HU206029B - Composition comprising trichoderma strains as active ingredient and process for producing the active ingredient - Google Patents

Abstract

Biological fungicides, and/or growth stimulants and/or crop quantity and quality improving compsns. contain as active ingredients novel strains of Trichoderma harzianum, Trichoderma harmatum and Trichoderma koningic. - Compsns. contain at least one of these strains to counteract pathogenic fungi; and/or to promote growth and improve quality, and in addn. they contain carrier for liq. a or solid phase fermentation and usual additives.

Description

FIELD OF THE INVENTION The present invention relates to a biological fungicide and / or plant growth stimulant and / or a quantity and / or qualitative product enhancer comprising at least one novel selected fungal strain of the genus Trichoderma.

The present invention further relates to novel selected strains of the Trichoderma genus having antagonistic properties.

The present invention further relates to a method for antagonizing plant pathogenic fungi using novel antagonist Trichoderma strains.

A further object of the present invention is to provide a method for stimulating plant growth using novel Trichoderma strains.

It is a further object of the present invention to provide a method for improving the quantity and / or quality of the crop using the new Trichoderma strains.

The present invention further relates to a method for cultivating novel Trichoderma strains in a known solid and liquid medium, and to a new method for cultivating known and novel Trichoderma strains in a solid medium.

The control of phytopathogenic fungi is currently based on the use of fungitoxic substances. Some of the fungitoxic compounds are contact, while others, which are of greater importance in agriculture, have systemic effects.

The use of systemic fungicides with specific activity often results in the development of a fungicide-resistant strain of the pathogen which can be delayed by the use of a mixture of active ingredients. However, in many cases this is limited by the fact that most contact fungicides are wettable powders and most systemic agents are available in emulsifiable concentrates and are not always physically compatible.

Another common problem is that not only one but at least five (6) pathogenic fungal species are attacked simultaneously at the same time (eg beet mortality, root ulcer). In the latter case, the active ingredient must be active against all major pathogens, ie it must have a broad spectrum of activity.

In agriculture, recent attempts have been made to replace chemical fungicides, which in many cases are ineffective in controlling soil-dwelling plant pathogenic fungi, with biological pesticides.

Fungal strains of Trichoderma species have been known as antagonistic fungi for decades. These fungi have been found to be capable of various plant pathogenic fungi under laboratory conditions. It is also known that most of these species produce one or more antibiotics that are fungistatic, fungicidal, or antifungal agents. have a bacteriostatic effect.

Among the Trichoderma species, we investigated T. viride, T. harzianum, T. hamatum and T. koningii species, and in the case of potato, carnation, strawberry cultivars Rhizoctonia solani, cucumber, chrysatemum, tomato, egg fruit Fusarium oxisporum and other species and, in the case of onions, against Sclerotium cepivorum, which causes white rot (Vájná, L .: Phytopathogenic fungi, Agricultural Publisher, Budapest, 1981).

New strain Trichoderma harzianum is disclosed in European Patent Publication No. 0 124388, while strain T. viride resistant to benomylla is described in U.S. Patent 4,489,161.

Use of Trichoderma harzianum against R. solani (Phytopathology 69, [1] 64-68, 1969) and use of T. harzianum against S. rolfsii (Plant Dis. Rep. 63, [10], 823-6, 1979), against Schlerotinia minor, T. viride (Indian J. Agric. Schi. 41 [1], 29-37 (1971)), sunflower Fusarium, Phytium, Sclerotium and others. and T. viride (Phytopathology 73 [3], 407-411, 1983) have also been described.

Based on the experience of domestic and foreign experiments, it can be concluded that Trichoderma species as antagonist organisms play an important role in the control of many plant pathogenic fungi. However, antagonistic activity (mycoparasitism, antibiotic production, nutrient competition) is not attributable to species, but is inherent in some strains within naturally occurring populations. As a result, active strains should be selected from natural populations. The efficacy of strains selected in laboratory conditions, as in vitro tests do not allow the replication of naturally occurring antagonistic effects, is ultimately validated in field or propulsion experiments.

Methods for mass propagation of selected Trichoderma strains for the advantageous antagonist effect have not yet been developed. So far, most of the tests have been carried out in laboratory with small quantities of fungi.

The aim of the present invention was to select new Trichoderma strains with better activity in laboratory and field experiments, which, when used in agriculture, have a much greater protective effect than the known Trichoderma strains and strains. conventionally used chemical fungicidal agents; when using combinations.

It is a further object of the present invention to select strains of Trichoderma which not only antagonize plant pathogenic fungi but also stimulate plant growth, thereby also favoring the quantity and / or quality and / or early maturity of the product.

A further aim was to select new Trichoderma strains that have a broader spectrum of activity than previously known Trichoderma strains, i.e., they can be used effectively against only one plant pathogenic fungal strain.

It is a further object of the present invention to provide a biological fungicidal composition suitable for biological control of plant pathogenic fungi.

A further object of the present invention was to provide a method for controlling soil-dwelling plant pathogenic fungi1

By virtue of the gonistic effect, an effective biological protective effect can be achieved and a higher yield and / or revenue can be achieved by a plant growth stimulating effect.

It is a further object of the present invention to provide a novel method for efficiently propagating new and known Trichoderma strains by solid phase fermentation.

In our experiments it was found that the strains Tv-5, Tk-3, T-14 and Tha-2 isolated from the domestic soil have higher activity than the previously known Trichoderma strains and their agricultural application yields higher yield than known hitherto known Trichoderma strains; their action is equivalent to or better than known chemical fungicidal compositions; effect of hair combinations.

The new Trichoderma strains provide biological protection not only against Rhizoctonia solani, Fusarium oxisporum, Sclerotmia sclerotiorum, which has been demonstrated previously for the known Trichoderma strains, but also provide protection against several other plant pathogenic fungi such as Ph. , Verticillium, Cytospora, Eutypa, Diaporthe.

To date, foreign studies have investigated the efficacy of each Trichoderma strain against one or two of the major pathogens of a cultivated plant (U.S. Patent No. 4,489,161), but found that the new strains we selected simultaneously contained several plant pathogenic fungi (Fusarium oxisporus). cinerea, Phoma betae, Rhizoctonia solani, Stereum purpureum, Phytophthora sp., Pythium debaryanum, Sclerotinia sclerotiorum, Diaporthe helianthi, Cytospora cincta, Eutypa lata, Endothia parasitica, etc.). In some cases, not only antagonistic, but also fimgistatic and even fungicidal effects were observed.

As used herein, "antagonist effect" is understood to mean an effect that inhibits the growth, reproduction, suppression, restriction of populations of pathogenic fungi, and sometimes the death of these fungi. The antagonistic effect may be manifested by inhibiting certain important life processes of the plant pathogen fungus (eg by inducing increased cell membrane permeability) by antibiotic production or by simply parasitizing the plant pathogenic fungus (mycoparasitism).

The novel Trichoderma strains of the present invention are derived from domestic arable ornamental soil and / or. Isolated from sclerotia of Schlerotinia sclerotiorum in 1982-1985. Isolation from soil was carried out in a manner known per se for soil fungi: an aqueous suspension of the soil sample was plated on a plate of PDA (potato dextrose agar) or Trichoderma selective medium of known composition. (Papaviz, Phytopathology, 72, 121-125 [1982]). The plates were incubated at 25 ° C and the resulting Trichoderma fungal colonies were inoculated. After preparation of the pure culture, monoconidium cultures were prepared in known manner (dilution series, plate casting) and strain determinations were performed. The assay was performed on the basis of Rifai's gene monograph (Rifai, M.A., Revision ot the Genuus Trichoderma, Mycol. Papers 116.56, 1969).

The new Trichoderma strain Tv-5, deposited under NCAIM F (P) 001049, has the following morphological characteristics:

The conidium supports of the Trichoderma viride strain show irregular pine-like branching. On the main branch, lateral branches are formed singly or in groups of two or three. Secondary branches may also form on some larger side branches. The length of the side branches increases downward from the tip of the conidium support.

Conidium-forming cells (phialides) are found in triplicate groups (vermin) or in opposing pairs or individually on the conidium-bearing branches. According to our investigations, the phialospores are nearly spherical or broadly ellipsoidal, 3.7 x 3.4 μτη in size, their surface is rough in the light microscopic examination and it appears spiny.

The Tv-5 isolate is prone to mass production of chlamydia sheath. Chlamydospores are terminal or intercalar, generally spherical, less frequently ellipsoids, 6-8 µm in size.

Fungal growth on potato dextrose agar medium at 27 ° C at pH -7 is 6-8 mm / day. The colonies are colorless during the first 3-4 days, translucent, with poor mycelial formation. Sporulation in Figures 5-7. day, in annular zones. Conidium reservoirs are densified. Two-week-old cultures are dark green in color from the conidium mass formed.

Trichoderma koningii strain Tk-3, deposited under NCAIM F (P) 001052, is derived from sclerotia of Sclerotinia sclerotiorum. Conidium supports are slender, woody, and the lateral branches gradually increase in length from the apex of the support. The conidium-forming fialides have an average size of 10.0 x 3.0 mm, tapering towards the apex and are mostly grouped in the ventricle, but there are unique and irregular fialides.

The conidia are ellipsoidal or short cylindrical, smooth-walled, pale green, measuring 3.5-5.0 x 2.03.0 mm. The Tk-3 strain also forms chlamydospores, which are colorless, predominantly intercalar, spherical, up to 15 µm in diameter.

Colonies of Tk-3 strain grow rapidly on PDA media at 20-22 ° C under alternating illumination. The radial growth of the colony is daily

9-10 mm. The colon is translucent, colorless, with no aerial mycelium or barely formed. Spore formation occurs in annular, distinct, corset zones. No culture discolouration was observed.

The Tk-3 strain can be well grown on solid and liquid media as well as solid organic media commonly used and known for culturing Trichoderma strains.

The strain Tk-3 exhibited outstanding mycoparasitic activity against 10 plant pathogenic fungal species and furthermore some fungi against some fungi3

HU 206 029 also produces antibiotics with a potent effect. Studies have also shown efficacy against wound parasitic fungi infecting certain woody plants.

Trichoderma harzianum strain T-14, deposited under number NCAIM F (P) 001 051, is derived from ornamental soil. The conidium supports of the Τ-14 strain are slender, with a regular pine-like structure, and the lateral branches are increasingly long from the apex of the support. Conidium-forming fialides are usually grouped in regular vultures, measuring (5.0-8.0) x (3.0-3.5) pm.

The conidia are pale green, round or ovate, smooth-walled, measuring 3-4 x 3 µm. The strain also forms chlamydospores, which are hyaline (colorless), spherical, predominantly intercalar, 3-12 µm in diameter.

Colonies of ΑΤ-14 strain grow 9-10 ntr at 20-22 ° C. Spore formation is stimulated by illumination, sporulation occurs in annular zones, which are dark green in color. Strain Τ-14 does not cause media discoloration.

The Τ-14 strain can be cultured well on solid media, liquid culture, and strains commonly used and known for Trichoderma species. on organic solid media. Strain Τ-14 has an extremely high antagonistic property and produces fungistatic antibiotics against some fungi. Application experiments have also shown significant plant growth stimulating effects.

The Trichoderma hamatum strain Tha-2, deposited under NCAIM F (P) 001050, is derived from sclerotia of Sclerotinia sclerotiorum. It is characteristic of this strain that the conidium supports are thick, bulky, and the lateral branches of the support are short. The tip of the conidia support ends with a sterile hyphae. Atypical conidia holders are also common. The spore-forming fialides concentrate in dense clusters on the branches of the support, and no regularity can be found in their locations. The conidia average 3.2x2.7pm, pale green, ovate, cut at base. The strain forms chlamydospores, which are 5 to 9 µm in diameter, spherical or elongated in barrel shape.

This strain can be cultured well on well-known fungal media (e.g., potato dextrose agar, malt agar, Czapek medium). Colony growth is relatively slow at 6-7 mtn / day at 20 ° C. Initially, the colony is clear, translucent and does not form mycelium. Spore formation occurs when exposed to annular zones, and conidium supports form dense clusters. The sporulating culture is off-green and does not discolour the medium.

