WO1997016974A1 - Liquid formulation based on strains of the stringy fungi trichoderma harzianum and trichoderma viride - Google Patents

Liquid formulation based on strains of the stringy fungi trichoderma harzianum and trichoderma viride Download PDF

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Publication number
WO1997016974A1
WO1997016974A1 PCT/ES1996/000206 ES9600206W WO9716974A1 WO 1997016974 A1 WO1997016974 A1 WO 1997016974A1 ES 9600206 W ES9600206 W ES 9600206W WO 9716974 A1 WO9716974 A1 WO 9716974A1
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Prior art keywords
trichoderma
harzianum
imi
trichoderma harzianum
grows
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PCT/ES1996/000206
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Spanish (es)
French (fr)
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Enrique Monte Vazquez
Isabel Grondona España
Isabel Garcia Ancha
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Universidad De Salamanca
Consejo Superior De Investigaciones Cientificas
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
    • A01N63/38—Trichoderma
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/885—Trichoderma

Definitions

  • This invention relates to a formulation with Trichoderma spp. for its application as a biological control agent for the enemies of plants and plant material, biological entities causing biodeterioration and as a bioleaching agent.
  • the present invention relates to a liquid formulation, with organic and inorganic nutrients in solution, which contains in suspension vegetative and reproductive structures of a mixture of strains of the filamentous fungi Trichoderma harzianum Rifai and Trichoderma viride Pers.:Fr.
  • the formulation according to the invention is useful as a biological control agent, against diseases of plants and plant and biodeterioration material produced or transmitted by filamentous fungi and other enemies of plants, and as a bioleaching agent.
  • the chemical and biological components of the formulation are concentrated so that, during transport and storage, germination of Trichoderma spores does not occur. Once the formulation is diluted, immediately before its application to the soil, crops, etc., its chemical components can be assimilated by the spores of the fungus that will begin germination in a few minutes. In several hours the microorganism is multiplied.
  • Control diseases of plants and plant material taking advantage of the antagonistic capacity of four strains of T. harzianum and one of T.viride on other soil fungi.
  • Biological Control can be defined as the use of natural or modified organisms, genes or gene products, to reduce the effects of undesirable organisms, and to favor organisms useful to man, such as crops, trees, animals and beneficial microorganisms. More than half of the filamentous fungi that are used in the Biological Control of other plant pathogenic fungi. they belong to the genera Trichoderma and Gliocladium.
  • Trichoderma is a fungus widely distributed in nature, easy to isolate and cultivate, which grows rapidly on many substrates, does not affect man, animals and higher plants, acts as mycoparasite (parasite of other fungi), competes well for food and through space, it produces antibiotics and has an enzymatic system capable of attacking a wide range of pathogens.
  • the fungi of the genus Trichoderma, and in particular the species Trichoderma harzianum constitute a cleaner biological alternative, not cumulative in the food chain and respectful with the environment, as opposed to the pollutant chemical pesticides commonly used in Agriculture.
  • Trichoderma lignorum T. viride
  • Trichoderma lignorum was a parasite that curled around the hyphae of another fungus, Rhizoctonia solani, producing its death.
  • species such as Phytophthora, Pythium and Rhizopus as well as Sclerotium rolfsii could be parasitized by T. viride Based on these results, the possibility of using abundant inoculations with T. viride to control certain pathogens arose.
  • Trichoderma Although Trichoderma was recognized, in principle, as a fungal antagonist, it has also been described as an antagonist of certain pathogenic bacteria. Trichoderma has been studied extensively for its arvtibiotic properties of possible clinical use, for its enzymatic systems in food processes and for its biodegradation power of solid waste and wastewater. Other studies have established mycoparasi Trichoderma tism on many Oomycetes, Zygomycetes, Ascomycetes, Basidiomycetes and anamorphic fungi.
  • Trichoderma's competitive capacity and its success as a Biological Control agent, are affected by soil properties.
  • the pH had an influence on the relative value of Trichoderma as an antagonist of R. solani, affecting the growth and development of each fungus.
  • T. hamatum and T. pseudo-koningii are adapted to conditions of high soil moisture and that T. viride and T. polysporum are restricted to areas where low temperatures prevail, while T Harzianum is more frequent in temperate regions, and T. hamatum and T. koningii are widely distributed in areas of diverse climatic conditions.
  • the antagonism of the soil against Trichoderma is the most important factor in the regulation of the level of the possible inoculum of this fungus when applied in a natural environment.
  • aerial parts of the plant in leaves, in wounds of trees or at the time of pruning.
  • These formulations are usually liquid.
  • the treatment of soil with steam, solarization and other methods, can modify the ecosystem to the point of favoring the proliferation of Trichoderma.
  • the sub-warming of soils infected with A. mellea for example, stimulated the most heat-resistant resident, Trichoderma, who then proliferated and parasitized a weakened A. mellea.
  • Trichoderma sp. formulated in motor oil it has been used successfully to inoculate pines during the pruning period.
  • Trichoderma spores examples include the addition of Trichoderma spores to the soil along with a liquid fertilizer, in case the insulations used can tolerate a high osmotic pressure.
  • Culture media for liquid fermentation should include accessible and cheap agricultural products, such as glucose, starch, hydrolyzed corn and soy products, whey and molasses.
  • viable Trichoderma inoculums have been produced, in a fermentation tank, to obtain, after a period of incubation, a large amount of biomass composed of mycelium, clamidospores and some conidia.
  • Another formulation has also been prepared, air drying the biomass, grinding it and diluting the powder with Pyrax®, which is commercially available as a carrier for fungus inoculum.
  • Pyrax® which is commercially available as a carrier for fungus inoculum.
  • Other commercial supports that have been used frequently in Biological Control are Agro-Lig® and Pelgel®.
  • An improvement in application systems is the encapsulation of propagules, based on the ability of aqueous sodium alginate solutions to react with cations of certain metals (Ca 2+) and form gels.
  • a subsequent modification to this technique has been the incorporation of food transporters (eg bran) to alginate, in intimate contact with the antagonist, to provide the necessary nutrients.
  • Trichoderma's young mycelium on a partially consumed substrate has also been used, considering that the mycelium is much more resistant to soil fungistasis than conidia when germinating. Therefore, it may seem more effective to apply Trichoderma with a partially consumable food base, than to supplement Trichoderma with additional nutrients, at the time of its application.
  • Alginate granules have variability in quality and behavior in the soil.
  • Trichoderma is applied to the monolayer-shaped seeds around them, on a solid matrix, which can be Agro-Lig® or bituminous coal.
  • Trichoderma strain selection studies have been carried out for application as Biological Control agents in the control of diseases caused by soil fungi in vegetables, mainly beets.
  • a liquid formulation is now provided, with organic and inorganic nutrients in solution, which contains in suspension vegetative and reproductive structures of one, two, three or four strains of the filamentous fungus Trichoderma harzianum Rifai and / or Trichoderma viride Pers.:Fr. previously identified.
  • the formulation according to the invention is useful, as already indicated, as a biological control agent and as a bioleaching agent.
  • the present invention provides a liquid formulation whose quantitative composition (%, w / v) is as follows:
  • Trichoderma harzianum CECT 20179 csp 3 ⁇ 10 5 - 10 ⁇ 10 7 conidia / ml
  • the formulation according to the invention has chemical components that can be used as nutrients by T. harzianum and T.viride, while maintaining sufficient humidity for the germination of propagules of these fungi.
  • the liquid formulation of the invention allows antagonists to be used at any time and situation, without the need to limit the application at the time of sowing, as happens when Biological Control agents are formulated in solid media and substrates or by coating seeds and other materials of propagation.
  • the liquid formulation can be kept, concentrated, under refrigeration conditions, at 4 ° C, for at least 1 year and with 1 liter of it can be treated up to 30 Ha cultivated.
  • the formulation can be applied directly to soil and plant and / or animal and / or fungal material, living or dead; or mixed with substances used in Agriculture to vehicular other phytosanitary products such as fertilizers, fertilizers, microgranules, compost or those applied in the pilling, coating and encapsulation of seeds and embryos, etc.
