 b Titus moderate 0 0.24 d ASCCA+Titus moderate 75 0.088 e (mix) Untreated high 1 0 0.62 a ASCCA high 0 0.37 c Titus high 0 0.22 d ASCCA+Titus high 100 0.056 e (mix) 1 Moderate relative humidity was 75185%, high relative humidity was > 95%. 2 Treatment averages were separated with different letters according to a Least Significant Difference test (P < 0.05). Table 8. Mortality of Chenopodium album plants and biomass dry matter weight 14 days after treatment as affected by control agent applied, and relative humidity during the first 24 h after treatment in experiment 2. Entries are treatment averages. The control agents tested were Ascochyta caulina spores (ASCCA) and rimsulfuron (Titus). The experiment was carried in a climate chamber. Control agent(s) Relative Mortality Dry matter weight humidity (% dead plants) gramplant1 during first 24 h Untreated moderate 1 0 0.54 a 2 ASCCA moderate 0 0.46 a Titus moderate 0 0.23 b ASCCA+Titus moderate 25 0.19 b (mix) Untreated high 1 0 0.53 a ASCCA high 0 0.43 a Titus high 0 0.20 b ASCCA+Titus high 100 0.046 c (mix) 1 Moderate relative humidity was 75/85%, high relative humidity was > 95%. 2 Treatment averages were separated with different letters according to a Least Significant Difference test (P < 0.05). Example 3 Performance of Biocontrol Strains under Field Conditions Post-Emergence AnDlication [Example 3.1] Spray Application to 4-leaf stage plants: [Example 3.1.1] Observations are made on severity of infection, mortality of C album, growth of surviving C album and growth of the crops to be protected. Yields in treated plots is compared to yields of plots that are kept weed-free by hand to determine competition damage to the crops. The efficacy of A caulina to control juvenile C album is shown in the following table 9. The data provided in table 3 give a clear indication as to the potential of A caulina to be used as a mycoherbicide for controlling juvenile C album plants. Table 9 Summary of data of field experiments to control Chenopodium album by Ascochyta caulina Crop/year Environmental conditions Application Mortality Yield reduction shortly after application of herbicide C album (potential yield (artificially extended (growth reduction*) reduction*') wetness period) Maize '92 cloudy + some showers no 30% (79) 0% (19) idem (24 h) no 50% (95) 0% (19) Sugar cloudy + thundery no 5% (43) 50% (73) beet '93 shower idem (16 h) no 30% (66) 30% (73) idem (28 h) no 50% (80) 19% (73) Maize '93 cloudy + showers no 70% (90) 0% (20) idem yes 95% (99) 0% (20) 'reduction dry matter/mê C album, expressed in % of untreated "yield reduction of untreated, expressed in % of weed-free control Application to flowering plants [Example 3.1.2] Observations are made on seed production and seed infection. From the results achieved by field experiments described in Example 3.1.2 it can be demonstrated that flowers become infected and viable seeds are not produced, when spores are applied at the start of flowering and at mid time of flowering. When spores are applied at the other growth stages, seed are formed but they are infected with A caulina. The above results clearly demonstrate that A caulina can be suitably used for controlling Chenopodium album in crops. The two major considerations in the selection of plant pathogenic fungi for weed control are host-specificity and efficacy. The results of host specificity testing demonstrate that A caulina has a high specificity to the target weed Chenopodium album and is not pathogenic to the major crop plants such as maize, wheat, sugar beet, and pea. Efficacy of A caulina against Chenopodium album was demonstrated under a broad range of controlled condition both in the greenhouse and in the filed. The investigation did not show any major constraints on disease development. Mixed appilcation of A. caulina and Rimisulfuron and Prosulfuron [Example 3.2.4] Table 1. Description of treatments. Expt Trt Active ingredient applied Timing r Date and ti Weather con emerge. application ditions during first 24 h after application ASCCA Suifonylureun CHEAL 01 - - Untreated - 1 02 no - Pre/post 24-05, 19.00 Cloudy/rain 1 03 yesa - Pre/post 24-05, 19.00 Cloudy/rain 2 04 no - Post 01-06, 