The Tha-2 strain is well cultivated on liquid and various solid organic nutrient media.

The Tba-2 strain exhibits remarkably high antagonistic properties as a mycoparasite against a number of plant pathogenic fungi and produces a fungicidal antibiotic against some fungi.

Investigation of antagonist properties

Laboratory "in vitro" assays were performed to select the antagonist Trichoderma strains. These assays investigated the mycoparasitic property of the isolate in question. antibiotic-like effect. The assays were carried out by known methods (Dennis, C., Webster, J. Trans. Br. Mycol. Soc. 57, 25-39 and 363-369, 1971), with the exception that mycoparasitization was only taken into account. positive if microscopic lesions demonstrating parasitization were detected with a light microscope at 1200x magnification. In similar studies, it is usually considered sufficient to establish that the Trichoderma isolate under test has a colon overgrowth.

In vitro antagonistic activity of Trichoderma strains of the invention on certain plant pathogenic fungi

Mushroom tested Mikoparazitizmus Antibiotic effect TV-5 TK-3 T-14 Tha-2 TV 5 TK-3 T-14 Tha-2 Fusarium oxisporum f.sp. Diantha 4 - ++ +++ · 4-4 - ++ + ++ Fusarium oxisporum f.sp. melonis ++ fi ++ Fusarium graminearum + h Fusarium culmorum h ++ Verticillium dahliae H ++, Rhizoctonia solani +++ h 4th ++ +++ ++ +++ ⁺ +++ * +++ ⁺ Pythium debaryanum +++ h o 4-4- + Phytophthora sp. +++ h ++ ++ 4-4-4- ++ +++ ⁺ 4 + 4 + Sclerotinia sclerotiorum +++ h, and +++ 4-4- + 4-4- Botiytis cinerea h ++ +++ +++ ++ ++ +++ ⁺ X +++ Phoma betae +++ h, p. +++ ++ + X +++

HU 206 029 Β

Mushroom tested Mikoparazitizmus Antibiotic effect TV-5 TK-3 T-14 Tha-2 TV-5 TK-3 T-14 Tha-2 Helminthosporium sativum - ++ Diaporthe helianthi ++++ h +++ +++ Stereum woodpureum - - ++ X +++ Xytospora rubescens h ++ +++ Cytospora cincta - + Eutypa armeniacea +++ Endothia parasitica ++ Mycogone pemiciosa +++ Verticillium fungiciola +++

In the table, the notations are as follows:

Mikoparazitizmus:

- no signs of parasitism + signs of parasitism present, signs rarely ++ signs of parasitism common +++ signs of parasitism are common (for example, almost 100% of the spores formed are killed by parasitization) + h parasitization on hypha + o parasitization on oosphorus + s parasitization by sclerotia + p parasitization by picnidium

Antibiotic effect:

- no antibiotic effect + weak antibiotic effect ++ medium antibiotic effect +++ strong antibiotic effect +++ ⁺ fungistatic effect (100% growth inhibition) +++ x fungicidal effect

Examination of the Tv-5 strain showed that this fungus has a surprisingly broad range of fungi and, as a mycoparasitic strain, it is imperative to highlight it. In addition, some pathogens have fungistatic antibiotic activity.

It is surprising that the Tv-5 strain has a tendency to develop extreme chlamydospores. It massively parasitizes the young picnidiums and hyphae of Phoma betae and, by destroying them, forms masses of chlamydospores (dormant forms). The oospores of Pythium debaryanum are able to form massively parasitic chlamydospores inside them.

The isolate Tk-3 is distinguished by the fact that it has been surprisingly found that antibiotics and mycoparasites are simultaneously active against pathogenic fungi. In addition, several woody plants show strong antagonistic activity against parasitic fungi. Pathogenic fungi (eg Eutypa armeniacea, Diaporthe helianthi, Endothia parasitica), for which the antagonistic effect of the previously known Trichoderma strains is unknown, are present in the host fungus.

The isolate T-14 exhibits full growth inhibitory antibiotic activity against three major pathogenic fungi (Rhizoctonia solani,

Botrytis cinerea, Phytophthora sp.). In addition, several soil-borne fungi are parasitic to mycobacteria, and they are antagonistic to fungi of two cultivated mushrooms (Mycogone pemiciosa, Verticillium fungicola).

The Tha-2 strain is distinguished by its extremely potent antibiotic activity. It induces complete growth inhibition in most of the pathogenic fungi tested, and in three species it is fungicidal (Botrytis cinerea, Phoma betae, Stereum purpureum). The antibiotic effect on Tha-2 fungus is surprisingly coupled with the extremely pronounced mycoparasitic property that was found in most of the test fungi tested.

The novel Trichoderma strains of the present invention may be cultured by solid or liquid fermentation methods conventionally used to propagate Trichoderma species. (Papaviz, G.C., Review of Phytopath., 23,23-53,1985).

Liquid-phase culture results mainly in the production of mycelium, chlamydospores and less conidia.

The biological use of selected strains of Trichoderma species for plant protection purposes has long been based on the production of non-sexually produced reproductive cells (conidia). Conidia production, as is known, is more preferably solved by solid phase culture than by liquid phase culture. Most Trichoderma species also form chlamydospores. Chlamydospores are formed from certain cells of the fungal hyphae due to depletion of the substrate's nutrient sources or other biotic and abiotic factors that are unfavorable to the fungus, and are essentially used to ensure the survival of the species. Papavizas et al. (Pythopathology, 74, 1171-1175 (1984)) have found that the use of conidia for biological control is not necessarily the most advantageous solution, and that in some cases, the use of chlamydospores can provide better results.

According to the invention, the new Trichoderma strains are cultured in a liquid medium at a temperature of 8-34 ° C, preferably 20-28 ° C, preferably with aeration and agitation, preferably at a pH of 3.5-7, preferably 4, Adjust to 5-6.0.

HU 206 029 Β

Liquid-phase (submerged) culture can be done with standing or shaking cultures, in flasks or in a liquid culture medium.

The composition of the liquid medium corresponds to that commonly used for the cultivation of Trichoderma strains, i.e. one or more assimilable carbon sources and one or more suitable nitrogen sources and, if desired, one or more suitable inorganic salts and / or sulfur and / or phosphorus sources and / or contains trace elements.

Thus, the assimilable carbon sources include, for example, monosaccharides, disaccharides, complex polysaccharides, purines, pyrimidines, amino acids, condensed tannins and catechins, aldehydes and organic acids, especially long-chain fatty acids, methanol, methylamine, formates,

Examples of nitrogen sources include amino acids, urea, nitrates, nitrites, and materials containing these.

Preferably, the medium may also contain inorganic salts such as potassium, calcium, magnesium, sodium and bicarbonate salts as well as sources of sulfur and phosphorus.

As a source of carbon and nitrogen, the medium may be food or by-products, preferably glucose, maltose, sucrose, starch, soybean meal, other soy products, potatoes, potato flakes, hydrolyzed corn, corn steep liquor, whey, yeast, , sodium chloride, calcium chloride, potassium dihydrogen phosphate, ferrous (H) sulphate, magnesium sulphate, ammonium tartrate.

The concentration of the components in the medium may vary within wide limits and will always be selected according to the nature of the component and the nature and concentration of the other components present. When choosing this, we must always take into account the need to ensure optimal conditions for the cultivation of the given fungus, to promote and improve spore formation.

For example, the medium may contain from 5 to 500 g / l of assimilable carbon source, from 0.1 to 50 g / l of assimilable nitrogen source and, if desired, from 0.001 to 50 g / l of salt.

The cultivation is carried out under aerobic conditions, preferably with aeration and agitation. It may be advantageous to filter the air introduced into the medium. The mixing speed is selected to provide optimal conditions for the growth of the fungus, such as by shaking, for example, on a shaker at 100300 rpm, or by mixing, but also by culturing in a standing culture.

If desired, antifoam additives such as vegetable oils or conventionally used surfactants may be added to the medium. Preferred antifoam additives are Antifoam A from Sigma Chemical Co. (St. Louis, USA), but other antifoams commonly used for this purpose can be used.

The cultivation can be carried out at pH 3.5-7, preferably 4.5-6.0, 8-34 ° C, preferably 20-28 ° C, more preferably 23-26 ° C. 1-3 weeks. According to Lewis and Papavizas (Soil Bioi. Biochem., Vol. 15, No. 3, 351-357 (1983)), pH and agitation have little effect on spore formation.

The resulting spore suspension is separated from the liquid phase in a manner known per se and is preferably dried at 1540 ° C, preferably 30-40 ° C, more preferably 30-33 ° C and milled if desired.

The resulting spore mass can be stored at 3-5 ° C for a long time without deterioration in quality, preferably in a place protected from light and moisture.

The resulting spore mass (which contains chlamydospores, mycelium and conidia generally together or, if the fermentation conditions are selected, contains only chlamydospores) is used directly or with the addition of a suitable diluent and / or carriers and / or excipients. It is advantageous to use organic material as an excipient, which can be utilized for germination and germination even after the fungus has been released into the soil. if the soil nutrient content is inadequate.

The novel Trichoderma strains of the present invention may be propagated by solid phase fermentation by any method known in the art for culturing Trichoderma strains. The advantage of solid phase fermentation is that relatively inexpensive products or by-products can be used as the medium and the final product of the fermentation is the fungal mycelium-infused medium containing conidia of the Trichoderma strain. klamidospóráit. During cultivation, the fungus partially degrades and uses the solid medium.

When selecting media for solid-phase fermentation, three basic considerations are taken into account: it should be cheap, its composition should provide the optimum level of growth and spore formation of the fungus; available in sufficient quantities.

Various solutions have been proposed for the cultivation of Trichoderma strains on solid media.

Examples of nutrient media used are cereal seeds, e.g. barley, etc .; diatomaceous earth plus molasses; bark pellets; wheat bran; cereal seed meal plus sand; wheat bran plus sawdust, etc.

(Papaviz, G.C., Biological Control in Crop Production, Totowa, N.J. Allanheld, Osmum, pp. 305-322 [1981]).

In addition, experiments were conducted with quartz sand based solid media which, in addition to quartz sand, contain corn cobs, corn flour, urban sewage compost, coffee grounds, pine sawdust, coconut shredding material, hazelnut grits, sugarcane, 65) and also contained a liquid such as water, a mixture of molasses-corn syrup, glucose tartrate, sucrose nitrate. (Lewis, J. A., Papaviz, G. C. Soil Bioi. Biochem. Vol. 15, Vol.

HU 206 029 Β No. 351-357. (1983). From the point of view of chlamydospore and conidium formation, bran, maize flour and peanut meal proved to be the best. After inoculation with 10 ³ conidia / ml, the desired spore number (10 ⁸ spores / g) was achieved after culturing at 25 ° C for 3 weeks.

The novel Trichoderma strains of the invention may be cultured on solid phase media as known in the art.

In our experiments, it has been found that the new Trichoderma strains of the invention can be grown on solid medium according to known procedures at 8-34, preferably 2028 ° C, 90-100% relative humidity.

The solid medium may optionally be a suitably pretreated, high protein, high cellulose medium supplemented, if desired, with other excipients and / or additional nutrients.

For example, as crop media, crop products, food, agricultural or processing by-products, preferably cereal seeds, ground flour, molasses, wood bark or pellets, cereal bran, sawdust, vegetable shells, fodder plants and / or vegetables. It can be used.

The condition for the medium is generally that it has an appropriate C: N ratio, i.e. (25: 1) to (40: 1). If the C: N ratio in the medium is not the same, additional substances should be used. If the medium contains too few carbon sources for fermentation, then a suitable carbon source, such as sawdust, wood bark ground, sucrose, glucose (if desired in the form of an aqueous solution), molasses, corn steep broth, and the like. mixed. added. If more nitrogen is needed, a suitable nitrogen-containing material, such as urea, amines, ammonium salts, is added. If desired, magnesium, sodium, potassium, calcium, phosphorus and micronutrients may also be added.