  • dilution of the product favors the dispersion of T. harzianum and T.viride.
  • the formulation should be concentrated 30 and 60 times, as applied, respectively, on the plant or directly to the soil.
  • the invention can be developed on an industrial scale since the different fermentation systems available allow to obtain inoculum yields of T. harzianum and T.viride of more than 10 grams of biomass per 1 liter of fermentable substrate.
  • Trlc-boderma harzlamm IMI 352939 Trlc-boderma harzlamm IMI 352939:
  • conidiogenesis is phialidic enteroblastic, with conidiogenic cells with a length less than 10 mm; the conidia are subglobosous, with a diameter greater than 2.4 mm; and produces intercalar and terminal clamidospores with a diameter greater than 10 mm. According to the methodology used:
  • the colonies do not reach a diameter greater than 20mm in the glycerol-nitrate agar medium, after 10 days of incubation at 25 ° C.
  • alkaline phosphatase alkaline phosphatase, arylaminidase leucine, acid phosphatase, naphthol-AS-BI-phosphate, alpha-galactosidase, beta -galactosidase, alpha-glucosidase, beta-glucosidase, and N-acetyl-beta-glucosaminidase with maximum intensity; esterase, esterase lipase, lipase, arylaminidase valine, cystine arylamidase, trypsin, alpha-chymotrypsin, beta-glucuronidase and alpha-mannosidase, with moderate intensity; and alpha-fucosidase is not detected.
  • API ZYM system BiomérieuxS.A., Maroy-l'Etoile, France
  • esterase activity (Rf 0.82-0.84) variable
  • esterase activity (Rf 0.88) variable
  • Trichoderma harzianum IMI 352940 Trichoderma harzianum IMI 352940:
  • the conidiogenesis is fialidic enteroblastic, with conidiogenic cells with a length less than 10 mm; the conidia are subglobosous, with a diameter greater than 2.4 mm; and produces intercalar clamidospores with a diameter of less than 10 mm.
  • the colonies do not reach a diameter greater than 20mm and develop, in a variable way, an orange diffusible pigment in the glycerol-nitrate agar medium, after 10 days of incubation at 25 ° C.
  • alkaline phosphatase alkaline phosphatase, esterase lipase, leuclna arylaminidase, acid phosphatase, naphthol-AS-BI-phosphate, alpha-galactosidase and beta-glucosidase, with maximum intensity; esterase, lipase, arylamine valine, cystine arylamidase, trypsin, alpha-chymotrypsin, beta-galactosidase, alpha-glucosidase, N-acetyl-beta-glucosaminidase and alpha-mannosidase, with moderate intensity; and beta-glucuronidase and alpha-fucosidase are not detected.
  • esterase activity (Rf 0.63) weak and variable, esterase activity (Rf 0.67) variable
  • Tricboderma harzianua IMI 352941 Tricboderma harzianua IMI 352941:
  • conidiogenesis is phialidic enteroblastic, with conidiogenic cells with a length greater than 10 mm; the conidia are subglobosous, with a diameter greater than 2.4 mm; and produces terminal clamidospores with a diameter of less than 10 mm.
  • the colonies do not reach a diameter greater than 20mm in the glycerol-nitrate agar medium, after 10 days of incubation at 25 ° C.
  • alkaline phosphatase alkaline phosphatase, esterase, arylaminidase leucine, acid phosphatase, naphthol-AS-BI-phosphate, alpha-galactosidase, beta -galactosidase, alpha-glucosidase, beta-glucosidase, and N-acetyl-beta-glucosaminidase with maximum intensity; esterase lipase, lipase, valine arylaminidase, cystine arylamldasa, trypsin, alpha-chymotrypsin, beta-glucuronidase and alpha-mannosidase, with moderate intensity; and alpha-fucosidase is not detected.
  • API ZYM system Biomérieux SA, Marcy-1'Etoile, France
  • alkaline phosphatase activity (Rf 0.40-0.48);
  • Trlchoderma harzianum CECT 20179 Trlchoderma harzianum CECT 20179:
  • the conidiogenesis is phialidic enteroblastic, with conidiogenic cells with a length greater than 10 mm and a width slightly greater than 3 mm; the conidia are subglobosous, with a diameter greater than 2.4 mm; and produces terminal and intercalar clamidospores with a diameter greater than 10 mm.
  • alkaline phosphatase alkaline phosphatase, arylaminidase leucine and naphthol-AS-BI-phosphate esterase with maximum intensity
  • esterase lipase lipase, arylaminidase valine, cystine arylamidase, acid phosphatase, alpha-galactosidase, beta-galactosidase, alpha-glucosidase, beta-glucosidase, N-acetyl-beta-glucosaminidase and alpha-mannosidase with moderate intensity
  • esterase trypsin, alpha-chymotrypsin, beta-glucuronidase and alpha-fucosidase are not detected.
  • malate dehydrogenase activity (Rf 0.16-0.17), malate dehydrogenase activity (Rf 0.20-0.24), malate dehydrogenase activity (Rf 0.30-0.40); cellulase activity (Rf 0.28-0.33),
  • variable cellulase activity (Rf 0.42-0.54), cellulase activity (Rf 0.69-0.82);
  • Trichoderma viride CECT 20178 Trichoderma viride CECT 20178:
  • the conidiogenesis is phialidical enteroblastic, with conidiogenic cells with a length less than 10 mm and an brochure less than 3 mm; the conidia are oval, with a rough surface, with a diameter greater than 2.4 mm; and produces terminal and intercalar clamidospores with a diameter greater than 10 mm.
  • microorganisms used in the formulation object of patent are strains without editions or deletions of DNA, which have been isolated from Naturaleze and its genome has not been modified. The fact that they are natural strains hinders their cerecterization because there is no exogenous DNA that serves as a merchant for each of them.
  • ITS1-ITS4 region which encompasses the gene encoding the 5.8 S RNAr, and synthesize primers to amplify it by means of a polymerase chain reaction (PCR).
  • the 5.8S rRNA gene was used with the regions adjacent to it, using primers ITS1 and ITS 4, by PCR, obtaining fragments of approximately 0.6 kbp, for cepes of T. harzianum IMI 352939, IMI 352940 and IMI 352941, as observed by agerose gel electrophoresis, 1%.
  • the 5.8S rRNA gene (nucleotides from 212 to 358) has a great similarity with other filementous fungi, and, by temptation, among the 3 strains of T. harzianum. The differences were found in the regions adjacent to the gene, mainly in the sequences between nucleotides 100 and 200, and between nucleotides 408 and 440.
  • Tr ⁇ choderma harzianvm IMI 352939 (ITS1-ITS4 region of the 5.8S rRNA gene):
  • Trichodenaa harzianum IMI 352940 (ITS1 - ITS4 region of gene 5. 8S rRNA):
  • Tr ⁇ choderma harzianum IMI 352941 (ITS1-ITS4 region of the 5.8S rRNA gene):

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Abstract

Liquid formulation based on strains of the stringy fungi Trichoderma harzianum and Trichoderma viride. It comprises sources of carbon, mineral salts and traces of elements with organic and inorganic nutrients, and vegetative structures reproducing strains of the stringy fungus Trichoderma harzianum Rifai which have been identified as Trichoderma harzianum IMI 352940, Trichoderma harzianum IMI 352941, and Trichoderma harzianum CECT 20179; and Trichoderma viride Pers.: Fr identified as Trichoderma viride CECT 20178. The formulation is useful as a biological control agent for combatting diseases of plants and plant material and biodamage produced or transmitted by stringy fungi and other pests of plants, and as a biolixiviation agent.

Description

FORMULACION LIQUIDA A BASE DE CEPAS DE LOS HONGOS FILAMENTOSOS TRICHODERMA HARZIANUM Y TRICHQDERMA VTRIDE LIQUID FORMULATION BASED ON VINTAGE TRICHODERMA HARZIANUM AND TRICHQDERMA VTRIDE
Esta invención se relaciona con una formulación con Trichoderma spp. para su aplicación como agente de control biológico de los enemigos de las plantas y material vegetal, entidades biológicas causantes de biodeterioro y como agente de biolixiviación.  This invention relates to a formulation with Trichoderma spp. for its application as a biological control agent for the enemies of plants and plant material, biological entities causing biodeterioration and as a bioleaching agent.