07.00 Cloudy/no rain 2 05 yesa - Post 01-06, 07.00 Cloudy/no rain 2 06 yesa - Post 03-06, 20.00 Cloudy/rain 2 07 yesa 5.0 g rim./ha Post 01-06, 07.00 Cloudy/no rain 2 08 no 5.0 g rim./ha Post 01-06, 07.00 Cloudy/no rain 2 09 yesa 7.5 g pro./ha Post 01-06, 07.00 Cloudy/no rain 2 10 no 7.5 g pro./ha Post 01-06, 07.00 Cloudy/no rain 2 11 no 15 g pro./ha Post 01-06, 07.00 Cloudy/no rain 3 12 no - Post 06-06,14.00 Cloudy/rain 3 13 vesa - Post 06-06,14.00 Cloudy/rain a Suspension, 5x1012 spores/ha. Data are mostly presented as (treatment) averages. In some cases, variation was that large that presentation of an average is hardly reievant. In those cases, a min. - max. range is presented. The average of the two extremes can be a suitable estimate if the reader desires an (treatment) average. Statistical analyses were not presented. Species are sometimes indicated by five letter code, a composition of the first three letters of the Latin family name followed by the first two letters of Latin species indicator. Eperimental conditions The maize seedlings on the experimental site emerged rather fast as a result of the prevailing high temperatures during the first 6 days after sowing. They emerged from 6 May till 15 May. At 23 May, maize plant density on the site was circa 100.000 plants per ha. After the seedlings had emerged, growth and development of the maize plants was relatively slow due to adverse (cold and wet) weather conditions. During this cold period (15 May till 20 June) the maize plants really suffered. They looked pale most of the time. After 20 June the weather conditions changed (like in the whole of Westem Europe). During the whole summer of 1995, the weather conditions were warm and dry. The maize crop benefitted from these conditions until soil moisture on the site became limited. At the end of July and during August, the maize crop showed some signs of drought stress. Weeds emerged in the maize crop starting on 6 May. Species that reached moderate or high infestation levels were Chenopodium album, Solanum nigmm, Stellana media, Echinochloa crus-galli, Poa annua and Elymus repens. Weed species that were observed in low densities were Galium aparine, Polygonum spp., Matrecaria spp. and Sonchus spp. Crop yield loss by weeds was mainly caused by C. album, S. nigrum and E. crus-galli. Crop yield loss in untreated plots was between 10% and 70% (relative to weed free plots based on data of 16 August). With respect to the target weed, emergence of C. album plants occurred between 6 May and the beginning of June. This resulted in a large variation in seizes and development stages of the C. album plants. Variation in biomass of C. album in untreated plots was also large. Necrosis development after treatments Application of A. caulina to plots resulted in disease development of C. album, but not of other plant species. Toxic effects to the crop or to non-host weeds were not observed. For the post emergence treatments, disease development was as expected. Early disease symptoms were observed two to four days after application of A. caulina. Symptoms were necrotic (yellow-brownish) spots on leaves and/or on stems of plants. During the next 2 weeks, the amount of necrosis per plants increased till either plants died, or till a maximum, sublethal level of necrosis was reached. In the latter case, the plants were reduced in growth according to the amount of necrosis developed. Experiment 1: pre emergencel early post emergence application of A. caulina Summary of experimental conditions: The two treatments of experiment I (yes or no application of A. caulina spores) were applied on 24 May. On that date, a part of the C. album population had emerged, and plant density averaged 100 plants/m2 (min. max. range 44 to 188 plants/m2). C. album development stages ranged from 09 to 12. Soil cover by the C. album plants averaged 2 (min. - max. from I to 2.5). C. album was by far the most important weed on the experimental field. The weather conditions after application were suitable for infection (i.e. clouded and several rain showers during first 24 h after treatment). The application of A. caulina in this experiment resulted in significant disease