Use of Coniothyrium minitans for the protection of cultivated plants against infestation by pathogenic fungi of the genus Verticillium Spp.

Description

Field of the invention

[0001] The invention relates the area of specific soil decontamination and biological protection against pathogenic fungi in agriculture and horticulture, specifically the use of the Coniothyrium minitans strain for protection of cultivated plants against attacks by fungal pathogens, a preparation for protection of cultivated plants and a method of protection.Background of the invention

[0002] The Coniothyrium genus belongs to the Coelomycetes class, sub-group Deuteromycotina, identified for the first time in 1947 in the United States. The Coniothyrium minitans strain CON / M / 91-08 in combination with an appropriate carrier is used as a spray fungicide bio-preparation applied on soil or postharvest leftovers. The preparation contains spores of the Coniothyrium minitans strain which, after the application into soil, contaminate it and parasitize on present sclerotia of fungi from the Sclerotinia spp. genus that cause e.g. white rot of plant roots and degrade them relatively very quickly. Sclerotium is a formation created by certain types of organisms, most frequently fungi. The formations can have various shapes, sizes and colors and they are made up of hyphae. They make it possible to survive long harsh periods, for example drought or cold. Particularly after application of a preparation containing the Coniothyrium minitans strain on postharvest leftovers the hyperparasite fungus rapidly develops and it even spreads to adjoining sclerotia not directly hit by the spray. The Coniothyrium minitans fungus usually develops best in an aerated surface layer of soil ca. 10 cm deep at temperatures over 1 °C and in the presence of sclerotia. When the soil freezes Coniothyrium minitans suspends its growth but it does not die. Once the temperature increases the fungus starts to parasitize on sclerotia again and therefore the preparation can be used both in the fall, summer and in the spring months. The exact time point depends on the crop. A precondition for efficiency of the preparation is its mixing into the soil as deep as ca. 5 to 10 cm, immediately after the application.

[0003] This spray fungicide bio-preparation is used for protection of various cultivated plants, such as rapeseed, mustard, sunflower, poppy, vegetables, beans, decorative plants, aromatic and curative herbs or chicory, which are all attacked by Sclerotinia spp. fungi, particularly by Sclerotinia sclerotiorum and Sclerotinia minor.

[0004] Another common soil pathogen on cultivated plants is e.g. the Phoma genus which causes the so-called phoma blackening of stems. Black stains appear on the plant's hypocotyl, i.e. on the lowest part of a stem between the root and cotyledons, later the plant starts rotting and dies. The pathogen may also infect the leaves and cover them with irregular brown-grey stains. Fungi from the Phoma genus survive on infected postharvest leftovers and they spread in the seed. The only known protection against this pathogen is to remove or to plough in the postharvest leftovers, to sow healthy seed and to apply chemical fungicides.

[0005] The Verticillium genus hosts further pathogens for cultivated plants that is difficult to eliminate from soil. Pathogens belonging to this genus cause the so-called verticillium wilt accompanied by thin foliage of woody plants, partial necrosis of the crown, yellowing and drying of plant leaves, formation of long, oval grey or brownish stains on the stem, blackening of stem, blackening of roots etc. Infection of cultivated plants with this fungus may cause extraordinary losses. The only known protection of plants from this pathogen consists in prevention which includes a broad crop rotation and thorough liquidation of postharvest leftovers.

[0006] The purpose of the invention is to find a procedure which would effectively eliminate pathogens of the Verticillium genus found in Brassica spp. and which would be easy to apply. A bio-preparation is preferred.

[0007] Tomprefa et al., Journal of Applied Microbiology 106.6 (2009): 2048-2056, describes the antimicrobial activity of Coniothyrium minitans isolates and its macrolide antibiotic macrosphelide A against several fungi, yeast and bacteria, including several Verticillium species.

[0008] EFSA Journal 14.7 (2016): e04517 is a peer review of the pesticide risk assessment of the active substance Coniothyrium minitans strain CON / M / 91-8.Summary of the invention

[0009] The scope of the invention is defined by the claims. Any aspects and examples of the present disclosure which do not fall under the scope of the appended claims do not form part of the invention and are merely provided for illustrative purposes.