Más concretamente, la presente invención se relaciona con una formulación liquida, con nutrientes orgánicos e inorgánicos en disolución, que contiene en suspensión estructuras vegetativas y reproductoras de una mezcla de cepas de los hongos filamentosos Trichoderma harzianum Rifai y Trichoderma viride Pers.:Fr.  More specifically, the present invention relates to a liquid formulation, with organic and inorganic nutrients in solution, which contains in suspension vegetative and reproductive structures of a mixture of strains of the filamentous fungi Trichoderma harzianum Rifai and Trichoderma viride Pers.:Fr.
La formulación según la invención es útil como agente de control biológico, frente a enfermedades de vegetales y de material vegetal y biodeterioro producidos o transmitidos por hongos filamentosos y otros enemigos de las plantas, y como agente de biolixiviaciδn.  The formulation according to the invention is useful as a biological control agent, against diseases of plants and plant and biodeterioration material produced or transmitted by filamentous fungi and other enemies of plants, and as a bioleaching agent.
Los componentes químicos y biológicos de la formulación están concentrados para que, durante el transporte y almacenamiento, no se produzca la germinación de las esporas de Trichoderma. Una vez diluida la formulación, inmediatamente antes de su aplicación al suelo, cultivos, etc., los componentes químicos de la misma pueden ser asimilados por las esporas del hongo que iniciará la germinación en pocos minutos. En varias horas se consigue que el microorganismo se multiplique.  The chemical and biological components of the formulation are concentrated so that, during transport and storage, germination of Trichoderma spores does not occur. Once the formulation is diluted, immediately before its application to the soil, crops, etc., its chemical components can be assimilated by the spores of the fungus that will begin germination in a few minutes. In several hours the microorganism is multiplied.
A través del uso de la formulación según la invención, se pueden conseguir los siguientes efectos benefi- ciosos:  Through the use of the formulation according to the invention, the following beneficial effects can be achieved:
Controlar enfermedades de vegetales y material vegetal aprovechando la capacidad antagonista que tienen cuatro cepas de T. harzianum y una de T.viride sobre otros hongos del suelo.  Control diseases of plants and plant material taking advantage of the antagonistic capacity of four strains of T. harzianum and one of T.viride on other soil fungi.
Reducir la concentración de inoculo de microorga nismos patógenos y sus vectores, aprovechando la facilidad de colonización de las cinco cepas de Trichoderme. Reduce the concentration of microorga inoculum pathogenic earthquakes and their vectors, taking advantage of the ease of colonization of the five strains of Trichoderme.
Reducir la aplicación de pesticidas químicos polucionantes, con incidencia sobre la salud humana y sobre otros seres vivos, actualmente utilizados en Agricultura.  Reduce the application of polluting chemical pesticides, with an impact on human health and other living beings, currently used in Agriculture.
Controlar microorganismos causantes de biodeterioro de alimentos y sus envases, productos almacenados, materiales de construcción, postes, cercados, etc.  Control microorganisms causing biodeterioration of food and its packaging, stored products, construction materials, posts, fences, etc.
Obtención selectiva de material geológico por medio de la acción microbiana de las cepas de T. harzianum y de T.viride presentes en la formulación.  Selective obtaining of geological material through the microbial action of the strains of T. harzianum and T.viride present in the formulation.
En consecuencia, la formulación según la invención puede aplicarse específicamente para:  Consequently, the formulation according to the invention can be applied specifically to:
- el control de patógenos y/o vectores de patógenos, de origen íúngico y bacteriano, con importancia en Agricultura, Silvicultura, Jardinería, Praticultura y causantes de alteración y descomposición de material vegetal;  - the control of pathogens and / or vectors of pathogens, of unique and bacterial origin, with importance in Agriculture, Forestry, Gardening, Praticulture and causing alteration and decomposition of plant material;
- el control de microorganismos causantes de biodeterioro de alimentos y sus envases, materiales de construcción, materias primas y productos manufacturados;  - the control of microorganisms causing biodeterioration of food and its packaging, construction materials, raw materials and manufactured products;
la colonización de suelos que requieran el establecimiento de una nueva microflora, especialmente después de una desinfección y solarización del suelo;  colonization of soils that require the establishment of a new microflora, especially after disinfection and solarization of the soil;
- la extracción biológica de material geológico (Biolixiviación).  - the biological extraction of geological material (Bioleaching).
Puede definirse el Control Biológico como la utilización de organismos naturales o modificados, genes o productos génicos, para reducir los efectos de organismos indeseables, y para favorecer a organismos útiles para el hombre, tales como cultivos, árboles, animales y microorganismos beneficiosos. Más de la mitad de los hongos filamentosos que se utilizan en el Control Biológico de otros hongos patógenos de vegetales. pertenecen a los géneros Trichoderma y Gliocladium. Biological Control can be defined as the use of natural or modified organisms, genes or gene products, to reduce the effects of undesirable organisms, and to favor organisms useful to man, such as crops, trees, animals and beneficial microorganisms. More than half of the filamentous fungi that are used in the Biological Control of other plant pathogenic fungi. they belong to the genera Trichoderma and Gliocladium.
Trichoderma es un hongo ampliamente distribuido en la naturaleza, fácil de aislar y cultivar, que crece de forma rápida en muchos sustratos, no afecta al hombre, animales y plantas superiores, actúa como micoparásito (parásito de otros hongos), compite bien por el alimento y por el espacio, produce antibióticos y tiene un sistema enzimático capaz de atacar a una amplia gama de patógenos. Los hongos del género Trichoderma, y en particular la especie Trichoderma harzianum, constituyen una alternativa biológica más limpia, no acumulable en la cadena alimentaria y respetuosa con el medio ambiente, en contraposición a los pesticidas químicos polucionantes de uso común en Agricultura.  Trichoderma is a fungus widely distributed in nature, easy to isolate and cultivate, which grows rapidly on many substrates, does not affect man, animals and higher plants, acts as mycoparasite (parasite of other fungi), competes well for food and through space, it produces antibiotics and has an enzymatic system capable of attacking a wide range of pathogens. The fungi of the genus Trichoderma, and in particular the species Trichoderma harzianum, constitute a cleaner biological alternative, not cumulative in the food chain and respectful with the environment, as opposed to the pollutant chemical pesticides commonly used in Agriculture.
Desde muy antiguo se sabe que existen hongos capaces de controlar a otros hongos y en ello se ha sustentado el" equilibrio natural de los microorganismos existentes en el suelo. Así, se conoce el valor potencial de Trichoderma como agente de Control Biológico al haberse podido observar que Trichoderma lignorum (T. viride) era un parásito que se enroscaba en torno a las hifas de otro hongo, Rhizoctonia solani, produciendo su muerte. A este respecto, especies tales como Phytophthora, Pythium y Rhizopus así como Sclerotium rolfsii podían ser parasitadas por T. viride. En base a estos resultados, surgió la posibilidad de utilizar inoculaciones abundantes con T. viride para controlar ciertos patógenos.  Since ancient times it is known that there are fungi capable of controlling other fungi and in this the "natural balance of the microorganisms in the soil has been sustained. Thus, the potential value of Trichoderma as a Biological Control agent is known to have been able to observe that Trichoderma lignorum (T. viride) was a parasite that curled around the hyphae of another fungus, Rhizoctonia solani, producing its death.In this respect, species such as Phytophthora, Pythium and Rhizopus as well as Sclerotium rolfsii could be parasitized by T. viride Based on these results, the possibility of using abundant inoculations with T. viride to control certain pathogens arose.