development of C. album, and significant control of C. album. The C. album plants that had already emerged at the time of application of A. caulina, developed disease symptoms as previously described. The C. album plants that had not yet emerged at that time, also developed necrotic spots, but the spots were mainly observed on the basis of the stems. The amount of necrosis developed was considered moderately high (at 26 DAT, the average proportion of necrotic leaf area of C. album plants was estimated 0.45, proportions ranged from 0.1 and 1). The effect of the treatments on biomass of C. album is shown in Table 11. Biomass reduction was affected by the date of observation and the treatments. Near the end of the growth season (16 August), biomass reduction of C. album by A. caulina was between 50-70%. The amount of biomass in the untreated plots on 16 August was between 400 and 600 gram m¯2. The parameters plant density, soil cover and vegetation height were affected by the treatments in a similar way as biomass of C. album was affected in this experiment. Experiment 2: post emergence application of A. caulina and sulfonylureas Summary of experimental conditions: The treatments of experiment 2 (application of A. caulina spores and/or sulfonylureas) were applied on 1 or 3 June, post emergence of C. album. On 31 May, C. album plant density on the experimental field averaged 29 plants/m2, ranging from 3 to 136 plants/m2 (min. - max.). C. album development stage ranged from 10 to 15. Soil cover by the C. Album plants averaged 0. 7 (min. - max. from 0.1 to 2). An indication of the other weeds present on the experimental field is given in table 12. The weather conditions during the first ten days of June were suitable for infection (i.e. clouded and several rain showers), especially from 3 to 10 June. The conditions were more favourable for infection at 3 June than at 1 June. The various treatments in this experiment resulted in different levels of C. album control as well as in different control levels of other weeds. Application of A. caulina resulted in significant disease development. The amount of necrosis developed was higher for the application at 3 June than for the application at 1 June. At 13/11 DAT, respectively, The average proportion of necrotic leaf area of C. album plants was estimated 0.2 (0.1-0.8) for the application at 1 June and 0.6 (0.31) for the application at 3 June. Mixed application of A. caulina and a sufonylureum resulted in higher amounts of necrosis (0.8 for mixed application with rimsulfuron and 0.9 for mixed application with prosulfuron at 13 DAT) compared to application of A. caulina alone. The effect of the treatments on biomass of C. album is shown in Table 12. Biomass reduction was affected by the date of observation and the treatments. Near the end of the growth season (16 August), reduction of C. album biomass by application of A. caulina was 45% (applied on 1 June) or 70% (applied on 3 June). The sulfonylureum treatments reached > 90% C. album biomass reduction. The application of a mixture of A. caulina and a sulfonylureum (especially for rimsulfuron) resulted in higher levels of control compared to the application of A. caulina or a sulfonylureum alone. For the interpretation of these data it is important to keep in mind that weed infestation of plots differed between treatments, and that S. nigrum was not or hardly affected by any treatment. For instance, the hardly affected S. nigmm vegetation covered (a part of) the affected C. album plants, especially in the plots of the sulfonylureum treatments. This interference was observed from the last week of June (circa 28 DAT). The amount of biomass in the untreated plots (treatment 08) on 16 August was between 50 and 300 gram¯2. With respect to the target weed, the parameters plant density, soil cover and vegetation height were affected by the treatments in a similar way as biomass of C. album was affected in this experiment. Experiment 3: "late" post emergence application of A. caulina Summary of experimental conditions: The two treatments of experiment 3 (yes or no application of A. caulina spores) were applied on 6 June. On that date, C. album plant density