AT525817B1 - ORGANIC SEED TREATMENT - Google Patents

Abstract

The invention describes a novel biological seed treatment agent which has the fungus Fusarium oxysporum f. sp. strigae, strain DSM 33471 and is used to combat the parasitic weed Striga hermonthica on monocotyledonous crops and the use thereof.

Description

Description

The present invention relates to a treatment agent for seeds of monocotyledonous crop plants containing an active ingredient selected from a fungal strain from the Fusarium group and optionally a carrier substance and/or other auxiliary substances.

[0002] Parasitic weeds of the genus Striga lead to high yield and quality losses worldwide. Particularly in the sub-Saharan zone, the infestation of maize (Zea mays) and millet (e.g. Sorghum spp.) with Striga hermonthica leads to annual yield losses of around eight million tons (Gressel J., Hanafı A., Head G., Marasas W., Obilana A.B., Ochanda J., Soussi T., Tzotzos G., 2004: Major heretofore intractable biotic constraints to African food security that may be amenable to novel biotechnological solutions. Crop Protection, Vol. 23: 661-689.). These losses were estimated in 2003. Since then, the loss of yield caused by S. hermonthica has probably increased significantly, leading to a massive negative impact on the lives of around 300 million people through the parasitic plant (Pennisi E., 2010: Armed and dangerous. Science, Vol. 327: 804 -805).

Despite considerable efforts through chemical plant protection and plant breeding as well as through cultural measures, it has not yet been possible to prevent the further spread of the weed in this region. Measures taken include, for example, the cultivation of varieties with lower susceptibility to parasitic weeds, the use of the herbicide Imazapyr on Imazapyr-resistant maize (IR maize) or the pull-push cultivation method, in which the legume Desmodium intortum is sown between the rows of maize.

[0004] For this reason, there have been efforts to use a Striga wilt pathogen, in particular Striga hermonthica, the fungus Fusarium oxysporum, to combat the weed (Ndambi B., Cadisch G., Elzein A., Heller, A., 2011: Colonization and control of Striga hermonthica by Fusarium oxysporum f. sp. strigae, a mycoherbicide component: An anatomical study. Biological Control, Vol. 58: 149-159). Unfortunately, no success has been achieved with the wilt pathogen either. A recent development in this field, which uses the fungus Fusarium oxysporum, appears promising (Nzioki H., Oyosi F., Morris C.E., Kaya E., Pilgeram A.L., Baker C., Sands D.C., 2016: Striga Biocontrol on a Toothpick : A Readily Deployable and Inexpensive Method for Smallholder Farmers. Frontier in Plant Science, Vol. 7: Article 1121). Despite some successes, the main disadvantage of the method is the high level of manual effort required to use it. Up to now, the substrate covered with the fungus has been placed into the planting holes by hand, immediately before they are covered with the seeds and sealed with soil. Such a technology may still be applicable to maize with a plant number of around 60,000 plants per hectare (figures from Kenya), but not to millet or rice, where a significantly higher number of plants are grown per hectare. A solution to this problem that has been suggested is to attach the fungus to the seed. Experiments have already been carried out here (Elzein A., Kroschel J., Leth L; 2006: Seed treatment technology: An attractive delivery system for controlling root parasitic weed Striga with mycoherbicide. Biocontrol Science and Technology, Vol. 16: 3-26. ) which have not yet led to a commercial product. The reason for the limited success so far was, on the one hand, the use of fungal strains that had insufficient pathogenicity towards the weeds and, on the other hand, the storage stability of the fungus used in the seed treatment agent was too low.

[0005] From Dorah A. Oula, John M. Nyongesah, George Odhiambo, Samuel Wagai; The effectiveness of local strains of Fusarium oxysporium f. Sp. Strigae to control Striga hermonhica on local maize in western Kenya; food science nutrition; 2020:8.4352 ff., the effectiveness of five local Fusarium oxysprium f. Sp. Strigae strains FK1, FK2, FK3, FK4 and FK5 was examined to control Striga on maize crops. FK 5 was determined to be the most efficient strain based on risk and infection numbers and the maize yield.