Aunque Trichoderma se reconoció , en principio, como un antagonista de hongos, también se ha descrito como antagonista de ciertas bacterias patógenas. Trichoderma ha sido estudiado ampliamente por sus propiedades arvtibióticas de posible utilización clínica, por sus sistemas enzimáticos en procesos alimentarios y por su poder de biodegradación de residuos sólidos y aguas residuales. Otros estudios han establecido el micoparasi tismo de Trichoderma sobre muchos Oomicetos, Zigomicetos, Ascomicetos, Basidiomicetos y hongos anamórficos. Although Trichoderma was recognized, in principle, as a fungal antagonist, it has also been described as an antagonist of certain pathogenic bacteria. Trichoderma has been studied extensively for its arvtibiotic properties of possible clinical use, for its enzymatic systems in food processes and for its biodegradation power of solid waste and wastewater. Other studies have established mycoparasi Trichoderma tism on many Oomycetes, Zygomycetes, Ascomycetes, Basidiomycetes and anamorphic fungi.
Así, la omnipresencia de Trichoderma en suelos naturales y agrícolas de todo el mundo, es una prueba más de que es un excelente competidor por el espacio y por recursos nutritivos.  Thus, the omnipresence of Trichoderma in natural and agricultural soils around the world, is further proof that it is an excellent competitor for space and nutritional resources.
Por ejemplo, los hongos de la podredumbre de la madera han sido controlados en muchos casos por la presencia o el uso de Trichoderma spp., debido a la capacidad de Trichoderma para utilizar todos los carbohidratos no estructurales, evitando el establecimiento del Basidiomiceto destructivo y dejando la madera más permeable a la penetración de conservantes químicos.  For example, wood rot fungi have been controlled in many cases by the presence or use of Trichoderma spp., Due to Trichoderma's ability to use all non-structural carbohydrates, avoiding the establishment of destructive Basidiomycete and leaving the wood more permeable to the penetration of chemical preservatives.
La capacidad competitiva de Trichoderma, y su éxito como agente de Control Biológico, se ven afectados por las propiedades del suelo. Así, se ha observado que el pH tenía influencia sobre el valor relativo de Trichoderma como antagonista de R. solani, afectando al crecimiento y desarrollo de cada hongo.  Trichoderma's competitive capacity, and its success as a Biological Control agent, are affected by soil properties. Thus, it has been observed that the pH had an influence on the relative value of Trichoderma as an antagonist of R. solani, affecting the growth and development of each fungus.
Cuando las condiciones de sequía en el suelo se mantienen durante largos períodos de tiempo, las poblaciones de Trichoderma descienden. Sin embargo, ha de mencionarse que ciertas cepas de T. hamatum y T. pseudo-koningii están adaptadas a condiciones de elevada humedad del suelo y que T. viride y T. polysporum están restringidos a áreas donde prevalecen las bajas temperaturas, mientras que T. harzianum es más frecuente en regiones de clima templado, y T. hamatum y T. koningii están ampliamente distribuidos en áreas de condiciones climáticas diversas.  When the drought conditions in the soil are maintained for long periods of time, the Trichoderma populations descend. However, it should be mentioned that certain strains of T. hamatum and T. pseudo-koningii are adapted to conditions of high soil moisture and that T. viride and T. polysporum are restricted to areas where low temperatures prevail, while T Harzianum is more frequent in temperate regions, and T. hamatum and T. koningii are widely distributed in areas of diverse climatic conditions.
La disponibilidad de hierro es importante para el crecimiento y la capacidad competitiva de Trichoderma.  The availability of iron is important for the growth and competitive capacity of Trichoderma.
La competición por un determinado sustrato no excluye otros mecanismos de acción, como puede ser el micoparasitismo. También se ha observado que cuando se aplicaban fungicidas que eran tóxicos para Trichoderma, para controlar las manchas de las hojas de cacahuete, había un aumento de la enfermedad causada por S. rolfsii. The competition for a certain substrate does not exclude other mechanisms of action, such as mycoparasitism. It has also been observed that when fungicides that were toxic to Trichoderma were applied, to control peanut leaf spots, there was an increase in disease caused by S. rolfsii.
No obstante, no todos los fungicidas son tóxicos para Trichoderma. Así, el fungicida Tiram no tenía efecto sobre Trichoderma y Penicillium. En suelos tratados con Tiram, Trichoderma sobrevivía bien, se multiplicaba, y protegía, frente a Pythium, las plántulas de guisante, durante un tiempo considerable, después de que el fungicida alcanzara niveles no tóxicos para el patógeno. Igualmente, se han encontrado efectos beneficiosos similares con T. harzianum y el fungicida Metalaxyl ha proporcionado efectos interesantes, aplicado a la semilla de guisante, antes de pildorarla con conidios de T. harzianum.  However, not all fungicides are toxic to Trichoderma. Thus, the fungicide Tiram had no effect on Trichoderma and Penicillium. In soils treated with Tiram, Trichoderma survived well, multiplied, and protected, against Pythium, pea seedlings, for a considerable time, after the fungicide reached non-toxic levels for the pathogen. Similarly, similar beneficial effects have been found with T. harzianum and the fungicide Metalaxyl has provided interesting effects, applied to the pea seed, before firing it with conidia of T. harzianum.
El Benomilo así como otros fungicidas benzimidazólicos (cerno Tiatendazol y Metiltiofanato), inhiben fuertemente a Trichoderma. Captan, Procimidona y Vinclozolin no son inhibidores de Tríchoderma.  Benomyl, as well as other benzimidazole fungicides (ceria Thiatendazole and Methylthiophanate), strongly inhibit Trichoderma. Captan, Procimidone and Vinclozolin are not inhibitors of Tríchoderma.
El antagonismo del suelo frente a Trichoderma es el factor más importante en la regulación del nivel del posible inoculo da este hongo cuando se aplica en un ambiente natural. El conjunto de formas vivas del suelo y la naturaleza física y química del mismo, desempeñan el papel más importante en la reduección del número de propágulos de Trichoderma, después de su introducción como agente de Control Biológico.  The antagonism of the soil against Trichoderma is the most important factor in the regulation of the level of the possible inoculum of this fungus when applied in a natural environment. The set of living forms of the soil and the physical and chemical nature of it, play the most important role in the reduction of the number of Trichoderma propagules, after its introduction as a Biological Control agent.
Según el estado de la técnica, el Control Biológico con Trichoderma se puede llevar a cabo:  According to the state of the art, Biological Control with Trichoderma can be carried out:
-. Introduciendo el antagonista en el suelo con una base alimenticia, mediante formulaciones con conidios o en polvos, pasta o pildoras de alginato, que contienen biomasa obtenida por fermentación.  -. Introducing the antagonist into the soil with a food base, through formulations with conidia or in powders, paste or alginate pills, which contain biomass obtained by fermentation.
-.Por tratamiento de semillas. Se requiere menor cantidad da material biológico y el éxito dependerá de:-.For seed treatment. Minor is required amount of biological material and success will depend on:
- los aislamientos utilizados - the insulations used
- el tipo de enfermedad  - the type of disease
- el tipo de cultivo  - the type of crop
- la edad de los antagonistas  - the age of the antagonists
- la temperatura y pH del suelo  - soil temperature and pH
- el tipo de suelo y los microorganismos que existan en él  - the type of soil and the microorganisms that exist in it
- el estado nutricional del antagonista  - the nutritional status of the antagonist
- la densidad del inoculo en la semilla  - the density of the inoculum in the seed
- el potencial de inoculo del antagonista en el suelo  - the potential for inoculum of the antagonist in the soil
- el momento de la siembra  - the time of planting
-.Por aplicación a partes aéreas de la planta (en hojas, en heridas de árboles o en el momento de la poda). Estas formulaciones suelen ser líquidas.  - By application to aerial parts of the plant (in leaves, in wounds of trees or at the time of pruning). These formulations are usually liquid.
Los primeros resultados prácticos de control de enfermedades de plantas con Trichoderma fueron los obtenidos mediante esterilización parcial del suelo, en la lucha contra la podredumbre de la raíz de cítricos debida a A. mellea. Se demostró que la esterilización parcial del suelo con pesticidas selectivos debilitaba el crecimiento del patógeno y permitía una proliferación de Trichoderma, que era capaz de invadir y matar a A. mellea.  The first practical results of control of plant diseases with Trichoderma were those obtained by partial sterilization of the soil, in the fight against the rot of citrus root due to A. mellea. It was shown that partial soil sterilization with selective pesticides weakened the growth of the pathogen and allowed a proliferation of Trichoderma, which was able to invade and kill A. mellea.