on the experimental field averaged 184 plants/m2, ranging from 103 to 230 plants/m2 (min. - max.). C. album development stages ranged from 10 to 16. Soil cover by the C. album plants averaged 7.5 (min. - max. from 5 to 10). C. album was by far the most important weed on the experimental field. The weather conditions during the first ten days of June were suitable for infection (i.e. clouded and several rain showers) The application of A. caulina in this experiment resulted in significant disease development of C. album, and significant control of C. album. Disease development was as previously described. The amount of necrosis developed was considered high (at 13 DAT, the average proportion of necrotic leaf area of C. album plants was estimated 0.5, proportions ranged from 0.2 and 1). The effect of the treatments on biomass of C. album is shown in Table 15. Biomass reduction was affected by the date of observation and the treatments. Near the end of the growth season (16 August), biomass reduction of C. album by A. caulina was between 50-80%. The amount of biomass in the untreated plots (treatment 17) on 16 August was between 400 and 600 gram¯2. The parameters plant density, soil cover and vegetation height were affected by the treatments in a similar way as biomass of C. album was affected in this experiment. Levels of C. album control that were achieved by application of A. caulina as a mycoherbicide ranged from 45%-70% C. album biomass reduction (treatment averages, expressed as a %-difference with untreated). When biomass reduction was observed in plots treated with A. caulina, both the plant density and the plant size of the weed were affected. In experiments 1, 2, and 3, the control levels achieved (45-70% biomass reduction) were as expected. The efficacy of C. album control in these experiments was affected by the weather conditions shortly after application of A. caulina. Therefore, we can conclude that weather conditions after application of A. caulina determine to a large extend, how much necrosis will develop. On its tum, necrosis development determines how high the level of control will be. Weather conditions that are positive for necrosis development are conditions that prevent the applied A. caulina spores from desiccation. High relative humidity ( > 90%), rain showers and dew are therefore positive for necrosis development, and thus for efficacy of control. In experiment 1, circa 60% of the C. album plants on the date of treatment. Application of A. caulina in this experiment resulted in significant control of C. album. This points to both a pre and post emergence control effect. The levels of control of C. album that were achieved by mixed application of A. caulina and a sulfonylureum (experiment 2) were aiways higher than when the active ingredients were applied alone. The joint effect of the fungus and a sulfonylureum was demonstrated most obviously for rimsulfuron. Table 11. Effect of treatments on biomass reduction of Chenopodium album in experiment 1 on the experimental site near Wageningen, 1995. Entries are averages of 4 replicates. DAT is days after treatment. Biomass reduction (% difference with untreated) ExptlTrt Description of 15 DAT, 26 DAT, 44 DAT, 84 DAT, treatment 8 June 19 June 7 July 16 August 1/02 Formulation 0 0 0 0 check 1/03 ASCCA spores 40 53 61 60 Table 12. Effect of treatments on biomass reduction of Chenopodium album in experiment 2 on the experimental site near Wageningen, 1995. Entries are averages of 5 replicates. DAT is days after treatment. Biomass reduction (% difference with untreated) Expt/ Description of Species 13111 28/26 DAT, 48/46 76/74 Trt treatment DAT, 29 June DAT, DAT, 14 Jun 19 July 16 August e 2/04 Formulation check CHEAL 0 0 0 0 A4 SOLNI - 0 STEME - 0 ECHCR - 0 2/05 ASCCA A4 CHEAL 20 30 33 45 SOLNI - o STEME - o ECHCR - o 2/06 ASCCA A5 CHEAL 45 50 58 70 SOLNI - 0 STEME - o ECHCR - o 2/07 ASCCA A4 + CHEAL 60 90 98 100 rimsulfuron SOLNI - 10 STEME - 100 ECHCR - 90 2/08 Rimsulfuron A4 CHEAL 40 56 89 97 SOLNI - 10 STEME - 100 ECHCR - 90 2109 ASCCA A4 + CHEAL 70 95 100 100 Prosulfuron-l SOLNI - 0 - - STEME - 100 - - ECHCR - 0 2/10 Prosulfuron-1 A4 CHEAL 62 74 99 99 SOLNI - 10 - STEME - 100 - ECHCR - 0 - 2/11 Prosulfuron-2 A4 CHEAL 70 100 100 100 SOLNI - 10 - STEME - 100 - ECHCR - 5 - Table 13. Effect of treatments on biomass reduction of Chenopodium album in experiment 3 on the experimental site near Wageningen, 1995. Entries are averages of 4 replicates. DAT is days after treatment. Biomass reduction (% difference with untreated) Expt/Trt Description of 13 DAT, 31 DAT, 43 DAT, 71 DAT, treatment 19 June 7 July 19 July 16 August 3/12 Formulation 0 0 0 0 check 3/13 ASCCA spores 43 60 55 65 Mixed application of A. caulina and Atrazin [Example 3.2.5] The most relevant data of the experiment are presented in table 14. They clearly demonstrate a synergistic control effect of A. caulina and the herbicides. Table 14. Control of Chenopodium album in a maize crop on an experimental field of Droevendaal experimental farm near Wageningen, in 1993. Treatments were spray application of Ascochyta caulina spores (ASCCA; 1012 spores ha1), spray application of Atrazin plus Lentagran (Herbicide; 50 g atrazin ha'l and 45 g pyridate had', respectively), spray application of ASCCA plus Herbicide, and spray application of a control agent free solution. Treatment Mortality Biomass reduction (% dead plants) relative to control (%) 11 days after treatment 95 days after treatment Control 2 % a ASCCA 49 % c 86 % b Herbicide 22 % b 65 % a ASCCA+Herbicide 95 % d 98 % c 1 Treatment averages in a column were separated with different letters according to a Least Significant Difference test (P < 0.05). REFERENCES Morris et al (1989) Phytophylactica 21, 281-283 Trujillo ef al (1986) Plant Disease 70, 974-976 Eggers T and Thun K (1988) Zeitschrift für Pflanzenkrankheiten und Pflanzenschutz, Sonderheft 11, 225-237 Campbell and Madden (1990), Mlntroduction to plant disease epidemiology, John Wiley & Sons, New York, 532 pp Payne et al (1987) Genstat 5: reference manual, Clarendon Press, Oxford, 749 pp Zadoks and Schein (1979)2 Epidemiology and Plant Disease Management", Oxford University Press, New York, USA, 427 pp McCullagh and Nelder (1989) "Generalized linear models", Chapman and Hall, London, 511 pp Van der Aa and Van Kesteren (1979) Persoonia 10, 267-276

Claims: What is claimed is: 1. A strain of Ascochyta caullna which is capable of controlling Chenopodium album in crops under field conditions. 2. A biocontrol strain according to claim 1 which is pathogenic to Chenopodium album but does not negatively affect the crop plant to be protected. 3. A biocontrol strain according to any one of claims 1 or 2, which is capable of developing appressoria-like infection structures upon infection of a host plant. 4. A biocontrol strain according to any one of claims 1 or 3 which has the identification number AB 90-1 and a probe of which is deposited at the Centraalbureau voor Schimmelcultures, P.O.Box 273, 3740 AG Baam, The Netherlands, under accession number CBS 129.95. 5. Mutants and variants of a biocontrol strain according to claim 4 which still has the characteristic biocontrol properties of strain AB 90-1. 6. A method of controlling weeds of the family Chenopodiaceae, but especially Chenopodium album plants in crops, which comprises applying a herbicidally effective amount of one or more species of Ascochyta spp, but especially of the fungal pathogen Ascochyta caulina, to the said weed plants, the seeds or seedlings thereof or the crop area. 7. A method according to claim 6, wherein treatment of the weed plant is accomplished by applying the spores to the soil pre-emergently 8. A method according to claim 6, wherein treatment of the weed plant is accomplished by applying the spores to the soil post-emergently 9. A method according to claim 8, wherein treatment of the weed plant is accomplished when the plant has reached the 2-to 8-leaf stage, preferably the 2-leaf to the 6-leaf stage and most preferably the 3-leaf to 4-leaf stage. 10. A method according to claim 8, wherein treatment of the weed plant is accomplished at the start of flowering. at mid time of flowering, at the end of flowering or at the time the first seeds become mature. 11. A method according to any one of claims 6 to 10, wherein the treatment comprises applying spores of the biocontrol strain. 