[0006] From Muhammad Jamil, Boubacar A. Kountche and Salim Al-Babili; Curent progress in Striga management, Plant Physiology, Vol 185, 04,2021, S 1339 -1352 are among others

Microherbicides, such as some strains of F. oxysporum described which can produce large amounts of amino acids that are toxic to Striga, but not to the plants being cultivated, such as corn and millet.

The present invention now aims to provide a treatment agent for seeds of monocotyledonous crop plants which is both highly effective against weeds of the genus Striga, in particular Striga hermonthica, and on the other hand is stable in long-term storage under ambient conditions.

To solve this problem, the treatment agent according to the invention for seeds of monocotyledonous crop plants is essentially characterized in that the active ingredient is the fungus Fusarium oxysporum f. sp strigae, DSM 33471. Surprisingly, as part of special studies in Kenya, fungal strains belonging to the genus Fusarium were isolated that were very pathogenic towards Striga hermonthica. Just as surprisingly, in addition to their high pathogenicity, these strains were able to select for the ability to increase amino acid production (methionine). Certain amino acids have a specific herbicidal effect on plants (Fernandez-Aparicio M., Bernard A., Falchetto L., Marget P., Chauvel B., Steinberg C., Morris C.E., Gibot-Leclerc S., Boari A., Vurro M., Bohan D.A., Sands D.C., Reboud X., 2017: Investigation of Amino Acids as Herbicides for Control of Orobanche minor Parasitism in Red Clover. Front Plant Sci. Vol. 8: Article 842). Thanks to both properties, high pathogenicity and the production of methionine, the Fusarium strains are ideal for biological control of Striga hermonthica. Among the selected Fusarium strains, a special mutant, namely Fusarium oxysporum f. sp strigae, DSM 33471, is also resistant to the fungicide Captan WP50 (active ingredient Captan) in an unexpected manner. Not only the resistance to Captan WP50 but also the ability of Fusarium oxysporum f. sp strigae to form large quantities of spores in a substrate allow its use in a treatment agent for seeds of monocotyledonous crops.

A particularly efficient treatment of seeds is achieved if, as corresponds to a further development of the invention, the seed treatment agent is developed in such a way that the agent contains at least 1 x 10 ° and at most 1 x 10'%, preferably 1 x 10 ° to 1 x 10% colony forming units of the fungus Fusarium oxysporum f. sp. strigae, DSM 33471 per gram are included. Such active ingredient concentrations not only enable efficient control of the Striga wilt pathogen, in particular Striga hermonthica, but with such active ingredient concentrations, in particular the preferred number of colony-forming units, it is surprisingly possible to provide a long-term storage-stable composition. The storage of such a treatment agent is preferably carried out in a refrigerator at temperatures between 4 and 15 ° C, but the treatment agent can also be stored at ambient temperatures of 25 to 35 ° C for several months without any significant loss of activity. The spore powder according to the invention can be stored in the refrigerator for over a year without significantly reducing the number of colony-forming units present and without losing its excellent effect.

According to a further development of the invention, at least one group selected from conidia, chlamydospores or mycelial remains of Fusarium oxysporum f. sp strigae, DSM 33471 are included as colony-forming units. The use of chlamydospores in a seed treatment product overcomes the disadvantage that the preparation has poor storage stability on the seeds. This makes it possible to treat the seeds with a surprisingly small amount of fungal spore formulation per kilogram, depending on the plant species. Experiments known from the literature to treat seeds with fungal material regularly required significantly higher application rates. For example, Ciotola et al. (2000) 20 g of a spore powder for the treatment of only 1000 seeds of sorghum millet (Ciotola M., DiTommaso A., Watson A.K., 2000: Chlamydospore Production, Inoculation Methods and Pathogenicity of Fusarium oxysporum M12-4A, a Biocontrol for Striga hermonthica. Biocontrol Science and Technology, Vol. 10: 129-145.).