El tratamiento del suelo con vapor, la solarización y otros métodos, pueden modificar el ecosistema hasta el punto de favorecer la proliferación de Trichoderma. El calentamiento subletal de suelos infectados con A. mellea, por ejemplo, estimulaba al residente más resistente al calor, Trichoderma, que entonces proliferaba y parasitaba a un debilitado A. mellea.  The treatment of soil with steam, solarization and other methods, can modify the ecosystem to the point of favoring the proliferation of Trichoderma. The sub-warming of soils infected with A. mellea, for example, stimulated the most heat-resistant resident, Trichoderma, who then proliferated and parasitized a weakened A. mellea.
Como sustratos para la aplicación de Trichoderma en el Control Biológico, se han utilizado granulos de tierra de diatomeas impregnado con melazas, salvado de trigo, granulos de corteza de árbol, turba más salvado de trigo, granos de cebada, compost de corteza de madera y una formulación de granulos de material vegetal conteniendo T. harzianum y T. polysporum. Un Trichoderma sp. formulado en aceite de motor, ha sido utilizado con éxito para inocular pinos durante el periodo de poda. As substrates for the application of Trichoderma in Biological Control, granules of diatomaceous earth impregnated with molasses, wheat bran, have been used. tree bark granules, peat plus wheat bran, barley grains, wood bark compost and a formulation of plant material granules containing T. harzianum and T. polysporum. A Trichoderma sp. formulated in motor oil, it has been used successfully to inoculate pines during the pruning period.
Otros ejemplos incluyen la adición de esporas de Trichoderma al suelo junto con un fertilizante líquido, en caso de que los aislamientos utilizados puedan tolerar una presión osmótica alta.  Other examples include the addition of Trichoderma spores to the soil along with a liquid fertilizer, in case the insulations used can tolerate a high osmotic pressure.
Los medios de cultivo para la fermentación líquida deben incluir productos agrícolas accesibles y baratos, tales como glucosa, almidón, maiz hidrolizado y productos de soja, suero y melazas. Asi, se han producido inóculos viables de Trichoderma, en un tanque de fermentación, para obtener, después de un periodo de incubación, una gran cantidad de biomasa compuesta de micelio, clamidosporas y algún conidio. También se ha preparado otra formulación, secando al aire la biomasa, moliéndola y diluyendo el polvo con Pyrax® , que está disponible comercialmente como transportador para el inoculo del hongo. Otros soportes comerciales que se vienen utilizando con frecuencia en el Control Biológico son el Agro-Lig® y el Pelgel®.  Culture media for liquid fermentation should include accessible and cheap agricultural products, such as glucose, starch, hydrolyzed corn and soy products, whey and molasses. Thus, viable Trichoderma inoculums have been produced, in a fermentation tank, to obtain, after a period of incubation, a large amount of biomass composed of mycelium, clamidospores and some conidia. Another formulation has also been prepared, air drying the biomass, grinding it and diluting the powder with Pyrax®, which is commercially available as a carrier for fungus inoculum. Other commercial supports that have been used frequently in Biological Control are Agro-Lig® and Pelgel®.
Una mejora en los sistemas de aplicación es la encapsulación de propágulos, basada en la capacidad de las soluciones acuosas de alginato sódico para reaccionar con cationes de ciertos metales (Ca 2+) y formar geles. Una modificación posterior a esta técnica ha sido la incorporación de transportadores alimenticios (ej. salvado) al alginato, en intimo contacto con el antagonista, para proporcionar los nutrientes necesarios.  An improvement in application systems is the encapsulation of propagules, based on the ability of aqueous sodium alginate solutions to react with cations of certain metals (Ca 2+) and form gels. A subsequent modification to this technique has been the incorporation of food transporters (eg bran) to alginate, in intimate contact with the antagonist, to provide the necessary nutrients.
Igualmente, la adición de quitina o de paredes celulares a los inóculos, durante el pildorado de las semillas, aumenta la eficacia de T. hamatum como protec tor de guisantes y rábanos. El compost de corteza de árboles ha demostrado ser un medio excelente para el crecimiento y la liberación de Trichoderma spp. Likewise, the addition of chitin or cell walls to the inoculums, during the pill of the seeds, increases the efficacy of T. hamatum as a protec peas and radishes tor. Tree bark compost has proven to be an excellent medium for the growth and release of Trichoderma spp.
Como sistema de aplicación también se ha propuesto el "fluid drilling" para Control Biológico en plantas con semillas pequeñas. El salvado de trigo, junto con turba, constituye una mezcla óptima para la supervivencia de Trichoderma, como una fuente de inoculo de Control Biológico, para mezclar en suelo y macetas.  As an application system, fluid drilling for Biological Control in plants with small seeds has also been proposed. Wheat bran, together with peat, constitutes an optimal mixture for the survival of Trichoderma, as a source of Biological Control inoculum, for mixing in soil and pots.
También se ha recurrido a la aplicación de micelio joven de Trichoderma sobre un sustrato parcialmente consumido, al considerarse que el micelio es mucho más resistente a la fungistasis del suelo que los conidios al germinar. Por tanto, podría parecer más eficaz aplicar Trichoderma con una base alimenticia parcialmente consumible, que suplementar Trichoderma con nutrientes adicionales, en el momento de su aplicación.  The application of Trichoderma's young mycelium on a partially consumed substrate has also been used, considering that the mycelium is much more resistant to soil fungistasis than conidia when germinating. Therefore, it may seem more effective to apply Trichoderma with a partially consumable food base, than to supplement Trichoderma with additional nutrients, at the time of its application.
Los granulos de alginato presentan variabilidad en cuanto a calidad y comportamiento en el suelo. Actualmente, y como forma alternativa, se aplica Trichoderma a las semillas en forma de monocapa alrededor de las mismas, sobre una matriz sólida, que puede ser de Agro-Lig® o carbón bituminoso.  Alginate granules have variability in quality and behavior in the soil. Currently, and as an alternative, Trichoderma is applied to the monolayer-shaped seeds around them, on a solid matrix, which can be Agro-Lig® or bituminous coal.
A la vista de estos antecedentes, y de acuerdo con la presente invención, se han realizado estudios de selección de cepas de Trichoderma para su aplicación como agentes de Control Biológico en el control de enfermedades producidas por hongos del suelo en vegetales, principalmente remolacha.  In view of this background, and in accordance with the present invention, Trichoderma strain selection studies have been carried out for application as Biological Control agents in the control of diseases caused by soil fungi in vegetables, mainly beets.
Como resultado de estas investigaciones, se seleccionaron tres cepas de Trichoderma harzianum, las cuales han sido depositadas, al amparo del Tratado de Budapest, en la Colección de Cultivos del International Mycological Institute de Egham (Reino Unido), con los números de registro IMI 352939, IMI 352940 e IMI 352941, respectiva mente; y una cepa de Trichoderma viride y otra de Trichoderma harzianum que también se depositaron, al amparo del Tratado de Budapest, en la Colección Española de Cultivos Tipo de Burjasot (Valencia), con los números de registro CECT 20178 y CECT 20179, respectivamente. As a result of these investigations, three strains of Trichoderma harzianum were selected, which have been deposited, under the Budapest Treaty, in the Crop Collection of the International Mycological Institute of Egham (United Kingdom), with IMI registration numbers 352939 , IMI 352940 and IMI 352941, respectively mind; and a strain of Trichoderma viride and another strain of Trichoderma harzianum that were also deposited, under the Budapest Treaty, in the Spanish Type Culture Collection of Burjasot (Valencia), with the registration numbers CECT 20178 and CECT 20179, respectively.