[0010] The invention provides a method for controlling phytopathogenic fungi of the genus Verticillium spp. comprising applying Coniothyrium minitans strain CON / M / 91-08 in an effective amount to the root system of a plant, to harvest residues or to the soil on which a crop plant grows or is intended to grow, wherein said plant is of the genus Brassica spp.

[0011] The invention also provides use of Coniothyrium minitans strain CON / M / 91-08 for controlling phytopathogenic fungi of the genus Verticillium spp. in soil where a crop plant grows or is intended to grow, wherein said plant is of the genus Brassica spp.

[0012] The invention consists in methods of applying and the use of the Coniothyrium minitans strain CON / M / 91-08 for protection of cultivated plants against attacks by fungal pathogens Verticillium spp.. The presence of fungal pathogens from Verticillium genera poses a serious problem to cultivated crops because their elimination is very complicated and no elimination method has been known to protect crops. The Coniothyrium minitans CON / M / 91-08 fungal strain is commonly used against Sclerotinia sclerotiorum and the use of its effects against Verticillium genera is based on completely new findings about the effects of this strain. The Verticillium genera have different morphological cell structures from that of Sclerotinia sclerotiorum and therefore one cannot expect the Coniothyrium minitans CON / M / 91-08 strain to have antagonistic effects and to be efficient also against these types of fungal pathogens.

[0013] A preparation for protection of cultivated plants against attacks by fungal pathogens Verticillium spp. may contain the fungal strain Coniothyrium minitans CON / M / 91-08 on a nutritious carrier. The preparation may be in the form of spores, conidia, hyphae or a combination thereof. These forms of the fungal strain Coniothyrium minitans subsequently infect pathogens on cultivated plants, parasitize on them and subsequently degrade them. This removes the cause of the infection of cultivated plants and not only its consequences which means a major advantage in comparison with the existing methods.

[0014] It has surprisingly been found that the present C. minitans strain is also effective against verticillium wilt. With regard to the different morphology of Verticillium genera no analogy can be found with removal of the Sclerotinia genus pathogens. The preparation may be applied into the soil in the dose of e.g. 2 kg / ha before sowing of a cultivated plant and may be introduced into the soil up to 10 cm deep.

[0015] Advantages of the Coniothyrium minitans strain as mentioned above for protection of cultivated plants against attacks by fungal pathogens under this invention consist particularly in the fact that it is active not only against the known pathogen of cultivated plants from the Sclerotinia genus against which the Coniothyrium minitans strain has been commonly used but also against other pathogens usually occurring in soil used for cultivated plants, i.e. against Verticillium spp. The Coniothyrium minitans strain therefore generally improves physiological condition of cultivated plants and, particularly, it is convenient that application of a single bio-preparation can affect several pathogenic genera at a time.Detailed description

[0016] In one aspect, the invention relates to a method for controlling phytopathogenic fungi of the genus Verticillium spp. comprising applying Coniothyrium minitans strain CON / M / 91-08 in an effective amount to the root system of a plant, to harvest residues or to the soil on which a crop plant grows or is intended to grow, wherein said plant is of the genus Brassica spp.

[0017] Coniothyrium minitans strain CON / M / 91-08 is described in WO 96 / 21358. The strain was deposited with the German Collection of Microorganisms and Cell Cultures in Braunschweig, Germany under the accession number DSM 9660. The strain is available as Contans ®< WG from Bayer CropScience Biologics GmbH.

[0018] The strain may be cultured according to methods described in WO96 / 21358. For example the strain can be cultured on suitable substrates, such as seeds of grain, bran or other plant materials, or also with the help of agar culture media that are customary in mycology, such as potato dextrose agar, or malt peptone agar, or on suitable support materials to which a culture medium has been added, as well as in liquid nutrient media without the addition of agar.

