Coniothyrium minitans for protection of cultivated plants from attacks by fungal pathogens belonging to the genus phoma spp. or verticillium spp

The present invention relates to a method for controlling phytopathogenic fungi of the genus Phoma spp. and Verticillium spp. comprising applying Coniothyrium minitans strain CON/M/91-08 or a mutant having all identifying characteristics of the respective strain 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. Also disclosed is the use of said strain for controlling phytopathogenic fungi of the genus Phoma spp. and Verticillium spp.

CROSS-REFERENCE TO RELATED APPLICATION(S)

The present application is a 35 U.S.C. § 371 national phase entry of International Application No. PCT/EP2017/079192, filed Nov. 14, 2017, which claims the benefit under 35 U.S.C. § 119 of Czech Patent Application No. 2016-711, filed Nov. 14, 2016, the entire contents of which are hereby incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates the area of specific soil decontamination and biological protection against pathogenic fungi in agriculture and horticulture, specifically the use of theConiothyrium minitansstrain 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

TheConiothyriumgenus belongs to the Coelomycetes class, sub-group Deuteromycotina, identified for the first time in 1947 in the United States. TheConiothyrium minitansstrain 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 theConiothyrium minitansstrain which, after the application into soil, contaminate it and parasitize on present sclerotia of fungi from theSclerotiniaspp. genus that cause e.g. white rot of plant roots and degrade them relatively very quickly.Sclerotiumis 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 theConiothyrium minitansstrain on postharvest leftovers the hyperparasite fungus rapidly develops and it even spreads to adjoining sclerotia not directly hit by the spray. TheConiothyrium minitansfungus 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 freezesConiothyrium minitanssuspends 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.

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 bySclerotiniaspp. fungi, particularly bySclerotinia sclerotiorumandSclerotinia minor.

Another common soil pathogen on cultivated plants is e.g. thePhomagenus which causes the so-calledphomablackening 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 thePhomagenus 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.

TheVerticilliumgenus hosts further pathogens for cultivated plants that is difficult to eliminate from soil. Pathogens belonging to this genus cause the so-calledverticilliumwilt 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.

The purpose of the invention is to find a preparation or a procedure which would effectively eliminate pathogens of thePhomagenus andVerticilliumgenus and which would be easy to apply. A bio-preparation is preferred.

SUMMARY OF THE INVENTION

The invention consists in methods of applying and the use of theConiothyrium minitansstrain CON/M/91-08 for protection of cultivated plants against attacks by fungal pathogensVerticilliumspp. andPhomaspp. The presence of fungal pathogens fromVerticilliumandPhomagenera poses a serious problem to cultivated crops because their elimination is very complicated and no elimination method has been known to protect crops. TheConiothyrium minitansCON/M/91-08 fungal strain is commonly used againstSclerotinia sclerotiorumand the use of its effects againstVerticilliumandPhomagenera is based on completely new findings about the effects of this strain. TheVerticilliumandPhomagenera have different morphological cell structures from that ofSclerotinia sclerotiorumand therefore one cannot expect theConiothyrium minitansCON/M/91-08 strain to have antagonistic effects and to be efficient also against these types of fungal pathogens.

The subject of this invention is also a preparation for protection of cultivated plants against attacks by fungal pathogensVerticilliumspp. andPhomaspp. The invention consists in the fact that the preparation contains the fungal strainConiothyrium minitansCON/M/91-08 on a nutritious carrier. In a convenient version of the invention the strain in the preparation is in the form of spores, conidia, hyphae or a combination thereof. These forms of the fungal strainConiothyrium minitanssubsequently 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.

Another subject of the invention is a method of protection of cultivated plants from attacks by the fungal pathogensVerticilliumspp. andPhomaspp. The invention consists in the fact that the above-described preparation is applied, e.g., on the root system of a cultivated plant, in any case into the soil. It has surprisingly been found that the presentC. minitansstrain is also effective againstphomablackening of stem andverticilliumwilt. With regard to the different morphology ofVerticilliumandPhomagenera no analogy can be found with removal of theSclerotiniagenus pathogens. In a convenient version of the invention the preparation is applied into the soil in the dose of e.g. 2 kg/ha before sowing of a cultivated plant and it is introduced into the soil up to 10 cm deep.

Advantages of theConiothyrium minitansstrain 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 theSclerotiniagenus against which theConiothyrium minitansstrain has been commonly used but also against other pathogens usually occurring in soil used for cultivated plants, i.e. againstVerticilliumspp. andPhomaspp. TheConiothyrium minitansstrain 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 OF THE INVENTION

In one aspect, the invention relates to a method for controlling phytopathogenic fungi of the genusPhomaspp. andVerticilliumspp. comprising applyingConiothyrium minitansstrain 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.

Coniothyrium minitansstrain CON/M/91-08 is described in WO 96/21358 which is incorporated herein by reference. The strain was deposited with the German Collection of Microorganisms and Cell Cultures having the address Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstraβe 7B, 38124 Braunschweig Germany under the accession number DSM 9660 on Mar. 8, 2000. The strain is available as CONTANS® WG from Bayer CropScience Biologics GmbH.

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.

Usually the strain is formulated on a carrier, preferably a water-soluble sugar carrier, in a concentration of between 1×107and about 3×109, preferably viable, spores/g of carrier, preferably between 1×108and about 2×109spores/g carrier. Most preferably, the concentration lies between about 5×108and about 1.5×109spores/g of carrier, such as at about 1×109viable spores/g carrier. In particular the water-soluble sugar is glucose.