La eficacia de la aplicación de estos microorganismos en una pildora recubriendo la semilla, como alternativa a los pesticidas químicos habitualmente utilizados, ha sido demostrada en ensayos realizados a nivel de campo, en ambiente natural. Sin embargo, el pildorado de semillas limita el espectro de actuación de Trichoderma, al restringir su aplicación al momento de la siembra.  The effectiveness of the application of these microorganisms in a pill covering the seed, as an alternative to the chemical pesticides commonly used, has been demonstrated in field tests, in a natural environment. However, the seed piling limits the spectrum of action of Trichoderma, by restricting its application at the time of planting.
Sería por tanto interesante poder aportar una formulación liquida que sirviera de vehículo al inoculo de Trichoderma.  It would therefore be interesting to be able to provide a liquid formulation that would serve as a vehicle for Trichoderma's inoculum.
Partiendo de las premisas de que no existe un agente de Control Biológico universal y que no todas las cepas de Trichoderma presentan la misma capacidad antagonista cuando son utilizadas como agentes de Control Biológico, ya que dentro de la especie T. harzianum existen diferentes tipos fisiológicos que pueden ser eficaces o ineficaces en cada caso concreto frente a un determinado patógeno, la utilización de una mezcla con cuatro cepas de T. harzianum y una de T.viride seleccionadas frente a distintos patógenos, amplía el espectro de actuación de la formulación. Por ello, su aplicación en patosistemas en los que se vean implicados diversos microorganismos (como pueden ser los diferentes hongos que intervienen en las caídas de plántulas o los patógenos causantes de colapsos facilitados por un estrés hidrico, etc.), resulta ventajosa al no existir un único agente, ya sea químico o biológico, que los controle.  Starting from the assumptions that there is no universal Biological Control agent and that not all Trichoderma strains have the same antagonistic capacity when used as Biological Control agents, since within the T. harzianum species there are different physiological types that they can be effective or ineffective in each specific case against a certain pathogen, the use of a mixture with four strains of T. harzianum and one of T.viride selected against different pathogens, broadens the spectrum of action of the formulation. Therefore, its application in patosystems in which various microorganisms are involved (such as the different fungi that are involved in seedling falls or the pathogens causing collapses facilitated by water stress, etc.), is advantageous as there is no a single agent, whether chemical or biological, that controls them.
Dado que cada cepa de Trichoderma es sumamente específica frente a unos patógenos concretos, la aplica ción de las cinco cepas en una mezcla, dentro de la misma formulación, incrementa las posibilidades de éxito del producto bajo condiciones ambientales y patógenos diferentes. Since each strain of Trichoderma is highly specific against specific pathogens, it is applied tion of the five strains in a mixture, within the same formulation, increases the chances of success of the product under different environmental conditions and pathogens.
Por tanto, de acuerdo con la presente invención, se proporciona ahora una formulación líquida, con nutrientes orgánicos e inorgánicos en disolución, que contiene en suspensión estructuras vegetativas y reproductoras de una, dos, tres o cuatro de las cepas del hongo filamentoso Trichoderma harzianum Rifai y/o Trichoderma viride Pers.:Fr. anteriormente identificadas.  Therefore, according to the present invention, a liquid formulation is now provided, with organic and inorganic nutrients in solution, which contains in suspension vegetative and reproductive structures of one, two, three or four strains of the filamentous fungus Trichoderma harzianum Rifai and / or Trichoderma viride Pers.:Fr. previously identified.
La formulación según la invención es útil, como ya ha sido indicado, como agente de control biológico y como agente de biolixiviación.  The formulation according to the invention is useful, as already indicated, as a biological control agent and as a bioleaching agent.
Concretamente, la presente invención proporciona una formulación líquida cuya composición cuantitativa (%, p/v) es la siguiente:  Specifically, the present invention provides a liquid formulation whose quantitative composition (%, w / v) is as follows:
Figure imgf000012_0001
Figure imgf000012_0001
Componentes biológicos  Biological components
Trichoderma harzianum IMI 352939 c.s.p. 3×105- 10×107 conidios/ml Trichoderma harzianum IMI 352939 csp 3 × 10 5 - 10 × 10 7 conidia / ml
Trichoderma harzianum IMI 352940 c.s.p. 3×105-10×107 conidios/ml Trichoderma harzianum IMI 352940 csp 3 × 10 5 -10 × 10 7 conidia / ml
Trichoderma harzianum IMI 352941 c.s.p. 3×105-10×107 conidios/ml Trichoderma harzianum CECT 20179 c.s.p. 3×105- 10×107 conidios/ml Trichoderma harzianum IMI 352941 csp 3 × 10 5 -10 × 10 7 conidia / ml Trichoderma harzianum CECT 20179 csp 3 × 10 5 - 10 × 10 7 conidia / ml
Trichoderma harzianum CECT 20178 c.s.p. 3×105- 10×107 conidios/ml Trichoderma harzianum CECT 20178 csp 3 × 10 5 - 10 × 10 7 conidia / ml
La formulación de acuerdo con la invención posee componentes químicos que pueden ser utilizados como nutrientes por T. harzianum y T.viride, a la vez que mantienen la humedad suficiente para la germinación de propágulos de estos hongos.  The formulation according to the invention has chemical components that can be used as nutrients by T. harzianum and T.viride, while maintaining sufficient humidity for the germination of propagules of these fungi.
La formulación líquida de la invención permite utilizar los antagonistas en cualquier momento y situación, sin necesidad de limitar la aplicación al momento de la siembra, como sucede cuando se formulan los agentes de Control Biológico en medios y sustratos sólidos o recubriendo las semillas y otros materiales de propagación.  The liquid formulation of the invention allows antagonists to be used at any time and situation, without the need to limit the application at the time of sowing, as happens when Biological Control agents are formulated in solid media and substrates or by coating seeds and other materials of propagation.
La formulación liquida puede mantenerse, concentrada, en condiciones de refrigeración, a 4ºC, durante al menos 1 año y con 1 litro de la misma se pueden tratar hasta 30 Ha cultivadas. La formulación puede ser aplicada de forma directa sobre suelo y material vegetal y/o animal y/o fúngico, vivo o muerto; o mezclada con sustancias utilizadas en Agricultura para vehicular otros productos fitosanitarios como fertilizantes, abonos, microgránulos, compost o las aplicadas en la pildoración, revestimiento y encapsulación de semillas y embriones, etc. Como ventaja, la dilución del producto favorece la dispersión de T. harzianum y T.viride.  The liquid formulation can be kept, concentrated, under refrigeration conditions, at 4 ° C, for at least 1 year and with 1 liter of it can be treated up to 30 Ha cultivated. The formulation can be applied directly to soil and plant and / or animal and / or fungal material, living or dead; or mixed with substances used in Agriculture to vehicular other phytosanitary products such as fertilizers, fertilizers, microgranules, compost or those applied in the pilling, coating and encapsulation of seeds and embryos, etc. As an advantage, dilution of the product favors the dispersion of T. harzianum and T.viride.
Se ofrecen ahora ejemplos de formulaciones líquidas según la invención, en función de la via de aplicación de las mismas. Ha de entenderse que estos ejemplos se ofrecen sólo con carácter ilustrativo y de ningún modo limitativo del alcance de la invención.  Examples of liquid formulations according to the invention are now given, depending on the route of application thereof. It is to be understood that these examples are offered for illustrative purposes only and in no way limiting the scope of the invention.
Considerando que para tratar 1 Ha de cultivo se utilizan 3 litros de producto por via foliar o 6 litros de producto via suelo, habrá que concentrar la formulación 30 y 60 veces, según se aplique, respectivamente, sobre la planta o directamente al suelo. Whereas to treat 1 Ha of cultivation, 3 liters of product are used per foliar route or 6 liters of product via soil, the formulation should be concentrated 30 and 60 times, as applied, respectively, on the plant or directly to the soil.