[0019] Usually the strain is formulated on a carrier, preferably a water-soluble sugar carrier, in a concentration of between 1 x 10 7< and about 3 x 10 9< , preferably viable, spores / g of carrier, preferably between 1 x 10 8< and about 2 x 10 9< spores / g carrier. Most preferably, the concentration lies between about 5 x 10 8< and about 1.5 x 10 9< spores / g of carrier, such as at about 1 x 10 9< viable spores / g carrier. In particular the water-soluble sugar is glucose.

[0020] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. Hybridization reactions can be performed under conditions of different "stringency". In general, a low stringency hybridization reaction is carried out at about 40 °C in 10 X SSC or a solution of equivalent ionic strength / temperature. A moderate stringency hybridization is typically performed at about 50 °C in 6 X SSC, and a high stringency hybridization reaction is generally performed at about 60 °C in 1 X SSC.

[0021] The present Coniothyrium minitans strain may be applied to control plant pathogens of the genus Verticillium spp found in Brassica spp.. Members of this genus cause verticillium wilt which affects more than 350 species of eudicot plants. The most prominent plant pathogenic Verticillium species are V. dahlia, V. albo-atrum, V. longisporum, V. nubilum and V tricorpus (for an overview, see Barbara, D.J. & Clewes, E. (2003). "Plant pathogenic Verticillium species: how many of them are there?" Molecular Plant Pathology 4(4).297-305. Blackwell Publishing).

[0022] According to the present invention, plants are from the genus Brassica spp. such as Brassica napus (Oilseed rape, Rapeseed), Brassica napobrassica (Rutabaga, Rapeseed), Brassica oleracea var. botrytis (Cauliflower), Brassica oleracea var. capitata (Cabbage) and Brassica oleracea var. gemmifera (Brussel Sprouts), Citrullus vulgaris (Watermelon), Cucumis sativus (Cucumber), Cucurbita pepo (Pumpkin), Fragaria chiloensis (Strawberry), Gossypium spp. (Cotton), Helianthus spp. (Sunflower), Lycopersicon esculentum (Tomato), Solanum melongena (Eggplant) and Solanum tuberosum (Potato). Preferably, the Brassica spp. plant is Brassica napus, most preferably winter oilseed rape.

[0023] Coniothyrium minitans CON / M / 91-08 e.g. its conidia, are present in a formulation in a concentration of at least 10 5< viable spores per gram formulation (e. g. spores / g preparation), such as 10 5< - 10 17< viable spores / kg, preferably 10 7< - 10 15< viable spores / kg, more preferably 10 10< - 10 13< viable spores / kg at the time point of applying biological control agents on a plant or plant parts such as seeds, fruits or vegetables. Usual application rates into the soil are between 500g / ha and 20 kg / ha of a formulation comprising 1x10 9< viable spores / g. From this ratio, respective application rates of formulations with a different spore concentration may be calculated by the skilled person. Preferred ranges comprise an application rate of between approximately 1 and approximately 10 kg / ha, such as 8 or 9 kg / ha, furthermore preferred between approximately 1 and approximately 5 kg / ha, for example 1, 2, 3, 4 or 5 kg / ha or any value in between those values. Under certain circumstances and depending on the crop, the application rate may be about 8 kg, in particular if the crop is a vegetable such as lettuce. As a general rule, between 1 x 10 12< and 4 x 10 12< viable spores / ha are applied. These application rates are applicable both for application to the root system of a plant and to the soil on which a plant grows or is intended to grow, such as in-furrow or as spray. In any case, the conidia need to be worked into the soil.

[0024] Said Coniothyrium minitans strain may be applied in the form of spores, conidia, hyphae or a combination of any of the foregoing. It is preferred that said Coniothyrium minitans strain is applied in the form of conidia.