Suitable excipients are inorganic inorganic, solid or liquid carriers and / or diluents, for example those which improve the consistency of the medium, make the medium more relaxed, facilitate its passage through the fungus to be cultivated, or otherwise favorably for cultivation. provide. As excipients, for example, water, inorganic natural minerals such as quartz sand, artificial minerals such as perlite aluminosilicates, inorganic organic materials such as polymers and the like. can be used.

As a nutrient, any substance that satisfies the requirement to provide the conditions necessary for the fungus to be cultivated for growth, reproduction, spore formation, and does not in any way interfere with its life processes is suitable. do not facilitate the establishment of other micro-organisms which antagonize and / or compete with the fungus to be cultivated.

Suitable pretreatment may be, for example, shredding, sterilization and / or adjusting the pH of the medium.

The medium is preferably comminuted to a particle size of 0.1-200 mm, preferably 0.1-20 mm, more preferably 0.5-5 mm, before use.

The medium is preferably sterilized by heat treatment prior to culturing. The heat treatment can be carried out in a known manner, for example in an autoclave, e.g. In 30 minutes. The purpose of sterilization is to kill other microorganisms in the medium and to ensure exclusive growth of the Trichoderma strain. The procedure may also be carried out without sterilization, but the growth of the Trichoderma strain will be slower or reduced. it must compete with nutrients for other microorganisms present.

Prior to inoculation, the pH of the medium is preferably adjusted to a slightly acidic pH of 4.5 to 6.5, preferably 5 to 5.5, since the slightly acidic pH favors the growth of Trichoderma strains. The pH adjustment can be carried out in a manner and materials known per se. Any material which satisfies the requirement that it does not in any way inhibit the growth processes of the Trichoderma strains may be used in pH adjustment. Thus, dilute mineral acids such as 0.1 N hydrochloric acid, sulfuric acid, nitric acid, and suitable organic acids can be used to adjust the pH.

Solid state fermentation is carried out in a manner known per se, for example by using a suitable apparatus, such as a tray fermenter or a vertical tank, as described in British Patent No. 2166157.

The fermentation provides a temperature of 8-34 ° C, preferably 20-28 ° C, more preferably 24-26 ° C and a relative humidity of 90100%. Illumination is used during fermentation if desired. Daily exposure of 10-30 minutes, equivalent to natural light, is usually sufficient for proper spore formation. Generally, an incubation period of 18-21 days is required to obtain the appropriate number of spores (10 ⁸ 10 ⁹ spores / g).

Temperature Significantly Affects Growth of Trichoderma Strains and Spore Formation Deviation from optimal temperature slows down fungal growth. Growth was observed at 6-7 ° C. It stops at 3537 ° C. At higher temperatures, the fungus dies, while at lower temperatures it returns to a state of rest, preserving its viability.

Fungal growth and spore formation require close to 100% or 100% relative humidity. Decreased humidity leads to stoppage of fungal growth and spore formation. However, the spores formed (conidia, chlamydospores) remain viable at 40-60% relative humidity.

The culture is preferably illuminated daily for at least 10 to 30 minutes, at least equivalent to natural light. For this purpose, it is appropriate

The light may be artificially illuminated with sufficient light intensity or the culture may be placed in a diffused light room.

However, the disadvantage of known solid-phase industrial scale fermentation methods is that although the high protein, high cellulose media used are inexpensive, they can be used for other purposes and can cost up to 4000-6000 HUF per ton. A further disadvantage is that additional nutrients (e.g., inorganic salts, supplemental C or N sources) are required for proper propagation of Trichoderma strains. A sufficiently large number of spores (10 ^{7 to} 10 ⁹ ) over a relatively long period of time, approx. Available in 3 weeks.

It has been an object of the present invention to provide a method for more efficient propagation of the new and known strains of Trichoderma of the invention in a much shorter period of time, which is also suitable for commercial production.

Our other goal was to recycle materials that were either low-priced or have been treated as waste and had problems with their disposal.

In our experiments, it has been found that Trichoderma strains can be more advantageously propagated by culturing at 8-34 ° C, preferably 20-28 ° C, at a relative humidity of 90100%, if desired, well pre-treated, low protein, high cellulose medium, if desired. other nutrients and / or excipients.

During breeding, any material (pH adjuster, medium, supplemental nutrients, excipients, etc.) that meets the requirement of providing the conditions for the growth, reproduction, spore formation of the fungus to be cultivated, does not in any way interfere with its life processes. , or other microorganisms which antagonize and / or compete with the fungus to be cultivated.

Suitable pretreatment may be, for example, shredding, sterilization and / or adjusting the pH of the medium.

The medium is preferably comminuted to a particle size of 0.1-200 mm, preferably 0.1-20 mm, more preferably 0.5-5 mm, before use.

The medium is preferably sterilized by heat treatment prior to culturing. The heat treatment can be carried out in a known manner, for example in an autoclave, e.g. In 30 minutes. The purpose of sterilization is to kill other microorganisms in the medium and to ensure exclusive growth of the Trichoderma strain. The procedure may also be carried out without sterilization, but the growth of the Trichoderma strain will be slower or reduced. it must compete with other microorganisms present for nutrients.

Prior to the inoculation, the pH of the medium is preferably adjusted to a slightly acidic pH of 4.5 to 6.5, preferably 5 to 5.5. While pH adjustment is not necessary, Trichoderma strains can be well cultivated at neutral pH, but mildly acidic pH favors the growth of Trichoderma strains.

The pH adjustment can be carried out in a manner and materials known per se. However, substances that inhibit the life processes or growth of Trichoderma strains in any way should not be used during pH adjustment. Suitable materials are acids which meet this criterion, for example, mineral acids such as hydrochloric acid, nitric acid, sulfuric acid, etc., diluted appropriately, e.g. organic acids.

The solid medium is, if desired, a suitably pretreated low protein, high cellulose medium supplemented, if desired, with other excipients and / or additional nutrients.

Low-protein, high-cellulosic materials ideally contain C and N sources of Trichoderma strains for proper growth and growth, as well as micronutrients and other nutrients.

When low protein, high cellulose medium is used, it is sufficient to incubate the culture for 4-6 days to achieve a sufficiently high (10 ⁸ spores / g) spore count.

Preferred media for the process are by-products of crop production and processing, or mixtures thereof, such as poppy seed and / or corn pellets.

The corn pellet is made from the chopped stalks of corn and the corn. is a product made by pressing straw.

Poppy stump is understood to mean shredded stalk and tuber waste obtained from Papaver somniferum after harvesting the poppy. The method is suitable for the poppy seed or the poppy seed obtained after the recovery of the alkaloids. also waste of poppy stems. The extraction of the poppy is not interesting from the point of view of the feasibility of the process, it is only interesting from the point of view of the economics of production.

The utilization of the poppy pox for other purposes is not solved, its cost at the place where the by-product is produced is practically zero, and even its placement causes problems.

If desired, other nutrients and / or excipients may be added to the medium.

Additional nutrients include, for example, suitable sources of carbon, such as crop products, food or other by-products of the processing industry, preferably cereal seeds, ground flour, molasses, bark or ground pellets, cereal bran, sawdust, vegetable husks, fodder plants, etc. and / or a suitable nitrogen-containing material such as urea, amines, ammonium salts, etc. can be used. The media used in the previous methods are also suitable as supplementary nutrients. If desired, sources of magnesium, sodium, potassium, calcium, phosphorus and trace elements may be added. Low protein protein nutrients are preferred as supplemental nutrients.

HU 206029 Β

Suitable excipients are inorganic, inorganic, solid or liquid carriers and / or diluents, for example, which improve the consistency of the medium, wetting, loosening the medium, facilitating the passage of the fungus to be cultivated, or other means of cultivation. provide positive results. A wetting agent, preferably water, is always used if the medium has an inadequate moisture content for cultivation. Preferably, the wetting agent is used in an amount such that the weight of the wetting medium is from 5 to 80% by weight, preferably from 30 to 70% by weight. As an excipient, any material satisfying the above condition, such as water, inorganic natural minerals, artificial minerals, inorganic organic materials such as polymers, and the like, may be used. can be used. Examples include diatomaceous earth, vermiculite, perlite, talc, kaolin, montmorillonite, foam stone, sepiolite, bentonite, calcite, sand, dolomite, natural and artificial silicates, such as aluminum silicates, and the like. water, vegetable oils or epoxidized vegetable oils such as epoxidized coconut oil or soybean oil.

Solid state fermentation is known per se, e.g. is performed using a suitable apparatus, such as a tray fermenter.

The fermentation provides a temperature of 8-34 ° C, preferably 20-28 ° C, more preferably 24-26 ° C and a relative humidity of 90100%. Illumination is used during fermentation if desired. Daily exposure of 10-30 minutes, equivalent to natural light, is usually sufficient for proper spore formation.

Temperature significantly influences growth and spore formation of Trichoderma strains. Deviation from the optimum temperature slows the growth of the fungus. Growth was observed at 6-7 ° C. It stops at 35-37 ° C. At higher temperatures, the fungus dies, while at lower temperatures it returns to a state of rest, preserving its viability.

Ideal growth of the fungus and spore formation require close to 100% or 100% relative humidity. Decrease in humidity leads to stoppage of fungal growth and spore formation. However, the spores formed (conidia, chlamydospora) retain their viability even at 40-60% relative humidity.

Preferably, the culture is illuminated daily for at least 1030 minutes, at least equivalent to natural light. For this purpose, artificial lighting of sufficient light intensity may be used, or the culture may be placed in a scattered light room.

At higher intensities or longer exposure, no spore formation occurs, but no adverse effects occur. If the illumination is less than previously described, the growth of the fungus remains unchanged, but the spore formation is weaker.

The biopreparation obtained as a final product of solid phase fermentation can be used directly, but optionally after the addition of diluents and / or other excipients, for plant protection purposes. In some cases, it may be advantageous to add only inorganic material (s) to the biopreparation to achieve proper dilution, since this will prevent stimulation of pathogenic fungi already present in the soil.

The biological fungicidal and / or plant growth promoting and / or quantitative and / or qualitative crop enhancing composition of the present invention contains at least one of the novel Trichoderma strains of the present invention in an amount suitable for antagonizing plant pathogenic fungi and / or stimulating crop growth. optionally together with one or more biocompatible, agriculturally acceptable solid or liquid carriers and / or excipients.

The biopreparation of chlamydospores, conidia, or chlamydospores and / or conidia and / or hyphae of the novel Trichoderma strains of the present invention is used alone or preferably with excipients commonly used in formulation practice.

When the active ingredient is prepared by liquid-phase fermentation, a dry spore mass is obtained at the end of the process. When the active ingredient is prepared by solid-phase fermentation, a biopreparation containing chlamydospores and / or conidia and / or hyphae is obtained. By shaking the final product of the solid phase fermentation with water, the 3-7 µm spores remain in the aqueous phase to give an aqueous suspension of spores. These may be used effectively on their own, but may be suitably diluted and / or supplemented with excipients prior to use.

A composition may contain one or more new or known Trichoderma strains and / or other known Trichoderma strains as active ingredient.

Alternatively, additional treatments may be applied before or after treatment with Trichoderma strains. Complementary treatment may be with other known fungal strains or chemical agents. It is a requirement for the chemical substances used that the Trichoderma fungus (and, if applicable, the fungus used in combination with it and / or as an adjunctive treatment) does not reduce its effect or adversely affect its life processes or life cycle.

When treating with other fungal strains used as adjunctive therapy to Trichoderma strains, it is essential that the other fungal strain (s) do not compete with, or antagonize or antagonize the Trichoderma strain. in no other way hinders its life processes.

Treatment with Trichoderma strains is also a complementary treatment to other crop protection treatments al9

EN 206 029 Β, which also requires the above considerations.