By using the fungal strain according to the invention, it has also surprisingly been possible to produce a seed treatment agent which combines a very good effect with captan resistance, high storage stability and a very low application rate. In order to have a seed treatment agent that can be easily dosed despite the possible small quantities used, the treatment agent according to the invention for seeds of monocotyledonous crop plants is essentially developed in such a way that at least one solid powdery carrier selected from the group of zeolites, vermiculites, diatomaceous earths is also used as a carrier , kaolins, bentonites, peat, bran, preferably oat bran, wheat bran, flour from bamboo plants, wood flour, in particular hardwood flour, preferably beech wood flour or eucalyptus flour. Among the carrier materials, the organic carrier materials are preferred because they not only serve for an even distribution of the active ingredient in the seed treatment agent, but can also serve to support the germination of the fungus after sowing, so that it can as completely as possible the in the Seedlings present in the vicinity of the seeds can be affected by Striga hermonthica. In order to be able to achieve particularly good adhesion of the seed treatment material to the surface of the seed and thus a direct effect of the composition, the invention is further developed in such a way that the carrier has an average grain size of less than 500 μm, preferably less than 250 μm.

Particularly when using organic carrier materials in the seed treatment material, the invention is developed in such a way that a wood flour, in particular beech wood flour, with a grain size distribution between 200 μm and 20 μm is used as the carrier. A particle size distribution of between 200 µm and 20 µm of the carrier not only makes it possible to obtain a homogeneous mixture of carrier and active ingredient, but above all a fine distribution of the fungus Fusarium oxysporum f. sp strigae, DSM 33471 on the seed, where it grows after sowing with its fungal hyphae germinates and spreads through these hyphae into the surroundings of the treated seed in the soil. The germinating seeds and seedlings of Striga hermonthica present in the vicinity of the seed are attacked or killed by the effect of the amino acid and are therefore no longer able to attack the roots of the susceptible plant germinating from the seed. Finally, the treatment agent can contain further auxiliary substances selected from: binders, dyes or coating agents.

The invention is explained in more detail below using 7 examples.

EXAMPLE 1 Isolation of Fusarium oxysporum f. sp. strigae from wilting Striga plants

Wilting Striga plants were removed from corn and millet fields and stored in paper bags. Approximately 4 cm long segments were cut from the stem area of the plants and superficially sterilized for 5 minutes in a 1.2% sodium hypochloride solution. The segments were then rinsed thoroughly in sterile tap water and their ends were cut off with a sterile scalpel. The segments treated in this way were then placed on a semi-selective Nash & Snyder agar medium (Nash S.M., Snyder W.C., 1962: Quantitative estimation by plate counts of propagules of the bean rot Fusarium in field soil. Phytopathology 52, 567 - 572) and at 25 ° C incubated until fungal mycelium grew from the ends of the stem segments. With the aim of purifying the cultures, small agar blocks were then cut out from the edge of the growing colonies and transferred to fresh Nash & Snyder medium. This procedure was repeated once. Small pieces of agar were again cut out from the edge area of the colonies that then grew out and transferred to water agar. After 2 weeks, the agar plates were covered with sterile tap water and the resulting conidia were rubbed off using a Drigalski spatula and thus transferred into a suspension. From this, individual conidia were transferred either to a clove leaf agar medium (CLA) or to a potato dextrose agar medium. Colonies of Fusarium oxysporum were identified by conidial morphology in the clove leaf agar medium and by the typical discoloration of the fungal colony in the potato dextrose agar medium. The Fusarium oxysporum colonies formed on the clove leaf agar medium formed the characteristic

macro- and microconidia as well as chlamydospores. The Fusarium oxysporum colonies on the potato dextrose agar medium were characterized by a typical purple discoloration of the mycelium and the agar medium.

A total of 48 individual spore isolates of F. oxysporum were obtained with this experiment.

EXAMPLE 2 Selection of a Fusarium oxysporum strain with increased methionine production

[0016] Amino acid analogues can be used to select Fusarium oxysporum strains with increased amino acid production (Sands C.D., Pilgram A.L., 2009: Methods for selecting hypervirulent biocontrol agents of weed: why and how. Wiley Interscience, https:// onlinelibrary.wiley.com/action/doSearch?AllField=10.1002%2Fps. 1739).