Las formulaciones resultantes tendrán la siguiente composición:  The resulting formulations will have the following composition:
Ejemplo 1  Example 1
Vía foliar (formulación 30 veces concentrada):  Foliar path (30 times concentrated formulation):
Figure imgf000014_0002
Figure imgf000014_0002
Componentes biológicos  Biological components
Trichoderma harzianum IMI 352939 c.s.p. 9×106- 3×109 conidios/ml  Trichoderma harzianum IMI 352939 c.s.p. 9 × 106- 3 × 109 conidia / ml
Trichoderma harzianum IMI 352940 c.s.p. 9×106-3×109 conidios/ml  Trichoderma harzianum IMI 352940 c.s.p. 9 × 106-3 × 109 conidia / ml
Trichoderma harzianum IMI 352941 c.s.p. 9×106-3×109 conidios/ml.  Trichoderma harzianum IMI 352941 c.s.p. 9 × 106-3 × 109 conidia / ml.
Trichoderma harzianum CECT 20179  Trichoderma harzianum CECT 20179
Trichoderma harzianum CECT 20178  Trichoderma harzianum CECT 20178
Ejemplo 2  Example 2
Aplicación sobre suelo (formulación 60 veces concentrada):  Application on soil (60 times concentrated formulation):
Figure imgf000014_0001
Figure imgf000015_0001
Figure imgf000014_0001
Figure imgf000015_0001
Componentes biológicos  Biological components
Trichoderma harzianum IMI 352939 c.s.p. 1.8×107-6×109 conidios/ml  Trichoderma harzianum IMI 352939 c.s.p. 1.8 × 107-6 × 109 conidia / ml
Trichoderma harzianum IMI 352940 c.s.p, Trichoderma harzianum IMI 352940 c.s.p,
1.8×107-6×109 conidios/ml 1.8 × 107-6 × 109 conidia / ml
Trichoderma harzianum IMI 352941 c.s.p, 1.8×107-6×109 conidios/ml.  Trichoderma harzianum IMI 352941 c.s.p, 1.8 × 107-6 × 109 conidia / ml.
Trichoderma harzianum CECT 20179 c.s.p, 1.8×107-6×109 comidios/ml Trichoderma harzianum CECT 20179 csp, 1.8 × 10 7 -6 × 10 9 meals / ml
Trichoderma harzianum CECT 20178 c.s.p. 1.8×107-6×109 comidios/ml Trichoderma harzianum CECT 20178 csp 1.8 × 10 7 -6 × 10 9 meals / ml
La invención puede desarrollarse a escala industrial ya que los diferentes sistemas de fermentación disponibles permiten obtener rendimientos de inoculo de T. harzianum y de T.viride de más de 10 gramos de biomasa por 1 litro de sustrato fermentable.  The invention can be developed on an industrial scale since the different fermentation systems available allow to obtain inoculum yields of T. harzianum and T.viride of more than 10 grams of biomass per 1 liter of fermentable substrate.
A continuación, se ofrece una descripción de los microorganismos usados en las formulaciones líquidas según esta invención.  A description of the microorganisms used in the liquid formulations according to this invention is given below.
a) Características morfológicas, fisiológicas y bioquímicas:  a) Morphological, physiological and biochemical characteristics:
Trlc-boderma harzlamm IMI 352939:  Trlc-boderma harzlamm IMI 352939:
Posee los atributos morfológicos característicos de la especie Trichoderma harzianum Rifai, la conidiogénesis es enteroblástica fialídica, con células conidiógenas con una longitud inferior a 10 mm; los conidios son subglobosos, con diámetro superior a 2,4 mm; y produce clamidosporas intercalares y terminales con diámetro superior a 10 mm. De acuerdo con la metodología usada: It has the characteristic morphological attributes of the Trichoderma harzianum Rifai species, conidiogenesis is phialidic enteroblastic, with conidiogenic cells with a length less than 10 mm; the conidia are subglobosous, with a diameter greater than 2.4 mm; and produces intercalar and terminal clamidospores with a diameter greater than 10 mm. According to the methodology used:
crece en la superficie del medio, con esporulación variable, cuando se cultiva en glucosa 1% como fuente de carbono;  it grows on the surface of the medium, with variable sporulation, when grown in 1% glucose as a carbon source;
crece muy bien en la superficie del medio, con esporulación variable, cuando se cultiva en etanol 1% como fuente de carbono;  It grows very well on the surface of the medium, with variable sporulation, when grown in 1% ethanol as a carbon source;
crece en la superficie del medio, con esporulación, cuando se cultiva en ácido cítrico 1% y en ácido láctico 1% como fuentes de carbono;  it grows on the surface of the medium, with sporulation, when grown in 1% citric acid and 1% lactic acid as carbon sources;
crece en la superficie del medio, con esporulación variable, cuando se cultiva en glicina 0.2% como fuente de nitrógeno;  it grows on the surface of the medium, with variable sporulation, when grown in 0.2% glycine as a source of nitrogen;
crece muy bien en la superficie del medio, y esporula débilmente, cuando se cultiva en oxalato amónico 0.2% como fuente de nitrógeno;  It grows very well on the surface of the medium, and sporulates weakly, when grown in 0.2% ammonium oxalate as a source of nitrogen;
crece en la superficie del medio, con esporulación, cuando se cultiva en urea 0.2% como fuente de nitrógeno; no crece en la superficie del medio cuando se cultiva en creatina 0.2% como fuente de nitrógeno;  it grows on the surface of the medium, with sporulation, when it is grown in 0.2% urea as a source of nitrogen; it does not grow on the surface of the medium when grown in 0.2% creatine as a source of nitrogen;
crece a 4ºC;  grows at 4 ° C;
no crece en medios adicionados con cristal violeta a una concentración de 10mg/litro;  does not grow in media added with violet crystal at a concentration of 10mg / liter;
no esporula pero crece, formando agrupaciones de hifas aéreas en forma de rosetón, cuando se cultiva en medios adicionados de selenito sódico a una concentración de 320mg/litro;  it does not sporulate but it grows, forming groups of aerial hyphae in the form of a rosette, when it is grown in media added sodium selenite at a concentration of 320mg / liter;
desarrolla colonias de diámetro inferior a 25mm en el medio agar extracto de malta adicionado de 200 mg/litro de sulfato de cobre II, después de 24 horas de incubación a 25ºC;  develops colonies of diameter less than 25mm in the agar medium added malt extract of 200 mg / liter of copper sulfate II, after 24 hours of incubation at 25 ° C;
hidroliza la gelatina y esporula en ese mismo medio de cultivo;  hydrolyzes the gelatin and sporula in that same culture medium;
crece, con esporulación variable, en agar Czapek-amonio después de 10 días de incubación a 25ºC; crece, con fuerte esporulación, en agar nitrito- sacarosa después de 10 dÍas de incubación a 25ºC; grows, with variable sporulation, on Czapek-ammonium agar after 10 days of incubation at 25 ° C; grows, with strong sporulation, in nitrite-sucrose agar after 10 days of incubation at 25 ° C;
las colonias no alcanzan un diámetro superior a 20mm en el medio agar glicerol-nitrato, después de 10 dias de incubación a 25°C.  the colonies do not reach a diameter greater than 20mm in the glycerol-nitrate agar medium, after 10 days of incubation at 25 ° C.
Produce las siguientes actividades enzimáticas extracelulares, medidas con ol sistema API ZYM (BiomérieuxS.A., Maroy-l'Etoile, Francia): fosfatase alcalina, leucina arilaminidasa, fosfatase acida, naftol-AS-BI-fosfato, alfa-galactosidasa, beta-galactosidasa, alfa-glucosidasa, beta-glucosidasa, y N-acetil-beta-glucosaminidasa con intensidad máxima; esterasa, esterasa lipasa, lipasa, valina arilaminidasa, cistina arilamidasa, tripsina, alfa-quimiotripsina, beta-glucuronidasa y alfa-manosidasa, con intensidad moderada; y no se detecta alfa-fucosidasa.  It produces the following extracellular enzymatic activities, measured with the API ZYM system (BiomérieuxS.A., Maroy-l'Etoile, France): alkaline phosphatase, arylaminidase leucine, acid phosphatase, naphthol-AS-BI-phosphate, alpha-galactosidase, beta -galactosidase, alpha-glucosidase, beta-glucosidase, and N-acetyl-beta-glucosaminidase with maximum intensity; esterase, esterase lipase, lipase, arylaminidase valine, cystine arylamidase, trypsin, alpha-chymotrypsin, beta-glucuronidase and alpha-mannosidase, with moderate intensity; and alpha-fucosidase is not detected.