Formulations containing carriers and / or excipients may be formulated according to methods well known in the art and may include, for example, powders, granules, pellets, suspensions which may be sprayed or diluted prior to use, wettable powders and encapsulated products such as sodium alginate. Depending on the nature of the formulations, the method of application may be, for example, seed dressing, incorporation into soil, dusting, spraying, spraying or irrigation, which may be chosen depending upon the desired purpose and the particular circumstances.

Compositions of the novel Trichoderma strains of the present invention, optionally containing one or more solid or liquid carriers, may be prepared by known methods, for example, by homogeneous mixing of the active compounds and extenders, e.g., solvents, solid carriers and, if necessary, surfactants; grinding.

Organic and / or inorganic carriers may be used as solid carriers. Examples of inorganic carriers include diatomaceous earth, vermiculite, perlite, talc, kaolin, montmorillonite, foam stone, sepiolite, bentonite, calcite, sand, dolomite, aluminosilicates, artificial minerals, .

Examples of organic solid blowing agents include, if desired, pre-granulated plant ground, residues such as wheat bran, sunflower husk, ground corn pellets, poppy seed chips, pretreated compost, peat, sawdust. The organic carrier also serves as a nutrient for the antagonist fungus. However, care must be taken to ensure that the organic material used as the carrier for the antagonist fungus does not stimulate the pathogenic fungus and thus cause no exacerbation of the disease.

Examples of liquid carriers include water, mineral oils, vegetable oils, epoxidized vegetable oils such as epoxidized coconut oil or soybean oil, and the like. mention may be made of which water is most preferred.

Other excipients, such as emulsifiers, suspending agents, wetting agents such as anionic surfactants such as alkylsulfate salts, alkylarylsulfonates, dialkylsulfosuccinates, condensed products and the like, are optionally included in the composition of the invention. and nonionic surfactants such as polyoxyethylene alkyl ether, polyoxyethylene-polyoxypropylene block copolymer, polyoxyethylene sorbitan fatty acid esters, etc. and the like. other excipients such as adhesives, dispersants such as lignin sulfonates, alginates, xanthan gum, polyvinyl alcohol, gum arabic, molasses, casein, gelatin, carboxymethyl cellulose, agar, pine oil and the like. It contains.

It is a requirement for carriers and excipients that the fungus's life processes and reproduction should not be inhibited in any way, and should not contain such by-products.

The solid compositions generally contain from 10'-10 ¹¹ spores / g, preferably from 10 ⁸ to 10 ¹⁰ spores / g, more preferably from 10 ⁸ 10 ⁹ spores / g of conidia and / or chlamydospores of the Trichoderma fungus (s).

The liquid preparations generally preferably 1O -1O ^{9 5,} containing 10 ⁴ -10 °, more preferably ΙΟ ⁶ -10 ⁷ conidia / ml of spores.

Usually 1-10, preferably 2-8, more preferably 2-5 g of solid composition per m ² of soil are used. The liquid composition is generally applied in an amount of 200-1000 ml, preferably 300-800 ml, more preferably 400-600 ml per m ² of soil. The treatment may be repeated twice or more if desired. When the Trichoderma strain formulation is used for seed dressing, it is usually 2-10 g solid / kg of seed per kg of seed. 1050 ml of liquid preparation was used.

The dose applied will depend on the type of soil, the species of plant to be treated, the species composition and distribution of the pathogens, the composition and composition of the preparation, the Trichoderma strain used and the weather conditions which will be known to those skilled in the art.

The lower dose limit is also determined by these parameters and, therefore, general guidance cannot be given. One Trichoderma strain of the present invention may be effective in a given dose at a given dose and another Trichoderma strain of the present invention may not. Therefore, the dose should always be adjusted according to the circumstances.

The applied concentration and dose limit are mainly influenced by economic considerations, since the application of larger quantities of Trichoderma fungi usually does not cause adverse side effects, does not adversely affect the life cycle of the crop, nor does it cause environmental pollution when entering the waters.

The novel Trichoderma strains of the present invention are effective in protecting vegetables and fruits such as peas, lettuce, peppers, tomatoes, watermelons, strawberries, and the like. against pathogenic fungi which cause root diseases and wilt diseases, but can be effectively applied to woody plants and plants. for example, to prevent diseases of the mushroom.

Preferably, the Trichoderma fungal composition is used to prevent the disease before it develops.

The spores and strains of the new Trichoderma strains. the compositions containing them are preferably applied to the soil in solid form before or at the same time as sowing or planting.

After sowing or seeding, as a supplementary treatment, it is preferable to use a composition according to the invention in liquid form such as suspension, emulsion, etc. we can water the plants or. their root is a desire many times.

HU 206 029 Β

The novel Trichoderma strains of the present invention, respectively. the compositions containing them can be used for seed dressing, for example pea, sugar beet, paprika seed dressing.

The application of the new strains not only resulted in an increase in yield due to the antagonism of plant pathogenic fungi, but also in some cases a plant growth stimulating effect, as well as an improvement in the quality of the crop and accelerated plant development.

The invention is illustrated by the following examples, but the invention is not limited to the examples.

A) Preparation of new Trichoderma strains by a known liquid phase procedure All. Example 1 Aqueous media containing g / l glucose, 2 g / l potassium dihydrogen phosphate, 1 g / l magnesium sulfate, 1 g / l ammonium tartrate and 0.001 g / l ferric sulfate were prepared. 60-60 ml of the medium was weighed into 250 ml Erlenmeyer flasks and autoclaved at 80 ° C. Subsequently, a suspension of 2.5 ml conidia of 10 ³ conidia / ml of Tv-5 strain was added to each flask and the cultures were kept at 25 ° C for 3 weeks. The cultures are shaken on a shaker at 150 rpm. If the pH of the culture rises above 5.6 (close to 7), the pH is restored by the addition of 0.1 N hydrochloric acid.

The resulting product contains 5 x 10 ⁸ conidia and 2.6 x 10 ⁷ chlamydospores per milliliter.

At the end of the culture period, the aqueous spore suspension was filtered as a filter pad through a Buchner funnel containing muslin tissue and dried for 3 days at room temperature.

AI2-4. example

The procedure of Example 1 was repeated except that the medium was inoculated with a suspension of Tha-2, T-14 and Tk-3.

The results are shown in Table I.

A / 5th example

300 g of potatoes are cleaned and sliced and boiled in 1 liter of water. After boiling, decant the decoction, break the potatoes through a sieve and add to the filtrate. The media is then pH adjusted to 3.9-4.0 with 0.1 N sulfuric acid, and sterilized for 30-40 minutes at 10 ⁵ Pa overpressure.

The medium, cooled to 30-35 ° C, was then placed in a sterile glass vial at 5 to 6 mm thickness and inoculated with 10 ⁵ conidia / ml of spore suspension of T-14 strain using 10 ml of inoculum per 11 media. After inoculation, the vats were covered with a glass slide and incubated for 8 days at 30 ° C. At the end of the incubation, the plate formed from the fungal mycelium is removed and dried at 30-35 ° C in a ventilated room. The dried mushrooms are ground in a ball mill. 1.8 x 10 ⁷ spores / ml of biopreparation medium are obtained.

The procedure was repeated with the exception that a 1: 1 mixture of the above medium and molasses was used as the medium. so will 3,7xl0 ⁸ spore the resulting product 1 m! -Lunch.

The horse. example

The procedure of Example 5 was repeated, except that 15 g of potato flakes, 10 g of glucose and 1 liter of water were used as medium and cultured at 8 ° C under 12 hours of illumination for 21 days while maintaining the pH of the culture. was maintained at 6.0 and the medium was inoculated with a 10 ⁴ spore / ml suspension of the Tk-3 fungal strain.

Thus, the resulting culture containing 3.7 x 10 ⁶ spores per ml.

AI7. example

For the experiment, a 20 liter autoclavable polypropylene vessel was used, with a hole approximately 3 cm in diameter cut at the neck and sealed with a perforated rubber stopper. A glass tube, 5 cm from the bottom of the vessel, is introduced through it and air is bubbled into the medium. The air is obtained from a central power supply and filtered through 3 µm hair before being introduced into the medium. During the culture, the medium is continuously aerated and mixed. Antifoam A (Sigma Chemical Co., St. Louis, Mo. 63178) was used as an antifoam.

Aqueous media containing 30 g / l molasses and 5 g / l baking yeast are used as fermentation and starter media.

15 L of culture medium was added to each culture vessel and autoclaved at 100 ° C for 1 to 1 hour for two consecutive days.

The Tv-5 strain was first inoculated into a 500 mL starter medium in a 1 L flask and shaken on a rotary shaker for 5 days at room temperature. Inoculum concentration of ⁵ 2110 spores / ml suspension was used to inoculate a 15 1 culture medium in the culture vessel, and thus the medium in the culture vessel 171. The total volume of the medium was stirred at 25 ° C with aeration for 10 days. During the fermentation, the pH of the medium was maintained at 6.5 by addition of 0.1 N hydrochloric acid. The resulting biopreparation contained 4.7 x 10 ⁸ spores per ml.

The fungal biomass is separated by filtration by filtration through a 18 cm diameter Büchner funnel fitted with a linen muslin filter insert. The resulting material was dried for 3 days, weighed and ground in a Wiley mill, then sieved through a 425 µm (40 mesh) screen.

A / 8th example

The procedure of Example 1 was repeated except that 25 g / l glucose, 25 g / l sodium chloride, 5 g / l corn steep broth, 50 g / l molasses in aqueous medium at pH 5 were used. .

HU 206 029 Β

The medium is inoculated with a 10 ³ spore / ml suspension of Tk-3 strain at 1 ml / 60 ml medium. The culture was carried out at 25 ° C, pH 5 for 21 days.

The product obtained contains 3.5 x 10 ⁸ spores per ml.

, 4/9. example

The procedure of Example 8 was repeated except that the cultures were 20 g / l sucrose, 6 g / l sodium nitrate, 1.5 g / l potassium dihydrogen phosphate, 0.5 g / l magnesium sulfate and 25 g / l calcium chloride aqueous medium, pH

Adjust to 4.5 with 0.1 N phosphoric acid solution.

The medium was inoculated with a 10 ³ spore / g spore suspension of Tha-2 strain and after 21 days of cultivation at 26 ° C, 7.3 x 10 ⁷ spores / ml was obtained.

AJ Example 10

The procedure of Example 1 was repeated, except that Czapek's medium was adjusted to pH 7 and cultured at 22 ° C for 14 days. The product obtained contains 4.7 x 10 ⁷ spores per ml.

/. spreadsheet

Method for growing new Trichoderma strains on liquid medium

Example number broth Tapto. heaven. Tribe t ° C pH Light Tcny. time The amount of spore in the product obtained First THE 3 x 60 ml TV 5 25 ° C 5.6 12 o'clock 21 days 5 x 10 ⁸ conidia / ml 2.6 x 10 ⁷ chlamydospores / ml Second THE 3 x 60 ml Tha-2 25 ° C 5.6 12 o'clock 21 days 1.0 x 10 ⁸ conidia / ml 2.5 x 10 ⁷ chlamydospores / ml Third THE 3 x 60 ml T-14 25 ° C 5.6 12 o'clock 21 days 7.0 x 10 ⁸ conidia / ml 0.9 x 10 ⁷ chlamydospores / ml 4th THE 3 x 60 ml TK-3 25 ° C 5.6 12 o'clock 21 days 3 x 10 ⁸ conidia / ml 2x 10 ⁶ chlamydospores / ml 5th B B / L 3 x 1000 mL T-14 30 ° C 4 1 hour 8 days 1.8 x 10 ⁷ spores / ml 3.7 x 10 ⁷ spores / ml 6th C 3 x 1000 mL TK-3 8 ° C 6 12 o'clock 21 days 3.7 x 10 ⁶ spores / ml 7th D 3x 1000 ml TV 5 25 ° C 6.5 1 hour 10 days 4.7 x 10 ⁸ spores / ml 8th E 3 x 1000 mL TK-3 25 ° C 5 24 hours 21 days 3.5 x 10 ⁸ spores / ml 9th F 3 x 1000 mL Tha-2 26 ° C 4.5 20 hour 21 nsp 7.3 x 10 ⁷ spores / ml 10th G 3 x 1000 mL TV 5 22 ° C 7 0 hours 14 days 4.7 x 10 ⁷ spores / ml

B1. Fermentation of new Trichoderma strains by known solid phase 40

BH. example

400 g of wheat bran, 1 kg of quartz sand and 800 ml of water are mixed and the pH is adjusted to 5.0 by the addition of 0.1 N hydrochloric acid solution and placed in a sterilized plastic container 45. The medium is then inoculated with 10 ⁵ conidia / ml of spore suspension of T-14 strain in ml / 1 g of medium, the boxes are covered and the culture is incubated for 21 days at 50 ° C in a thermostated room at 90% relative humidity.