[0017] A Czapek-Dox agar (Difco, St Louis, MO) was mixed with ammonium sulfate (0.5 g L), uracil (20 mg L*), thiamine (4 mg L*) and 100 mg L* of powdered multivitamin tablets added. The agar medium was poured into 90 mm Petri dishes. After cooling, 100 hl of a Fusarium oxysporum conidia suspension (approx. 1 x 10° cfu per plate) were plated onto the surface of the agar medium using a Drigalski spatula. After the agar surface had dried, an agar cylinder (diameter 6 mm) was punched out of the middle of the medium using a sterile cork borer, so that a cylindrical hole was created. 0.1 ml of a sterile selenomethionine solution was filled into this. The concentration of the solution was 1000 ppm. Selenomethionine is an analogue of the amino acid methionine. The plates were then incubated for 4 hours under the laminar flow of a safety cabinet. Afterwards, 0.1 ml of the selenomethionine solution was filled into the cylindrical well again before the plates were incubated in an incubator at 28 °C. An inhibition zone emerged around the cylindrical depression in which only very isolated, if any, fungal growth was recorded. Only individual colonies could be detected. These were assumed to be mutants with increased methionine production. A total of 18 such colonies were found on a total of 50 Petri dishes at different distances from the cylindrical well in the center of the Petri dish. The closer the respective colony grew to the cylindrical depression, the higher the mutant's methionine production should be. The colonies created in this way were removed and further cultured on potato dextrose agar. Increased methionine production was demonstrated using a special bacterial strain (Leuconostoc mesenteroides, ATCC 4043) that is auxotrophic for methionine. The bacterial strain cannot therefore develop on a methionine-free agar medium. Bacterial growth was only detectable in the presence of some of the selected Fusarium oxysporum isolates, which therefore had to be assumed to excrete methionine into the agar medium. This was observed in 13 of the 18 newly obtained isolates. This provided evidence that the selected isolates were actually mutants that increased their excretion of methionine into the agar medium.

A further 4 selection cycles were carried out with the selected Fusarium oxysporum isolates, with the concentration of the selenomethionine solution being doubled in each case.

[0019] At the end of the selection process, 3 new Fusarium oxysporum strains were selected in which a particularly high methionine production could be demonstrated. EXAMPLE 3

Selection of an isolate with increased captan resistance

Five captan-containing potato dextrose agar media were prepared by adding 0.04; 0.1; 0.25; 0.6 and 1.5 g/l Captan 50WP prepared. First, the medium with the lowest captan concentration was used. 25 ml of each was poured into 90 mm Petri dishes. After cooling, 100 µl of a Fusarium oxysporum conidia suspension (approx. 1 x 10° cfu per plate) of the previously selected (see Example 2) methio-

nin-producing Fusarium oxysporum strains were plated on the surface of the agar medium. 10 Petri dishes per strain were used.

After 7 days, isolated fungal colonies became visible on the surface of the agar medium, which had to be assumed to be capable of development at the captan concentration used, i.e. to have already achieved a certain degree of resistance to the fungicide. These were removed using a sterile scalpel and further cultured on an identical medium in other Petri dishes. The selected isolates were then transferred to the media with the next higher captan concentration using the same method. Once again, colonies were found that grew on the medium containing captan. The fungal isolates obtained from this were further cultivated and subjected to the selection process again. This was continued until a captan concentration of 0.75 mg/l agar medium or 1.5 mg/l Captan 50WP was achieved (the product Captan 50WP contains 50% of the active ingredient captan). An isolate was obtained from this medium and was given the internal designation DSSN 6. This was deposited with the DSMZ Braunschweig under the master name DSM 33471.

EXAMPLE 4

Microconidia suspension as a prerequisite for the production of a Fusarium oxysporum spore powder

200 ml of potato dextrose broth were placed in a 1 liter Erlenmeyer flask. This was sealed with a cotton plug and aluminum foil and then autoclaved. The cooled broth was inoculated with 8 agar pieces (approx. 6x6 mm) covered with the DSM 33471 strain from the edge area of a fungal colony growing on a PDA culture medium.

The culture was then shaken at 90 rpm for 7 days at 26 ° C. As a result of the shaking culture, approximately 3.2 x 10' microconidia per milliliter were counted.