[0025] Conidia or conidial spores are asexually formed spores of fungi. Conidia are formed either by morphologically undifferentiated hyphae or frequently by upright hyphae the conidia carriers, or special structures of the fungi, the receptacles. The conidia of Coniothyrium minitans are formed in receptacles, the so-called pycnidia. They are therefore also frequently referred to as pycnidia spores. Pycnidia are hollow, usually spherical or pear-shaped asexual receptacles of fungi. Conidia are formed at the inner wall of the pycnidia and frequently develop on short conidia carriers, which are present in large masses.

[0026] Hypha (plural - hyphae) are thread-like organs of certain fungi. In their totality, the hyphae form the fungal mycelia by means of longitudinal growth and branching. A fungus essentially consists of the fungal mycelia. The mycelium, in turn, can assume particular forms, which then constitute the organ of the fungus, such as the receptacles.

[0027] Said Coniothyrium minitans CON / M / 91-08, preferably its conidia, may be present in a WG (wettable granule) or a liquid formulation. It is preferred that the formulation is a WG formulation.

[0028] Suitable applications include an application on harvest residues, and applications to the soil, e.g. during the pre-planting process or to the root system of a plant. Normally, with the exception of harvest residues, the respective formulation is worked into the soil to a depth of between 2 and 15 cm, preferably between 5 and 10 cm.

[0029] Application to harvest residues: After cultivating a plant, such as a susceptible crop plant, before the initial tilling of the soil, a formulation, preferably a WG formulation, of Coniothyrium minitans CON / M / 91-08 is applied on infested harvest residues with an applied amount per hectare as described above. For a subsequent summer crop, treatment with preferably takes place in the spring. The formulation is preferably applied immediately before the initial tilling of the stubble into the soil. After the application, the formulation is incorporated with the stubble in the soil. Where cultivation takes place without a plough, the application should be applied where possible in the late autumn when temperatures do not exceed 20°C and soil stays moist. Where this arises, subsequent rainfall will additionally promote distribution of the spores.

[0030] Application to the soil: Before planting, Coniothyrium minitans CON / M / 91-08 is applied and should be incorporated in the top layer of the soil, advantageously during seedbed preparation or with a combined seed drill. As an alternative to the pre-sowing application, Coniothyrium minitans CON / M / 91-08 may also be applied during post-emergence. For example, in the case of rapeseed, treatment can take place up to the 4 to 6 leaf stage.

[0031] The method may further comprise applying simultaneously or subsequently at least one further plant protection agent to the root system of a plant, to harvest residues or to the soil on which a crop plant grows or is intended to grow. Preferably, said at least one plant protection agent is a fungicide. It is even more preferred that said fungicide is effective against at least one fungal pathogen selected from of the genus Sclerotinia spp., Phoma spp. and Verticillium spp., preferably at least Phoma spp. and Verticillium spp.. Exemplary fungicides include Azoxystrobin, Pyraclostrobin, Picoxystrobin, Trifloxystrobin, Fluoxastrobin, Isopyrazam, Boscalid, Fluxapyroxad, Fluopyram, Bixafen, Isoflucypram, Penthiopyrad, Pydiflumetofen, Benzovindiflupyr, Prothioconazole, Tebuconazole, Difenoconazole, Flutriafol, Cyprodinil, Fludioxonil, B. Subtilis strain QST713, B. Pumilus strain QST2808, Carbendazim, Thiophanat-methyl, Procymidone, Iprodione or a mixture of any of the above. Preferably, the fungicide is Prothioconazole or Fluopyram or a mixture thereof.

[0032] In another aspect, the present invention relates to the use of Coniothyrium minitans strain CON / M / 91-08 for controlling phytopathogenic fungi of the genus Verticillium spp. in soil where a crop plant grows or is intended to grow, wherein said plant is of the genus Brassica spp.

[0033] The preferred embodiments described for the method of the invention may equally be applied to the use of the invention.Example 1: Effect of Coniothyrium minitans on Verticillium and Phoma in oilseed rape.

[0034] The preparation / formulation for protection of cultivated plants against attacks by fungal pathogens Verticillium spp. and Phoma spp. contains 1x10 12< of active spores of the fungal strain Coniothyrium minitans CON / M / 91-08 in 1 kg of the preparation. The preparation was applied in the course of August into the soil before sowing of winter rapeseed in the dose 2 kg / ha and introduced into soil 5 cm deep at 20 °C.