The product was dried at room temperature for 3 days. The product contains 5.6 x 10 ⁸ conidia and 0.9 x 10 ⁶ chlamydospores per gram.

BIB. example

The barley seeds were sterilized at 100 ° C for 2 hours and then cooled to pH 5.0 with 0.2 N phosphoric acid. Water is then added and filled into sterile breeding cans

The Tv-5 strain is inoculated with 10 ⁵ spores / ml of spore suspension and sealed with a transparent plastic film. The culture was carried out for 15 days at 26 ° C and 100% relative humidity with 24 hours illumination.

At the end of the culture time, 1.9 x 10 ⁸ spores / g are obtained.

Following the above procedures, B / l-13 was performed. example cultures.

The results are shown in Table II. Table.

The media used in the examples are as follows:

wheat bran (Examples 1-6):

quartz sand 1 kg wheat shoe 0.4 kg water 800 ml sawdust (example 7):

a 3: 1: 4 by volume mixture of wheat bran, sawdust and water (Example 8):

sterilized barley kernel moistened with 33% water by weight

HU 206 029 B

corn cob (example 9): NaNO ₃ 6 g / l silica sand 1 kg KH ₂ PO ₄ 13 g / l corncob 0.4 kg CaCl ₂ 25 g / l M-CSL 1.6 1 MgSO ₄ x 7H ₂ O · 0.5 g / l M-CSL has the following composition: 5 bark meal (Example 11): glucose 25 g / l oak bark meal 350 g NaCl 25 g / l water 650 ml corn soup 5 g / l beet (example 12) molasses 5 g / l beet slices 40g corn flour (Example 10): 10 urea 2g cornflour 40g water 100 ml SN 200 ml peanuts (Example 13) SN has the following composition: peanut shells, composted soil and perlite saccharose 20 g / l 3: 3: 1 by volume

II. spreadsheet

Cultivation of new Trichoderma strains on solid media

Example number Medium quality Media volume. Tribe sign t ° C PH Light (h) Breeding time (days) Inoculation with spores / ml Spore concentration in 1 g of product obtained First wheat bran 2.2 kg TV 5 26 5.0 0 21 10 ⁴ 4x to 10 x 10 ⁶ 9 ⁸ conidia klamidospóra Second wheat bran 2.2 kg T-14 26 5.0 0 21 10 ⁵ 5.6 x 10 ⁸ 0.9 x 10 ⁶ conidia klamidospróba Third wheat bran 1.1 kg Tha-2 26 5.0 0 21 10 ⁵ 2.0 x 10 ⁸ conidia 0.9 x 10 ⁵ chlamydospore OSPORA conidium 4th wheat bran 2.2 kg TK-3 26 5.0 0 21 10 ⁵ 3.2 x 10 ⁸ conidia for 5.0 x 10 ^s chlamydospore 5th wheat bran 1.1 kg TV 5 10 5.0 0 21 10 ⁵ 2.0 x 10 ⁴ conidia 1.2 x 10 ³ chlamydospore 6th wheat bran 1.1 kg TV 5 32 5.0 0 21 10 ⁵ 3.6 x 10 ⁴ conidium 1.7 x 10 ³ chlamydospore 7th sawdust 3 x 500g T-14 30 7.0 12 14 10 ⁵ 2.8 x 10 ⁷ spores 8th barley seed 3xl000g TV 5 26 4.0 24 15 10 ⁵ 1.9 x 10 ⁸ spores 9th corn cob 3xl000g TK-3 20 5.0 12 16 10 ⁵ 3.7 x 10 ⁷ spores 10th cornflour 3xl000g Tha-2 23 6.0 6 15 10 ⁵ 3.2 x 10 ⁷ spores 11th bark 3xl000g TV 5 25 5.5 6 14 10 ⁵ 8 x 10 ⁶ spores 12th sugar beet 3xl000g TK-3 22 7.0 2 20 10 ⁵ l, 2x10 ⁸ spores / g 13th hazelnut 2 x 250g TV 5 22 6.4 12 22 10 ⁴ 7.6 x 10 ⁶ spores / g

C. Fermentation of New Ttichoderma Strains by a New Solid Phase Procedure Example Ctl g air-dried poppy seed chips are placed in a 20 cm diameter Petri dish and sterilized by autoclaving at 130 ° C for 30 minutes. Check the pH of the medium and, if necessary, adjust the pH to 5.0-5.5 with 0.1 N hydrochloric acid. The sterile medium is then allowed to cool and, when cooled to 22-25 ° C, is inoculated with a 10 ⁴ conidia / ml spore suspension of Tv-5 isolate. For inoculation, we washed the solid conidium mass suspended in sterile water from solid Czapek medium (Z. Király, Z. Klement, Z. Solymosi, J: Reds Methods in Plant Pathology, Academy of Sciences, Budapest, 1970, p. 285). Sterile conditions are maintained during inoculation.

The inoculated medium was incubated at 25 ° C and 90% relative humidity for 5 days while being exposed to natural light for 12 hours daily.

After incubation, the medium containing the fungal spores was dried at 35 ° C to air-dry.

The composition thus obtained contains 9 x 10 ⁸ conidia per g.

HU 206 029 Β

The procedure was repeated using Tk-3 strain and Tha-2 strain. After 5 days of incubation, 10 ⁸ spores / g of poppy pox or Biopreparations containing 1.4 x 10 ⁸ spores / g of poppy toxin were obtained.

Example 02

1000 g of corn pellet were moistened with distilled water and sterilized by autoclaving at 100 ° C for 2 x 60 minutes. After cooling, pour into sterile 30 x 70 cm plastic sterilized 10 x 10 cm plastic boxes in a 20% Hypo solution and spread in a thin layer of 2-3 cm.

An aqueous suspension of Tv-5 isolate at 10 ⁴ conidia / ml is prepared in 10 cm Petri dishes by shredding cultures on malt Czapek agar.

Spray 50 ml of aqueous conidia suspension onto 1000 g of corn pellet in the crate and mix the inoculated medium evenly. The boxes inoculated in this manner were sealed with a thin, transparent plastic film and incubated at room temperature for 12 days at 25 [deg.] C. and 100% relative humidity for 12 days.

After incubation, the culture is dried at room temperature to give a product having a spore concentration of 10 ⁹ spores / g.

The procedure was repeated using T-14 strain. After 6 days of incubation, 5.7 X 10 ⁸ spores / g are obtained.

Example 03

The procedure of Example 1 was repeated except that during incubation no illumination was used, and culturing was performed in the dark at 22 ° C.

Thus, after inoculation with Tv-5 strain and incubation for 5 days, a biopreparation of 6.7 x 10 ⁸ spores / g is obtained.

Example 04 40

The procedure of Example 1 is repeated except that the poppy seed bag is not sterilized before use and moistened with 20 ml of a 20 g / l sucrose aqueous solution and 10 quartz sand is added. 45

Using the Tha-2 strain, after 5 days incubation, a biopreparation of 1.2 x 10 ⁸ spores / g is obtained.

Example 05

The procedure of Example 2 is repeated, except that the corn pellet is moistened with 1.5 L of the following medium:

1,000 g CaCl ₇ 1,000 g kno ₃ 0.250 g MgSO ₄ x 7H ₂ O 0.250 g H ₂ KPO ₄ 0.125 g hk _{2 after 4} 0,125g water To 1000 ml. Tk-3 strain 3.7 x 10 ⁸ spores / g

containing a bio-preparation.

Example 06

The procedure of Example 1 was repeated, except that the incubation was carried out at a temperature of 8 ° C or at a temperature of 8 ° C. b) It is carried out at 32 ° C and 10 g of artificial aluminosilicate is added to the poppy box.

The use of the Tv-5 strain gives a product of 6.8 x 10 ⁶ spores / g and 7.3 x 10 ⁸ spores / g of b).

Example 07

The procedure of Example 2 is repeated, except that the medium used is a) ground meal which is dampened with 4 l of medium 5 or b) A 3: 1 weight ratio of sunflower stalk and molasses is applied and the medium is moistened with 1.5 l of the medium of Example 5.

For the Tv-5 strain, after 3 days of incubation, 3.8 x 10 ⁸ , respectively. 1.2 x 10 ⁹ spores / g are obtained.

Example 08

The procedure of Example 2 was repeated, except that a 1: 1 mixture of corn pellets and poppy seed chips was used as the medium, to which 250 g of perlite and 250 g of wheat bran were added.

Using the Tv-5 strain, after 6 days of incubation, 1.3 x 10 ⁹ spores / g are obtained.

The results are shown in Table III. are summarized in Table.

Table Hl

A method for growing new Trichoderma strains on a new solid medium

Example number Medium quality Supply heaven Tribe t ° C PH Light (clock) Fact. time Product spore / g First máktokszecska 50g TV 5 TK-3 Tha-2 25 5.0 12 5 days 9x 10 ⁸ 10 ⁸ 1.4 x 10 ⁸ Second corn pellets 1000g TV 5 T-14 25 7.0 12 6 days 10 ⁹ 1.4 x 10 ⁸

HU 206029 Β

Example number Medium quality Tapto. heaven. Tribe t ° C PH Light (clock) Fact. time Product spore / g Third máktokszecska 50g TV 5 22 5.0 0 5 days 6.77 x ^{10 8} 4th poppy seeds + sucrose + quartz sand 60 g Tha-2 25 5.0 24 5 days 1.2 x ^{10 8} 5th corn pellet + nutrient solution 1000g TK-3 25 7.0 12th 6 days 3.7 x ^{10 8} 6 / a. 6 / b. poppy seeds + Al silicate 60 g TV 5 8 32 5.0 12 5 days 7.8 x 10 ⁶ 7.3 x 10 ⁸ 7 / a. shredded straw + nutrient solution 1000g TV 5 25 7.0 12 6 days 3.88 x ^{10 8} 7 / b. sunflower meal + molasses + nutrient solution 1000g TV 5 25 7.0 12 6 days 1.2 x ^{10 9} 8th corn pellets + poppy seed + perlite + wheat bran 1500g TV 5 25 7.0 12 6 days 1.3 x ^{10 9}

D / Propagation of Known Trichoderma Strains by Fermentation in New Solid Medium In the examples, the strain Trichoderma viride (lignorum) (T-1) described in US 736925 and the following deposition number strains were used:

Tk-1 (ATCC 64262) Trichoderma koningii

Th-14 (ATCC 64263) Trichoderma hamatum

Tv-12 (NRLL 5243) Trichoderma viride 30

Tv-14 (NRRL 5242) Trichoderma viride

Inoculation was always performed with an aqueous spore suspension of 10 ⁴ conidia / ml.

The procedures of the Example were performed in multiple replicates, and the results obtained are averaged. 35

DH. Example 50 x 50 g of air-dried poppy seed chips are placed in a 20 cm diameter Petri dish and the pH of the medium is adjusted to 5.0 with 0.1 N hydrochloric acid. The medium 40 is then inoculated with Tl isolate with 10 ⁴ conidia / ml spore suspension. Use 0.4 ml of inoculum per gram of medium.

Sterile conditions are maintained during inoculation. 45

The inoculated medium was incubated at 25 ° C and 90% relative humidity for 6 days while being exposed to natural light for 12 hours daily. After incubation, the medium containing the fungal spores was dried at 30 ° C to air-dry 50.