EXAMPLE 5 Production of a product sample

A crystallization dish was filled with 20 g of beech wood flour with a grain size of approximately 40120 μm, such as the product “Lignocel® HB 120” from J. Rettemaier & Söhne GmbH & Co. KG, as well as 40 ml of an aqueous soil extract filled. The two substances were mixed well together. The crystallization dish, closed with aluminum foil, was then sterilized in an autoclave at 121 ° C for 15 minutes.

The previously prepared microconidia suspension (Example 4) had been stored in the refrigerator for about 24 hours. During this time the microconidia settle to the bottom. The supernatant (approx. 100 ml) was poured off so that the conidia concentration increased to approximately 6 x 10' conidia per milliliter. 20 ml of this suspension were placed in the crystallization dish filled with the wood flour and the aqueous soil extract and mixed well with the wood flour-soil extract mixture using a sterile long-handled spoon. The result was a loose, hand-moist mixture. This was placed in an incubator in the closed crystallization dish and incubated at 25 °C for 14 days.

After incubation, the substrate was dried in the laminar flow of a sterile workbench at approx. 23 ° C for seven days in the now opened crystallization dish. During the drying process, the substrate was thoroughly mixed daily with a sterile long-handled spoon to ensure that the drying was as even as possible. After drying, a fine, homogeneous powder was formed.

The number of colony-forming units (cfu) per gram was determined immediately after drying. For this purpose, 100 mg of the resulting powder were suspended in 1000 ml of sterile tap water in a beaker and homogenized for two minutes using an Ultra Turrax from IKA®-Werke GmbH & Co. KG. Even during the homogenization

During the process, 100 μl of the resulting suspension were removed from the beaker using a Varipipette from Eppendorf SE and spread on a PDA culture medium. After 24 hours, clear mycelial growth could be seen on the surface of the culture medium, so that the fungal colonies or germinating cfu could be counted under the microscope. After counting and converting, the figure was 5.2 x 10' cfu per gram of substrate.

Microscopic examinations of the powder revealed that large amounts of conidia and chlamydospores were present in the product.

Product samples were produced in the same way as described above, in which the carrier material Lignocel® HB 120" from J. Rettemaier & Söhne GmbH & Co. KG was replaced by flour or ground oat bran obtained from bamboo plants The counted fungal colonies contained in the powder and the amounts of conidia and chlamydospores did not differ significantly from the numbers determined when using the flour from Buchholz.

EXAMPLE 6 Storage stability

A product produced as described in Example 5 was sealed in aluminum-coated plastic bags in portions of 10 g each and then either in the incubator at 25 ° C, in the refrigerator at 5 ° C or in the freezer at -10 ° C stored. The number of germinable units per gram of product was determined after 3, 6, 9 and 12 months. The result of the investigation is shown in Table 1.

Table 1: Storage stability of a fungal spore powder, produced using the fungal strain DSM 33471, with an initial germination capacity of 5.2 x 10' cfu/g and storage at various temperatures

Storage temperature Number of cfu after storage of 3 months 6 months 9 months 12 months -10 °C 5.1 x 107 4.9 x 107 5.0 x 107 4.8 x 107 +5 °C 4.9 x 107 4.9 x 107 4.3 x 107 4.1x 107

+25°C 3.9 x 107 2.2 x 107 1.1 x 107 3.7 x 10°

[0032] Surprisingly, it could be shown that the product has very good storage stability in relation to the germination capacity of the formulated fungal cfu when stored in the refrigerator (+5 ° C) and when stored in the freezer (-10 ° C).

EXAMPLE 7 Effect of fungal strain on Striga hermonthica

The product produced using the fungal strain according to the invention was bound to corn seeds using a sugar solution. One gram of spore powder (Example 5) was used per kilogram of corn seed. The powder had a germination capacity of 5.2 x 10' cfu per gram. A total of 17 trials were conducted in 4 different districts in Kenya. The tests, which were carried out by farmers living at the test sites, consisted of one treated and one untreated plot of 100 m² each. The test locations can be specified as follows:

- Bungoma (6 farmers)

* Kakamega (5 farmers)

- Siaya (4 farmers)

- Vihiga (2 farmers)

In all cases these were highly contaminated with Striga hermonthica

Areas. The test plots were planted in March during the long rainy season of 2021 and harvested in September. The number of fully developed Striga plants per plot was determined in June. Only those plants that had already formed seed capsules were taken into account. The results of the counting are shown in Table 2. The seed treatment led to a significant reduction in Striga infestation.