12. A method according to any one of claims 6 to 11, wherein the treatment comprises spray application. 13. A method according to claim 11, wherein the treatment comprises applying granulates comprising spores of Ascochyta caulina. 14. A method according to claim 11, wherein the rate of application is in a range of about 105 spores to about 1012 spores/l, preferably of about 107 spores to about 10'0 spores and more preferably of about 103 to about 108 sporeslcm2 15. A method according to claim 11, wherein the spore densities are in a range of about of about 103 to about 108. spores/cm2, preferably of about 105 to about 107 spores/cmê and more preferably of about 105 to about 106 spores/cmê. 16 A herbicidal composition for controlling weeds of the family Chenopodiaceae, but especially of Chenopodium album, in crops comprising one or more species of Ascochyta spp, but preferably the fungal pathogen Ascochyta caulina, in a herbicidallyeffective amount together with a suitable carrier. 17. A herbicidal composition according to claim 16 comprising further compounds including fertilizers or micronutrient donors or other preparations that influence plant growth of the crop plant to be protected; or selective herbicides, insecticides, fungicides, bactericides, nematicides, molluscides or mixtures of several of these preparations. 18. A herbicidal composition according to claim 16 comprising a selective herbicide. 19. A herbicidal composition according to claim 18 comprising a species of Ascochyta spp, but especially the fungal pathogen Ascochyta caulina and a herbicide in a synergistically-effective amount. 20. A herbicidal composition according to any one of claims 16-19, which comprises the fungal pathogen in form of spores. 21. A herbicidal composition according to any one of claims 16 to 20, wherein the fungal pathogen is an Ascochyta biocontrol strain according to claim 3 as well as mutants and variants thereof. 22. A method of controlling weeds of the family Chenopodiaceae, but especially Chenopodium album plants in crops, which comprises applying a composition according to any one of claims 16 to 21 to the said weed plants, the seeds or seedlings thereof or the crop area. 23. A method of controlling Chenopodium album plants in crops, which comprises applying to the weed plants, the seeds or seedlings thereof or the crop area a composition comprising the fungal pathogen Ascochyta caulina and a herbicide in a synergistically-effective amount. 24. A method according to any one of claims 21 or 23, wherein the rate of application is in a range of between 108 to 1014 spores/ha, preferably between 10'0 and 1013 spores/ha of biocontrol agent and 0.01 to 2 kg/ha, preferably 0.02 to 1 kg/ha herbicide. 25. A method according to any one of claims 6 to 15 and 22 to 24, wherein the biocontrol strain is applied to the crop area or plant to be controlled, simultaneously or in succession, with other compounds. 26. A method for the preparation of a herbicidal composition according to any one of claims 16 to 21 by intimately mixing the biocontrol strain with an extender, with or without a surface-active auxiliary. 27. Use of a biocontrol strain according to any one of claims 1 to 4 or of a composition according to any one of claims 16 to 21 for controlling the weed Chenopodium album in crops of cultivated plants. 28 A method of producing pycnidiospores of Ascochyta caulina comprising (a) cultivating Ascochyta caulina on an oat meal agar or a wheat bran medium; (b) harvesting the spores by flooding the medium with an ample use of water or another suitable solvent for between 2 and 5 hours (c) optionally supporting the spore release mechanically.

Inventors: KEMPENAAR CORNELIS, LOTZ LAMBERTUS ARIE PHILIP, SCHEEPENS PETRUS CAROLUS, KEMPENAAR, CORNELIS, LOTZ, LAMBERTUS, ARIE, PHILIP, SCHEEPENS, PETRUS, CAROLUS
Applicants: CIBA GEIGY AG, INSTITUUT VOOR AGROBIOLOGISCH, KEMPENAAR CORNELIS, LOTZ LAMBERTUS ARIE PHILIP, SCHEEPENS PETRUS CAROLUS, CIBA-GEIGY AG, INSTITUUT VOOR AGROBIOLOGISCH EN BODEMVRUCHTBAARHE, KEMPENAAR, CORNELIS, LOTZ, LAMBERTUS, ARIE, PHILIP, SCHEEPENS, PETRUS, CAROLUS
Application year: 1996
Main classification: A01N63
IPC codes: A01N63