According to the inventionConiothyrium minitansstrain CON/M/91-08 also encompasses mutants having all identifying characteristics of the respective strain. Such identifying characteristics include an activity against at least one plant pathogen of the genusPhomaspp. orVerticilliumspp., in combinations with an activity againstSclerotiniaspp., such asSclerotinia sclerotiorumand/orSclerotinia minorand/orSclerotium cepivorum.

The term “mutant” refers to a variant of the parental strain as well as methods for obtaining a mutant or variant in which the pesticidal activity is greater than that expressed by the parental strain. The “parent strain” is defined herein as the original strain before mutagenesis. To obtain such mutants the parental strain may be treated with a chemical such as N-methyl-N′-nitro-N-nitrosoguanidine, ethylmethanesulfone, or by irradiation using gamma, x-ray, or UV-irradiation, or by other means well known to those skilled in the art.

A “variant” is a strain having all the identifying characteristics of the respective Accession Numbers as indicated in this text and can be identified as having a genome that hybridizes under conditions of high stringency to the genome of the respective Accession Numbers.

“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×SSC or a solution of equivalent ionic strength/temperature. A moderate stringency hybridization is typically performed at about 50° C. in 6×SSC, and a high stringency hybridization reaction is generally performed at about 60° C. in 1×SSC.

A variant of the indicated Accession Number may also be defined as a strain having a genomic sequence that is greater than 85%, more preferably greater than 90% or more preferably greater than 95% sequence identity to the genome of the indicated Accession Number. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 80%, 85%, 90%, or 95%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example, those described in Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987) Supplement 30, section 7. 7. 18, Table 7. 7. 1.

Equally, the presentConiothyrium minitansstrain may be applied to control plant pathogens of the genusVerticilliumspp. Members of this genus causeverticilliumwilt which affects more than 350 species of eudicot plants. The most prominent plant pathogenicVerticilliumspecies areV. dahlia, V. albo-atrum, V. longisporum, V. nubilumandV tricorpus(for an overview, see Barbara, DJ. & Clewes, E. (2003). “Plant pathogenicVerticilliumspecies: how many of them are there?” Molecular Plant Pathology 4(4).297-305. Blackwell Publishing).

Coniothyrium minitansCON/M/91-08 e.g. its conidia, are present in a formulation in a concentration of at least 105viable spores per gram formulation (e. g. spores/g preparation), such as 105-1017viable spores/kg, preferably 107-1015viable spores/kg, more preferably 1010-1013viable 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 solid are between 500 g/ha and 20 kg/ha of a formulation comprising 1×109viable 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 1×1012and 4×1012viable 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.

In a preferred embodiment, saidConiothyrium minitansstrain is applied in the form of spores, conidia, hyphae or a combination of any of the foregoing. It is preferred that saidConiothyrium minitansstrain is applied in the form of conidia.

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 ofConiothyrium minitansare 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.

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.

In another preferred embodiment, saidConiothyrium minitansCON/M/91-08, preferably its conidia, are present in a WG (wettable granule) or a liquid formulation. It is preferred that the formulation is a WG formulation.

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.

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, ofConiothyrium minitansCON/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.

Application to the soil: Before planting,Coniothyrium minitansCON/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 minitansCON/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.

In one embodiment, the method of the invention further comprises 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 genusSclerotiniaspp.,Phomaspp. andVerticilliumspp., preferably at leastPhomaspp. andVerticilliumspp. Exemplary fungicides include Azoxystrobin, Pyraclostrobin, Picoxystrobin, Trifloxystrobin, Fluoxastrobin, Isopyrazam, Boscalid, Fluxapyroxad, Fluopyram, Bixafen, Isoflucypram, Penthiopyrad, Pydiflumetofen, Benzovindiflupyr, Prothioconazole, Tebuconazole, Difenoconazole, Flutriafol, Cyprodinil, Fludioxonil, B.Subtilisstrain QST713, B.Pumilusstrain 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.

In another aspect, the present invention relates to the use ofConiothyrium minitansstrain CON/M/91-08 for controlling phytopathogenic fungi of the genusPhomaspp. andVerticilliumspp. in soil where a crop plant grows or is intended to grow.

The preferred embodiments described for the method of the invention may equally be applied to the use of the invention.

The following example illustrates the invention without limiting it to particular embodiments. Those skilled in the art will find or will be able to provide, based on routine experimenting, one or more equivalents of the embodiments of the invention disclosed herein.

The preparation/formulation for protection of cultivated plants against attacks by fungal pathogensVerticilliumspp. andPhomaspp. contains 1×1012of active spores of the fungal strainConiothyrium minitansCON/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.

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 byPhomaspp. genus, based on cross-cuts of postharvest leftovers and plants attacked byVerticilliumspp. genus based on erticillium wilt of roots and root crowns.

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 comprisingC. minitansCON/M/91-08 i.e. the strain used in accordance with this invention, the roman numbers “I, II, HI” 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 thePhoma, VerticilliumandSclerotiniumgenera 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 under this invention, 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 under this invention, i.e. a reduction by three fifths. Similar results were obtained for root crowns and roots as shown in Table 1. In all the casessclerotiniawere eliminated from the stems, root crowns/stalk basis and roots and also the risk of attacks by other diseases extremely decreased, such asphomablackening of stem on the plant's section to the extent leading to dying or premature ripening of the plant caused byPhomaspp., while application of the preparation under this invention 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 byVerticilliumspp. on the roots, while the application of the preparation under this invention 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).