The grain yields per plot were determined in September. The yields shown in Table 3 were achieved.

Table 2: Influence of the treatment of corn seeds with a mushroom powder containing the cfu of the fungal strain Fusarium oxysporum DSM 33471 on the number of plants of the parasitic weed Striga hermonthica

Location Repeats | x Number of Striga plants per plot Untreated control | Seeds treated with DSM 33471 Bungoma 6 195.5 a* 44.8 b Kakamega 5 184.0 a 67.6 b Siaya 4 160.6 a 28.8 b Vihiga 2 220.8 a 107.8 a

*) Differences in control success between the untreated and treated plots of a location are statistically significant with a probability of error of 5% if the numbers of the plants identified are marked with different letters.

Table 3: Influence of a seed treatment with a mushroom powder containing the cfu of the fungal strain Fusarium oxysporum DSM 33471 on the maize yield when the test plots were heavily contaminated with Striga hermonthica

Location replicates x maize yield per plot in kg Untreated control | Seeds treated with DSM 33471 Bungoma 6 13.4 a* cn 19.1 b Kakamega 5 11.0a 18.2 b Siaya 4 11.3a 21.4 b Vihiga 2 11.6a 14.8 a

*) Differences in yield between the untreated and treated plots of a location are statistically significant with a probability of error of 5% if the respective yield values are marked by different letters.

By treating the corn seeds with the fungus Fusarium oxysporum DSM 33471, a significantly increased corn yield per plot could be achieved at all locations. The difference was statistically significant at 3 of 4 sites.

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Claims (8)

Patent claims 1. Treatment agent for seeds of monocotyledonous crops containing an active ingredient selected from a fungal strain from the Fusarium group and optionally a carrier substance and / or other auxiliary substances, characterized in that the active ingredient is the fungus Fusarium oxysporum f. sp strigae, DSM 33471. 2. Treatment agent for seeds of monocotyledonous crop plants according to claim 1, characterized in that in the agent at least 1 x 10 ° and at most 1 x 10'%, preferably 1 x 10 ° to 1 x 10% colony-forming units of the fungus Fusarium oxysporum f. sp. strigae, DSM 33471 per gram are included. 3. Treatment agent for seeds of monocotyledonous crop plants according to claim 2, characterized in that at least one group selected from conidia, chlamydospores or mycelial remains of Fusarium oxysporum f. sp strigae, DSM 33471 is contained as colony-forming units. 4. Treatment agent for seeds of monocotyledonous crops according to claim 1, 2 or 3, characterized in that as a carrier at least one solid powdery carrier selected from the group of zeolites, vermiculites, diatomaceous earths, kaolins, bentonites, peat, bran, preferably oat bran , wheat bran, flour from bamboo plants, wood flour, especially hardwood flour, preferably beech wood flour or eucalyptus flour. 5. Treatment agent for seeds of monocotyledonous crop plants according to claim 4, characterized in that the carrier has an average grain size of less than 500 μm, preferably less than 250 μm. 6. Treatment agent for seeds of monocotyledonous crop plants according to claim 4 or 5, characterized in that a wood flour, in particular beech wood flour, with a grain size distribution between 200 μm and 20 μm is used as the carrier. 7. Treatment agent for seeds of monocotyledonous crops according to one of claims 1 to 6, characterized in that the further auxiliaries are selected from: binders, dyes or coating agents. 8. Use of a treatment agent for seeds of monocotyledonous crops according to one of claims 1 to 7, characterized in that 0.1 - 1 kg / t of the treatment agent to the seeds of in particular Eleusine coracana, Eragrostis tef, Hordeum vulgare, Oryza satvia, Oryzy glaberrima , Panicum spp., Pennisetum glaucum, Saccarum officinarum, Sorgum bicolor, Triticum aestivum and Zea mays is bound or deposited in a shaking container. No drawings for this