The composition thus obtained contains 1.2 x 10 ⁸ conidia per g.

D / second EXAMPLE 1 55 x 300 g of corn pellet were moistened with distilled water and sterilized in an autoclave for 2 x 60 minutes at 100 ° C. After cooling, the pH is adjusted to 5.0 with 0.1 N sulfuric acid solution, and then poured into a 30 x 70 cm plastic tray in sterile 20% Hypo solution and spread in a thin 2-3 cm layer. .

An aqueous suspension of Tl isolate at 10 ⁴ conidia / ml was prepared in 10 cm Petri dishes by dissolving the cultures on malt Czapek agar.

15 ml of aqueous conidia suspension are sprayed onto 300 g of corn pellet in a box and the inoculated medium is uniformly mixed. The boxes inoculated in this manner were sealed with a thin transparent plastic film and incubated at room temperature for 12 hours at room temperature under artificial light equivalent to natural light for 6 days at 25 ° C and 100% relative humidity.

After incubation, the facture was dried at room temperature to give a product containing 4.6 x 10 ⁸ conidia / g spore.

DI3-9. example

The procedure of Example 1 or 2 was followed except that different amounts of quality media, temperature, pH, illumination period and culture time were used.

The results are shown in Table IV. listed in the table.

In Example 4, a 1: 1 mixture by weight of poppy seeds and corn pellets was used, while in Example 7 a 3: 1 by weight mixture of sunflower seeds and molasses was used.

Example 6 uses shredded straw meal which is moistened with 500 g of the following medium per 500 g:

Ca (NO ₃ ) ₂ x 4 H ₂ O l, 000g CaCl ₂ l, 000g kno ₃ 0,250g MgSO ₄ x 7H ₂ O 0.250 g H ₂ KPO ₄ 0.125 g hk _{2 after 4} 0.125 g water To 1000 ml.

HU 206 029 Β

ARC. spreadsheet

Method for cultivating known strains of Tirchoderma on a new solid medium

- Example number Medium quality broth The amount i deer t'C pH light (clock ': being cool I would be conodium / g First máktokszecskc 5 x 50g T l 25 5.0 12 6 days 1.2 x 10 ^sec Second kukoricapellct 3 x 300g T l 25 5.0 12 6 days ^And 4.6 x 10 Third máktokszecska 3 x 100g T l 7.0 1/2 5 days ^And 0.7 x 10 4th poppy seeds + corn pellets 1: 1 t / h 3 x 100g T l 25 7.0 24 5 days 1.2 x 10 ⁸ 5th máktokszecska 3xl00g TV-12 8 5.0 18 14 days 1.1 x 10 ⁶ 6th shredded straw meal + nutrient solution (500 g / 21) 3xl00g TV-12 32 5.0 1 8 days 5.2 x 10 ⁸ 7th sunflower meal + molasses 3; 1 t / h 3 x 100g L-tk 25 5.0 12 7 days 6.7 x 10 ⁷ 8th máktokszecska 3x 100g Tk 4 20 6.0 1 6 days 5.3 x 10 ⁸ 9th corn pellets 3 x 500g TV-14 26 5.5 1 6 days 6.3 x 10 ⁸

The efficacy of the compositions of the present invention is illustrated by the following experimental examples.

The chemical fungicides used in the experimental examples contain the following active ingredients:

Orthocid: 50% Capt captan: N-trichloromethylthio-tetrahydrophthalimide Agráció Dithane 50% benomyl M-45: 80% mancozeb mancozeb: (manganese + zinc) ethylene bis-dithiocarbamate fundazol: 50% benomyl benomyl: 1-butylcarbamoylbenzimidazole- 2-methyl-carbamate Quinolate V-4-X: 50% carboxin + 15% copper oxoquinolate Tachigaren 50 WP: 50% himexazole himexazole: 3-hydroxy-5-methylisoxazole Chinofur 40 FW: 40% carbofuran carbofuran: 2,3-dihydro-2,2-dimethyl-7 - M-benzofuranyl-methyl-carbamate Sabet 72 EC: 72% cycloate Cycloate: S-ethyl-N-ethyl-N-cyclohexylthiocarbamate Pyramin FL: Chloridazone (1-phenyl-4-amino-5-chloropyridazone) Kolfugo 25 FW: 25% carbendazim carbendazim: 2- (methoxycarbonylamino) benzimidazole

Rowral

Flow: Ipridione [1-i-propylcarbamoyl-3- (3,5-dichlorophenyl] hydantoin]

Sumilex: 50% procymidone procymidone: N- (3,5-dichlorophenyl) -1,2-dimethylcyclopropanecarboxamide

Previcur: 70% Propamocarb Hydrochloride Propamocarb Hydrochloride: Propyl N- (3dimethylaminopropyl) carbamate Monohydrochloride

Ronil

WP: 50% vinclozalin vinclozalin: 3- (3,5-dichlorophenyl) -5-methylvinyl-1,3-oxazolidine-2,4-dione

Basamid

G: 98% diazomet diazomet: 3,5-dimethyl-4,6,2H-tetrahydrol, 3,5-thiadiazine-2-thione

In the experimental examples, chemical pesticides have always been shown to be the most effective against the given plant disease. we used the combination in comparison.

Further comparisons were made with the known Soviet T-1 Trichoderma viride (lignorum) strain disclosed in U.S. Patent No. 736,925. U.S. Pat.

Experimental Example 1

Pea seed dressing

Pea seed was seeded with a 10 ⁷ conidia / ml suspension of Tv-5 and Tk-3 strain. During the dressing we used 8.0 liters of comb juice per 1 t of seed. The juice and seed were rotated in a concrete mixer for 10 minutes.

The effect of the preparation is 1.0 kg / t Qrthocid

HU 206 029 B

WP '+ 1.0 kg / t was compared with Agrocit chemical fungicide formulation.

Seeds were sown on April 12, 21 days after sowing, and the height and green weight of the plants were measured in greenhouse experiments, while on July 23, seedlings per hectare and yield per hectare were measured. The greenhouse experiments were performed in 4 replicates and the field experiments in 2 replicates. The results listed in the table are average values.

Table 1

Greenhouse examination Field investigation Plant height cm Green mass g / 10 plants cardinal number db / m Product result t / ha 1. TV-5 13.0 9.6 8.34 2.54 2. Tk-3 12.8 9.4 8.74 2.26 3. Orthocit + agrocit 12.4 8.7 8.54 2.18 4. Untreated 14.3 10.3 7.82 2.08

It can be seen from the table that the new strains showed the same effect as chemical fungicides in greenhouse experiments, while in the field experiments the treatment with Tv-5 strain was about 15% and the Tk-3 strain about 15% compared to the chemical fungicide treatment. A 5% yield resulted in an increase. The treatment with the new strains did not cause a noticeable change in the development of the plants, their rate of development,

Experimental Example 2

Sugar beet seed dressing - greenhouse test

The result of biological control of germinal diseases in case of artificial infection

Sugar beet seed was seeded with Tk-3 strain and a suspension containing xanthan gum. The conidia concentration of the composition was 10 ⁶ conidia / ml, containing 0.5% xanthan gum, and 15 ml of the composition of the invention was used per kg of seed.

The effect of the preparation was obtained with a suspension containing the known Tl strain and xanthan gum in the above concentration, which was used in 15 ml per kg of seed and 2.0 g / kg seed + 2.0 g / kg Agrocit + Dithane M-45 chemical fungicide. compared with a composition which has proved to be most effective so far.

Sugar beet seed was sown in 30 x 30 x 18 cm plastic boxes in the garden soil used for seedling cultivation. Four days before sowing, on April 15, the soil was inoculated with the following pathogens:

- Phomabetae

- Fusarium oxisporum

- Fusarium culmorum

- Rhizoctonia solani • - Pythium debaryanum.

The seeds were sown on April 18, using 100-100 seeds per plant pot. The experiments were performed in four replicates. The number of healthy and diseased plants was determined twice per treatment and repeat. First, when the fungicide-treated plots of plants were hatched (April 25) and second, on the 25th day after the first count (May 20). The mean of the results is shown in Table 2.

Table 2

Treatment Hatched plants as% of infected control April 25 May 20 Infected control 100 100 Untreated control 90.55 89.11 Agrocit + Dithane M-45 108.58 110.48 xanthan 100.85 106.04 T-l + Xanthan 97.85 101.61 Tk-3 + Xanthan 117.59 112.50

Table 2 shows that treatment with Tk-3 strain produced extremely good results, while treatment with the known T-1 strain showed worse results than no treatment.

Experimental Example 3

Sugar beet dressing - field experiment

Monopoly-N-1 beet seed was seeded with 10 ⁶ conidia / ml in 20 ml / kg aqueous Trichoderma suspension. The effect of the preparation was compared with the combination of Fundazol 50 WP (2 kg / t) + Quinolate V-4-X (2.0 kg / t) + Tachigaren 70 WP (2.0 kg / t). Further comparisons were made with an aqueous suspension of the known T1 strain used at 10 ⁶ conidia / ml at 20 mg / kg seed.

The sowing was carried out on April 15 at 190000 germs / ha at 4 cm depth on 0.5 ha plots. Prior to application, soil disinfection was performed with Chinufur 40 FW (4.0 l / ha) and pre-sowing weed control with Sabet 72 EC (5.0 1 / ha) + Pyramin FL (6.0 1 / ha).

The evaluation was done in cotyledon (May 5) and then in two true leaf (May 19) conditions. The number of emerged plants and the proportion of diseased plants were determined on the basis of 10 x 5 m ² sample areas per treatment. At harvest (November 5) we measured the yield achieved. The results in the table show the average of 3 replicates.

HU 206 029 Β

Table 3

treatments 10 x 5 m ^{2 of} hatching plants Yield L'ha Untreated control in Tea Proportion of diseased plants in T V.5 V. 19 λ '. 5 Y. A T l 100.3 101.4 0 0.79 31.5 Tha-2 106.0 100.8 0 1.75 33.1 TK-3 109.6 114.0 0 0.70 33.7 TV 5 104.5 104.5 0 1.41 32.6 untreated control 100 100 0 2.27 31.1 Chemical fungicide 88.3 102.5 0 1.05 32.5

Among the new Trichoderma strains, treatment with Tk-3 was the best, significantly better than treatment with the known T-1 strain and chemical fungicide combination. The new strains tolerated chemical disinfection and weed control well.

Experimental Example 4

Paprika seed dressing - greenhouse test

Experiment 2 was performed under the same conditions except that pepper seed was used instead of sugar beet seed and control plants were infected with Altemaria alternata sand culture instead of Phoma betae.

Table 4

Treatment Hatched plants are contagious; % of control April 25 May 20 Infected control 100 100 Untreated control 75.28 96.76 Agrocit + Dithane M-45 98.50 108.99 xanthan 92.50 99.64 T-l + Xanthan 103.67 108.27 Tk-3 + Xanthan 113.85 116.54

No sick plants were observed after emergence. The germs have probably already died in the soil. Treatment with Tk-3 - Xantane proved to be most effective. On April 25, treatment with the known strain T-1 + Xantane showed a better result than the chemical combination, on May 20, essentially the same result, with ca. We achieved an 8% better result at both times when using the new strain.

I. Summary table

Effect of seed dressing (untreated control = 100%)

Treatment Pea field Sugar beet Pepper glasshouse glasshouse field toast crop hatched plant hatched plant crop hatched plant Untreated Cont. 100 100 100 100 100 100 Infected Cont. 112.2 103.3 T l 114.0 101.4 101.3 111.8 TV 5 106.6 122.1 104.5 104.8 Tha-2 100.8 106.4 TK-3 111.8 108.7 126.2 114.0 108.3 120.4 Orthocid + Agrocit 109.2 104.8 Agrocit + Dithane M-45 124.0 Fundazol + Quinolate + Ta- chigaren 102.5 104.5

Experimental Example 5

Watermelon Wilting Disease Control - Field Study To control the watermelon fusarium downy mildew, an aqueous suspension of the new Trichoderma 55 strains of the present invention was treated before seedling, at seedling, and after planting. the plants. Trichoderma strains were propagated on poppy seeds.