[0035] After harvesting the crop, all postharvest leftovers were collected, i.e. leftovers of stems and roots collected from an accurately outlined square meter of the experimental field and the results were evaluated. The evaluation consisted in counting of green, i.e. live, postharvest leftovers, such as stems, root crowns and roots, and also monitoring of plants attacked by Phoma spp. genus, based on cross-cuts of postharvest leftovers and plants attacked by Verticillium spp. genus based on erticillium wilt of roots and root crowns.

[0036] Table 1 shows the evaluation of postharvest leftovers. The stem part, root crown and roots were each evaluated separately. Table 1 in the first column indicates the ordinal number of repeated evaluation of Contans, a publicly available granular formulation comprising C. minitans CON / M / 91-08 i.e. the strain used in accordance with this invention, the roman numbers "I, II, III" mean the individual repetitions. Subsequently, the physiological condition of the individual parts of the plants were evaluated which were subsequently attacked by fungal pathogens of the Phoma, Verticillium and Sclerotinium genera and the percentage share is subsequently indicated in the table. In the lower part of the table the average values are indicated. The share of green stems increased on average from 19.3 % on the untreated control plants to the average of 68.6% on plants treated with the preparation, which represents an increase by about two thirds. The share of prematurely ripened stems decreased on average from 80.7 % on untreated plants to the average of 31.4 % on plants treated with the preparation, i.e. a reduction by three fifths. Similar results were obtained for root crowns and roots as shown in Table 1. In all the cases sclerotinia were eliminated from the stems, root crowns / stalk basis and roots and also the risk of attacks by other diseases extremely decreased, such as phoma blackening of stem on the plant's section to the extent leading to dying or premature ripening of the plant caused by Phoma spp., while application of the preparation reduced the attacks on average from 62.5 % to 28.7 %, i.e. by more than one half, in comparison with the control sample not treated by the preparation. The application also reduced attacks by other dangerous diseases, specifically by Verticillium spp. on the roots, while the application of the preparation reduced the attacks on average from 58.9 % to 19,7 %, i.e. by 39 % of the original value of the control sample, which means a decrease by two thirds (see Table 1).

Claims

1. Method for controlling phytopathogenic fungi of the genus Verticillium spp. comprising applying Coniothyrium minitans strain CON / M / 91-08 in an effective amount to the root system of a plant, to harvest residues or to the soil on which a crop plant grows or is intended to grow, wherein said plant is of the genus Brassica spp.

2. Method according to claim 1, wherein said Coniothyrium minitans strain is applied in the form of spores, conidia, hyphae or a combination thereof.

3. Method according to claim 1 or 2, wherein said Coniothyrium minitans strain is applied in the form of conidia.

4. Method according to claim 3, wherein said conidia are present in a WG or a liquid formulation.

5. Method according to claim 4, wherein said formulation is a WG formulation.

6. Method according to any one of claims 1 to 5, wherein said Coniothyrium minitans strain is applied pre-planting, during growth or post-harvest.

7. Method according to any claim 6, wherein said Coniothyrium minitans strain is applied pre-planting.

8. Method according to anyone of claims 1 to 7, wherein said plant is Brassica napus.

9. Method according to claim 8, wherein said Brassica napus plant is winter oilseed rape.

10. Method according to any one of claims 1 to 9, further comprising applying simultaneously or subsequently at least one further plant protection agent to the root system of a plant, to harvest residues or to the soil on which a crop plant grows or is intended to grow.

11. Method according to claim 10, wherein said plant protection agent is a fungicide effective against at least one fungal pathogen of the genus Verticillium spp..

12. Use of Coniothyrium minitans strain CON / M / 91-08 for controlling phytopathogenic fungi of the genus Verticillium spp. in soil where a crop plant grows or is intended to grow, wherein said plant is of the genus Brassica spp.