60 plots of 6 plots of 83 m ² each were used in the experiment, 49 seedlings were uniformly planted per plot on 19 May at 130 x 130 cm spacing. Spraying was performed with a back sprayer and a conical nozzle. Untreated plots were also sprayed each time with the same amount of water as the spray volume.

As a control, a suspension of Trichoderma strain, known as T-1, was used. Kolfugo 25 FW + Rowral Flow was used in combination at 0.2-0.2% concentration.

HU 206 029 Β

The time and method of treatment was as follows

Time of treatments way May 1, 19 before planting spraying with watering can 61 / nr spray or 10 ⁶ / ml aqueous conldium suspension May 2, 19 at planting a biopreparation (10 ⁸ spores / g) of 5 g maize pellets grown under a lawn in the planting pit June 3rd 4.3-4 true leaf state individual sprinkling of stems with 100 ml / sprayer or 10 ⁶ conidia / ml aqueous suspension June 4, 18 The beginning of Indian growth same as treatment 3 June 5, 29 flowering, fruiting same as treatment 3

Evaluation was performed four times, immediately prior to the treatments and 2 weeks after the last spraying.

Review time:

June 1, 4

June 2, 18

June 3, 29

July 4, 15

The rate of Fusarium dormancy was expressed in each case in relation to the number of plants grown and as a percentage of untreated plots. The mean of the results is listed in Table 5.

Table 5

Treatment Degree of fusarium drought Percentage of matured plants to be evaluated % Of untreated control is the date of evaluation Second Third 4th Second Third 4th Untreated control 4.15 27.5 73.7 100 100 100 TV 5 5.77 16.4 38.7 139.0 59.6 52.5 TK-3 3.47 18.9 45.2 83.6 68.7 61.3 Kolfugo 25FW + RowralFlow 4.80 8.9 22.0 115.7 32.4 29.8 T l 5.82 39.5 67.1 140.2 143.6 91.0

At the time of the first evaluation, the rate of drought erosion was the lowest in Tk-3-treated plots, with about 16% compared to untreated control. As the cycle progressed, the efficacy of fungicidal combination and Τν-5 treatment became increasingly pronounced. The strain known as T-1 has been shown to be virtually ineffective.

Experimental Example 6

Control of greedy mold and sclerotinous sprouted lettuce

Before the experiment, the number of colonies of Fusarium, Rhizoctonia, Penicillium and Trichodeima in the soil was determined, among other fungal species were Aspergillus, Botrytis, Mucor, Rhizopus. The number of bacterial colonies was not taken into account. Mycology was performed using the soil dilution method for Trichoderma species.

Fungal species. Dilution ratio 10 ' 10 ² 10 ³ 10 ⁴ 10 ⁵ 1234 1234 1234 1234 1234 Fusarium sp. 2442 1-1 .... .... .... Rhizoctonia sp. 1 -1 - --- 1 .... - .... Trichoderma sp. .... .... .... --- 1 .... Penicillium sp. -113 --- 1 - 1 - .... .... Other 3 3 10 13 3558 1212 -33- -1-3

The treatments were performed at the following times:

November 1, 25 T-14 20 g / m ² (7.18 x 10 ⁸ spores / g Tha-2 30 g / m ² (5.53 x 10 ⁷ spores / g) Tv-5 40 g / m ² (5.89 x 10 ⁶ spores / g) December 20, 2 T-14 1000 mL / m ² (1.6 x 10 ⁹ spores / mL) Tha-2 1000 mL / m ² (1.5 x 10 ⁹ spores / mL) Tv-5 1000 mL / m ² (3.6 x 10 ⁹ spores / ml) December 1, 7 Sumilex + Previcur N (0.1 + 0.3%) 10001 / ha December 2, 14 Rondan + Previcur N (0.1 + 0.3%) 10001 / ha December 3, 28 Sumilex + Previcur N (0.1 + 0.3%) 10001 / ha January 4th Rondan + Previcur N (0.1 + 0.3%) 10001 / ha January 5, 11 Sumilex + Previcur N (0.1 + 0.3%) 10001 / ha

Prior to treatment 1, the number of spores of Trichoderma strains grown on beet was determined in the laboratory and the biopreparation required per plot was measured beforehand. Before spreading, the barley biopreparation was mixed with 3-5 g of soil, homogenized in separate crates, then sprayed onto the soil surface and raked to a depth of 6-8 cm. The nutritious lettuce seedlings were planted within 1-4 days after treatment.

December 20 treatment was done by sprinkling an aqueous spore suspension of Trichoderma strains on the root of the plants.

As a control, the chemical fungicide combination which proved to be the best against lettuce was destroyed five times.

The first evaluation was made at week 3 after setting up, at which time no significant difference in plant phenology was observed between treatments.

HU 206 029 Β

Infections with Botrytis and Sclerotinia were observed in 1 plant.

The second evaluation was performed at week 4 after overheating. No significant infection occurred even on untreated plots. However, there was a significant difference in the maturity of the plants: 75-80% of the plants on Trichoderma strains were more developed than on untreated and chemically treated fungicide plots.

As a result of treatment with Trichoderma strains, almost all lettuce heads were sold on the experimental plots on 10 and 17 February, extra or Class I quality. Significantly later the lettuce heads became suitable for picking on the control plot and their quality was also lower. In terms of total numbers, the excess yield was 3.5-fold on plots treated with Trichoderma strains compared to plots treated with in-house combination.

Table 6

Crop production and sales

Treatment with Trichoderma strains per 100 m ² .

Chemical fungicide treated He treated me with Trichoderma Quantity turnover (EUR) Quantity turnover (EUR) Additional 102.50 1164.40 1331.67 15,314.21 I.O. 339.38 3149.0 996.67 9468.40

Chemical fungicide treated He treated me with Trichoderma Quantity turnover (Ef) Quantity turnover (EUR) ILO. 116.67 745.50 - - IIl.o. 90.63 245.60 - - Eladha- insoluble 183 - 5.0 - Altogether 649.18 5304.50 2328.34 24782.60

Experimental Example 7

Biological control of gray moldy and white moldy drought lettuce Trichoderma strains were propagated on poppy seed chips. The 10 ⁹ spores / g biopreparation thus obtained prior to transplanting the seedlings (11.2)

It was applied to the soil or to a 1: 1 mixture of soil and peat in a quantity of 10 g / m ^{2 for} 7-10 days.

One month after the basic treatment, the aqueous solution of Trichoderma strains was watered with 10 ⁹ spores / ml of the stem of the lettuce and 50 ml of the suspension was added to each lettuce.

After setting up the experiment, we examined the plant health of the lettuce every two weeks and determined the quality and market ability of the lettuce taken between 1.8 and 11.11.

71a. spreadsheet

treatments Botrytis and Schlerotinia infection (%) ' 11.16. 12.11. 12.29. 1.11. 1.25. 2.04. All(%) Experiment I l.T-14th - - - 6.25 1.25 - 7.50 2. Tha-2 - - - 6.25 2.5 - 8.75 3. untreated 1.25 1.25 3.75 8.75 18.75 25,10 58.85 II. experiment Peat 1 + T-14 - - - 7.94 0.79 - 8.73 Peat 2 + Tha-2 - - - 3.97 1.59 - 5.56 3. untreated (peat only) - - 2.5 28.75 8.75 - 40.0

* - Average of 3 repetitions

71b. spreadsheet

1:08 to 1:11. Quantity and quality of the lettuce harvested (The indications used in the treatment are the same as in Table 7a)

treatments Marketable pcs Additional I. o. P. Average weight g db % db % db % 1/1 16 12 75% 4 25% - - 16.9 1/2 16 11 69% 5 31% - ' 16.4 V3 11 3 27% 5 45%: 3 27% 13.6

HU 206 029 Β

treatments Marketable pcs Additional I. o. P. Average weight g db '% db % db % II / l 88 52 . 59% 36 41% - - 16.0 II / 2 47 6 13% 38 81% 3 6% 14.4 11/3 47 26 55% 15 32% 6 13% 15.6

The tables show that the T-14 strain was particularly good. Both the infection rate was significantly reduced (by 50% relative to the control), the crop became marketable earlier, its quality improved significantly (the number of extra heads increased approximately three-fold compared to the untreated control), and the average weight was significantly higher than the control. plants. When mixed with soil as a fungus-friendly organic material, we obtained almost the same results as the T-14 strain, while the effect of the Tha-2 strain was significantly reduced both in terms of early marketability and average weight of the crop. From this it can be filtered that the organic matter introduced into the soil should be selected according to the characteristics of the particular fungal strain. 25

Experimental Example 8

Biological control of forced lettuce gray mold and white mold tőpusztulása The experiment in greenhouse under plastic tent Ravell types of lettuce were performed 24 to 30 m ² plots three replications. In one part of the experimental area, the soil was disinfected with Basamid (66.7 g / m ² ) and in another part with formalin (66.7 cm ³ / m ² ).

On one half of the disinfected soils, T-14, Tv-5, Tha-35 2, and Th. a 10 ⁹ spore / g biopreparation containing the known T1 strain was also added to the soil in the planting zones at 10 g / m ² . As a control, contamination was examined on plots treated with Trichoderma biopreparation only, chemical disinfectant and untreated plots 40.

The biopreparation was mixed into the soil 4 weeks after the chemical disinfection of the soil and two weeks before the seedling was planted.

After planting, approx. A total of 45 four times biweekly treatments were performed on Trichoderma strains, irrigated with 10 ⁹ spore / ml aqueous suspensions of plants with 50 ml suspension per plant. Overheating was performed on November 19, December 2, 18, and January 2, 50. After setting up the experiment, we examined the incidence of white mold and gray mold disease every two to three weeks.

Trichoderma biopreparation only showed the lowest infection on the surface, with an average of 93% protection compared to untreated controls. The efficiency of Basamid G was 81.3% compared to factory control and 87.4% compared to formalin treatment. No significant difference was found between the individual Trichoderma strains, although T-1 and T-1 strains were found in the experiments. the T-14 strain proved to be most effective. The known T-1 strain generally produced less good results.

In the area treated with Trichoderma strains, the lettuce after planting is 57-60. days, while it was marketable in the field control and untreated control areas 10-14 days later.

The experiments also showed that Trichoderma strains had a relatively good tolerance to soil disinfection with strong chemical disinfectants, slightly worse after formalin treatment and better protection after treatment with Basamid G than when treated with Trichoderma strains alone. The latter is due to the fact that Basamid G kills soil microorganisms, but does not inhibit the life processes of Trichoderma strains that rapidly settle there.

Table 8

treatments Sclerotinia and Botrytis infection% 11.05 12.02 12.18 01.02 all infection.% I. Basamid G + l.T-14 - - * 0.3 - 0.3 2. TV-5 - 0.3 0.3 - 0.6 3. Tha-2 - - 0.3 - 0.3 4. T-l - 1.1 0.3 - 1.4 II. l.T-14 - 0.3 0.8 - 1.1 2. TV-5 - 1.1 1.1 - 2.2 3. Tha-2 - 0.3 0.6 - 0.9 4. T-l - 0.3 1.1 - 1.4 III. Formalin + l.T-14 - 0.6 0.3 0.3 1.2 2. TV-5 - 0.8 0.3 0.3 1.4 3. Tha-2 - 1.1 0.3 0.6 2.0 4. T-l - 1.1 0.3 - 1.4 ARC. factory inspection Basamid G - 0.8 1.4 4.2 6.4 formalin - 1.4 3.1 5.0 9.5 Sun unmanaged accounts! 1.4 4.7 5.3 5.8 17.2

HU 206 029 Β

Experimental Example 9

Biological control of peppercorns

The field inspections were carried out in a white teal pepper planted under a foil tent on plots of 750 rrr.

As a basic treatment, 10 ⁹ conidia / g hiopreparations were applied to the soil in the planting zone at a rate of 10 g / m ² one week prior to seedling planting (March 12). The supernatants were treated with 10 ⁹ conidia / ml aqueous suspension applied at 100 ml / m ² . During the vegetation period 6 over-treatments were carried out every 20-60 days (April 14, May 13, June 2, July 30, August 26, September 28).

On the factory control plot, Ronilan 50 WP (0.1 t%), Sumilex 50 WP (0.1 t%) 8 times every 10-20 days from April to the end of September. The plants were treated with 50 WP (0.2% w / w) fungicidal preparations.

After the soil treatment, the plant health status and the development of the peppers were evaluated every 2-3 weeks.

Sclerotinia infection was eliminated by Trichoderema strain T-14 with 66.7% and T-1 (known standard) with 52% efficiency. The protective effect of T-14 strain against Fusarium oxisporum was 43%, while the T-1 strain showed only 24% biological activity.

In the Τ-14 treated areas it was 41 days from planting to first harvest, 55 in the Tl strain areas and 60 in the field control area, ie 19 days earlier in the Τ-14 treated areas than in the field control. and 5 days earlier in T-treated areas.

As a result of Trichoderma treatments, the yield increased by 147% compared to the factory control, the T-14 strain had four times the extra quality than the control group, twice the extra quality.

9 / a. spreadsheet

Result of phytosanitary examination

Evaluation time Sclerotinia and Fusarium infection (%) T-14 T l factory inspection Fus. Scler. Fus. Scler. Fus. Scler. 4:28. 3.0 0 0 0 0 0 5/14/14 0 0 0 0 0 0 06/06/02 0 0.7 0 1.7 0 0 6/16/16 0 0 0 0 0 60 06/30 0 0 0 0 0 70 7/9/09 0 2.5 0 37 0 79 7:29. 0 13.0 0 315 10 80 8:26. 12.0 27.0 16 39 21 81

9 / b. spreadsheet

Test results until the end of the vegetation period

Pepper crop (kg) by quality category 750 no-no IJ ' T l factory inspection Additional 2008 1105 515 1st os / ki'yú 47.30 4500 2057 Grade 11 10263.5 11,100 6794.5 III. class A 194 199 1162 lower class 51 56 826 sunburn 12 24 2 industrial 41 84 785

Experimental Example 10

Biological control against gray moldy rot in strawberries

In the open field, 0.1 ha plots were experimented with Aikó strawberry T-14 strain Sumilex 50 WP chemical fungicide.

The flowering plants were sprayed at 800 l / ha with 10 ⁵ conidia / ml aqueous suspension at the second bloom and at the second bloom.

Spray was applied to Sumilex 50 WP at the same time on the control plot, applied in both applications at a rate of 1 kg / ha with Nonit (40% sodium dioctylsulfosuccinate) (500 L / ha).

Table 10

Treatment Infectivity expressed as a percentage of total fruit 06:15 06:18 06:22 06:25 06:29 together- sen T-14 4.0 7.89 4.5 6.5 6.7 6.1 Sumilex 50 WP 1.02 4.93 4.8 6.7 4.1 6.7 untreated 12.69 10.28 14.8 16.9 14.2 14.1

From the data in the table it can be seen that the efficacy of the treatments with T-14 strain reaches the level of chemical control and is significantly better than the untreated control.

Claims (45)

PATENT CLAIMS A biological or fungicidal and / or plant growth stimulating and / or qualitative and / or quantitative crop-improving composition, characterized in that the active ingredient is Trichoderma viride [NCAIM F (P) 001049], Trichoderma harzianum [NCAIM F (P) 001051]. , Contains at least one of the strains Trichoderma hamatum [NCAIM F (P) 001 050] and Trichoderma koningii [NCAIM F (P) 001052] in amounts of ΙΟ ^{6 to} 10 ¹¹ spores / g and ΙΟ ^{4 to} 10 ¹¹ spores / ml, respectively. compatible solid EN 206 029 Β and / or liquid carriers and / or excipients. 2. A composition according to claim 1 wherein the active ingredient is Trichoderma viride [NCAIM F (P) 001049]. Composition according to claim 1, characterized in that it contains Trichoderma harzianum [NCAIM F (P) 001051] as an active ingredient. Composition according to claim 1, characterized in that it contains Trichoderma hamatum [NCAIM F (P) 001050] as an active ingredient. 5. The composition of claim 1, wherein the active ingredient is Trichoderma koningii [NCAIM F (P) 001052]. 6. A solid preparation according to any one of the preceding claims, characterized in that the active substance contains 10 ⁸ 10 ^1θ, preferably ÍOMO ⁹ spores / g. 7. Liquid composition according to any one of claims 1 to 10, characterized in that the active ingredient is selected from the group consisting of 105-10 ⁹ . preferably ΙΟ ^{6 to} 10 ⁷ spores / ml. 8. Composition according to any one of claims 1 to 3, characterized in that it comprises an organic and / or inorganic carrier as a solid carrier. Composition according to claim 8, characterized in that the inorganic solid carrier used in the cultivation of the fungus is diatomaceous earth, vermiculite, aluminum silicates, perlite, talc, kaolin, montmorillonite, foam stone, sepiolite, bentonite, calcite, calcite. , minerals and / or soil. Composition according to Claim 8, characterized in that the organic solid carrier is the medium used for the cultivation of the fungus, the residue formed after the cultivation thereof and / or, if desired, pre-granulated plant by-products, residues or ground meal, preferably wheat bran, sunflower meal, contains ground pellets of corn, barley, poppy seed, pre-treated compost, sawdust and / or peat. 11. Composition according to any one of claims 1 to 3, characterized in that the liquid carrier is an organic and / or inorganic carrier. 12. A composition according to claim 11, wherein the inorganic carrier is water, the organic carrier is vegetable oils, mineral oils, epoxidized vegetable oils. A process for the preparation of a composition according to claim 1, wherein the Trichoderma viride [NCAIM F (P) 001049], Trichoderma harzianum [NCAIM F (P) 001051], Trichoderma hamatum [NCAIM F (P) 001050] and / or At least one of the strains of Trichoderma koningii [NCAIM F (P) 001052] suitable for their growth (a) on solid medium; or b) culturing in liquid medium and mixing the resulting culture with a biocompatible solid and / or liquid carrier and / or excipient to form a formulation at a concentration of ΙΟ ^{6 to} 10 ¹¹ spores / g and ΙΟ ^{4 to} 10 ¹¹ spores / ml. Process according to claim 13, variant a), characterized in that the culture is carried out at a temperature of 8-34 ° C, preferably 20-28 ° C. 15. The method of claim 13, variant a), wherein the culture is carried out at a relative humidity of 90-100%. Method according to claim 13, variant a), characterized in that the cultivation is carried out on pre-treated high protein, high cellulose medium supplemented with excipients and / or additional nutrients. 17. A16. The process of claim 1, wherein the medium is high protein, high cellulosic plant products, food, agricultural and / or other by-products of the processing industry. 18. The method of claim 16, wherein the nutrient medium is cereal grains, ground flour, molasses, bark or pellets, cereal bran, sawdust, vegetable hulls, fodder plants and / or vegetables or mixtures thereof. 19. A16. The process of claim 1, wherein the medium is wheat bran, corn flour, barley kernels, corn cobs, peanut flour, beet slices and / or sawdust. 20. A16-19. Process according to any one of claims 1 to 4, characterized in that the additional nutrient is a suitable source of carbon, preferably sawdust, bark powder, sucrose, glucose, molasses, corn steep liquor and / or a suitable nitrogen source, preferably nitrogenous material such as urea, ammonium or sources of potassium, calcium, phosphorus and / or trace elements are used. 21. A16-20. Process according to any one of claims 1 to 3, characterized in that water, minerals and / or inorganic organic materials, preferably water, perlite and / or quartz sand, are used as excipients. 22. A16-21. Process according to any one of claims 1 to 3, characterized in that the culture is carried out on sterilized and / or shredded medium, the pH of which is optionally slightly acidified. 23. The method of claim 22, wherein the sterilization is carried out at 75-150 ° C by heat treatment. 24. The method of claim 22, wherein the medium is comminuted to a particle size of 0.1-200 mm. 25. The method according to claim 22, wherein the pH of the medium is adjusted to 4.5-7.0, preferably 5.5-6.5. 26. A16-25. The method according to any one of claims 1 to 3, characterized in that the culturing is carried out at least 1030 minutes of light equivalent to natural light per day. 27. The method of claim 13, variant a), wherein the culturing is pretreated, low EN 206 029 Β protein is performed on high cellulose medium supplemented with excipients and / or additional nutrients. The method of claim 27, wherein low protein, high cellulosic crop byproducts or mixtures thereof. 29. A method according to claim 28, characterized in that. the medium being poppy seed and corn pellets. 30. A 26-29. The process according to any one of claims 1 to 3, characterized in that the culture is carried out on sterilized and / or shredded medium, the pH of which is optionally slightly acidified. 31. The method of claim 30, wherein the sterilization is carried out at 70-150 ° C by heat treatment. 32. The method of claim 30, wherein the medium is comminuted to a particle size of 0.1-200 mm. 33. The method of claim 30, wherein the medium has a pH of 4.5 to 6.5. preferably 5-5.5. 34. A 27-33. The method according to any one of claims 1 to 3, characterized in that the cultivation is carried out in daylight exposure equivalent to at least 1030 minutes per day. 35. A 27-34. The method according to any one of claims 1 to 3, characterized in that the culture is carried out for at least 4 days, preferably for 5 to 7 days. 36. A 27-35. Process according to any one of claims 1 to 5, characterized in that suitable carbon sources, preferably vegetable crops, food or other by-products of the food industry, preferably cereal seeds, flour, molasses, bark meal or pellets, cereal bran, sawdust, with suitable nitrogen sources, preferably urea, amines, ammonium salts and / or magnesium, sodium, potassium, calcium, phosphorus and / or trace elements. 37. A 27-36. Process according to any one of claims 1 to 4, characterized in that water, one or more mineral and / or inorganic organic substances, preferably a polymer, is used as an excipient. 38. The method of claim 13, variant b), wherein the culture is carried out on a liquid medium at 8-34 ° C, preferably 20-28 ° C, with aeration and agitation, and the pH of the medium. 3.5-7.0, preferably 4.5-6.0. The process according to claim 38, wherein the medium is one or more assimilable carbon sources and one or more suitable nitrogen sources and, if desired, one or more appropriate inorganic salts and / or sulfur and / or phosphorus sources and / or trace elements. containing liquid medium. 40. The process according to claim 38, wherein the assimilable carbon source is monosaccharides, disaccharides, complex polysaccharides, purines, pyrimidines, amino acids, condensed tannins and / or catechins, aldehydes and / or organic acids, preferably long chain fatty acids, amines, formates and / or materials containing them. 41. The method of claim 38, wherein the nitrogen source is one or more amino acids, urea, nitrate, nitrate, a substance containing them, or a mixture thereof. 42. A process according to claim 38 wherein the salt is water-soluble salts containing potassium, calcium, magnesium, sodium cations and nitrates, dihydrogen phosphate, hydrogen phosphate, sulfate and / or chloride anions. . 43. A process according to claim 38, wherein the carbon and nitrogen sources are food products or by-products, preferably glucose, maltose, sucrose, starch, soy flour, other soy products, potato, potato flakes, hydrolyzed corn, corn broth, lactic acid, and / or molasses. 44. A method for growing new strains of Trichoderma viride [NCAIM F (P) 001051], Trichoderma harzianum [NCAIM F (P) 001051] and Trichoderma hamatum [NCAIM F (P) 001050] and Trichoderma koningii [NCAIM F (P) 001052]. solid medium at 834 ° C and 90-100% RH, characterized by low protein and high cellulosic crop byproduct as a solid medium, optionally pretreated and supplemented with excipients and / or additional nutrients ( ke). 45. The method of claim 44, wherein the medium is sterilized poppy seed chips, corn pellets, sunflower stalk, and molasses or straw meal sterilized at 70-100 ° C.