De acuerdo con la metodología usada, presenta los siguientes bandas de isoenzimas, obtenidas mediante electroforesis en geles de poliacrilamida al 7.5% (10% para la actividad catalasa):  According to the methodology used, it presents the following isoenzyme bands, obtained by electrophoresis in 7.5% polyacrylamide gels (10% for catalase activity):
actividad fosfatase alcalina (Rf 0.43-0.51) variable;  alkaline phosphatase activity (Rf 0.43-0.51) variable;
actividad catalasa (Rf 0.16-0.34);  Catalan activity (Rf 0.16-0.34);
actividad fosfatasa acida (Rf 0.25-0.31),  acid phosphatase activity (Rf 0.25-0.31),
actividad fosfatasa acida (Rf 0.32-0.43);  acid phosphatase activity (Rf 0.32-0.43);
actividad celulasa (Rf 0.28-0.33),  cellulase activity (Rf 0.28-0.33),
actividad celulasa (Rf 0.45-0.48);  cellulase activity (Rf 0.45-0.48);
actividad esterasa (Rf 0.25) variable,  esterase activity (Rf 0.25) variable,
actividad esterasa (Rf 0.31) débil y variable, actividad esterasa (Rf 0.55) débil y variable,  weak and variable esterase activity (Rf 0.31), weak and variable esterase activity (Rf 0.55),
actividad esterasa (Rf 0.57) débil y variable, weak and variable esterase activity (Rf 0.57),
actividad esterasa (Rf 0.65) variable, esterase activity (Rf 0.65) variable,
actividad esterasa (Rf 0.74) variable,  esterase activity (Rf 0.74) variable,
actividad esterasa (Rf 0.78) variable,  esterase activity (Rf 0.78) variable,
actividad esterasa (Rf 0.82-0.84) variable, y actividad esterasa (Rf 0.88) variable. esterase activity (Rf 0.82-0.84) variable, and esterase activity (Rf 0.88) variable.
Trichoderma harzianum IMI 352940:  Trichoderma harzianum IMI 352940:
Posee los atributos morfológicos característicos de la especie Trichoderma harzianum Rifai, la conidiogénesis es enteroblástica fialídica, con células conidiógenas con una longitud inferior a 10 mm; los conidios son subglobosos, con diámetro superior a 2,4 mm; y produce clamidosporas intercalares con diámetro inferior a 10 mm.  It has the characteristic morphological attributes of the species Trichoderma harzianum Rifai, the conidiogenesis is fialidic enteroblastic, with conidiogenic cells with a length less than 10 mm; the conidia are subglobosous, with a diameter greater than 2.4 mm; and produces intercalar clamidospores with a diameter of less than 10 mm.
De acuerdo con la metodología usada:  According to the methodology used:
crece en la superficie del medio, con esporulación variable, cuando se cultiva en glucosa 1% como fuente de carbono;  it grows on the surface of the medium, with variable sporulation, when grown in 1% glucose as a carbon source;
crece en la superficie del medio, pero no esporula, cuando se cultiva en etanol 1% como fuente de carbono; crece en la superficie del medio, con esporulación, cuando se cultiva en ácido cítrico 1% como fuente de carbono;  it grows on the surface of the medium, but does not sporulate, when grown in 1% ethanol as a carbon source; it grows on the surface of the medium, with sporulation, when grown in 1% citric acid as a carbon source;
crece débilmente en la superficie del medio, pero esporula, cuando se cultiva en ácido láctico 1% como fuente de carbono;  it grows weakly on the surface of the medium, but sporula, when grown in 1% lactic acid as a carbon source;
crece débilmente en la superficie del medio, con esporulación variable, cuando se cultiva en glicina 0.2% como fuente de nitrógeno;  it grows weakly on the surface of the medium, with variable sporulation, when grown in 0.2% glycine as a source of nitrogen;
crece débilmente en la superficie del medio, con esporulación, cuando se cultiva en oxalato amónico 0.2% como fuente de nitrógeno;  it grows weakly on the surface of the medium, with sporulation, when grown in 0.2% ammonium oxalate as a source of nitrogen;
crece débilmente en la superficie del medio, con fuerte esporulación, cuando se cultiva en urea 0.2% como fuente de nitrógeno;  it grows weakly on the surface of the medium, with strong sporulation, when grown in 0.2% urea as a source of nitrogen;
no crece en la superficie del medio cuando se cultiva en creatina 0.2% como fuente de nitrógeno;  it does not grow on the surface of the medium when grown in 0.2% creatine as a source of nitrogen;
no crece a 4ºC;  does not grow at 4 ° C;
no crece en medios adicionados con cristal violeta a una concentración de 10mg/litro;  does not grow in media added with violet crystal at a concentration of 10mg / liter;
no esporula pero crece, formando agrupaciones de hifas aéreas en forma de copos, cuando se cultiva en medios adicionados de selenito sódico a una concentración de 320mg/litro; no sporula but it grows, forming clusters of aerial hyphae in the form of flakes, when grown in sodium selenite-added media at a concentration of 320mg / liter;
desarrolla colonias de diámetro superior a 30mm en el medio agar extracto de malta adicionado de 200 mg/litro de sulfato de cobre II, después de 24 horas de incubación a 25ºC;  develops colonies with a diameter greater than 30 mm in the agar medium added malt extract of 200 mg / liter of copper sulfate II, after 24 hours of incubation at 25 ° C;
produce un pigmento amarillo difusiblβ en el medio de cultivo, cuando se incuba en agar extracto de malta adicionado de 200 mg/litro de sulfato de cobre II, después de 14 días de incubación a 25ºC, en oscuridad; hidroliza fuertemente la gelatina pero no esporula en ese mismo medio de cultivo;  produces a yellow diffusiblβ pigment in the culture medium, when it is incubated in agar extract added from 200 mg / liter of copper sulfate II, after 14 days of incubation at 25 ° C, in the dark; strongly hydrolyzes the gelatin but does not sporulate in that same culture medium;
crece, con poca esporulación, en agar Czapek-amonio y agar nitrito-sacarosa, después de 10 días de incubación a 25ºC;  grows, with little sporulation, on Czapek-ammonium agar and nitrite-sucrose agar, after 10 days of incubation at 25 ° C;
las colonias no alcanzan un diámetro superior a 20mm y desarrollan, de forma variable, un pigmento difusible de color naranja en el medio agar glicerol-nitrato, después de 10 días de incubación a 25ºC.  the colonies do not reach a diameter greater than 20mm and develop, in a variable way, an orange diffusible pigment in the glycerol-nitrate agar medium, after 10 days of incubation at 25 ° C.
Produce las siguientes actividades enzimáticas extracelulares, medidas con el sistema API ZYM (Biomérieux S.A., Marcy-1'Etoile, Francia): fosfatasa alcalina, esterasa lipasa, leuclna arilaminidasa, fosfatasa acida, naftol-AS-BI-fosfato, alfa-galactosidasa y beta-glucosidasa, con intensidad máxima; esterasa, lipasa, valina arilaminídasa, cistina arilamidasa, tripsina, alfa-quimiotripsina, beta-galactosidasa, alfa-glucosidasa, N-acetil-beta-glucosaminidasa y alfa-manosidasa, con intensidad moderada; y no se detectan beta-glucuronidasa y alfa-fucosidasa.  It produces the following extracellular enzymatic activities, measured with the API ZYM system (Biomérieux SA, Marcy-1'Etoile, France): alkaline phosphatase, esterase lipase, leuclna arylaminidase, acid phosphatase, naphthol-AS-BI-phosphate, alpha-galactosidase and beta-glucosidase, with maximum intensity; esterase, lipase, arylamine valine, cystine arylamidase, trypsin, alpha-chymotrypsin, beta-galactosidase, alpha-glucosidase, N-acetyl-beta-glucosaminidase and alpha-mannosidase, with moderate intensity; and beta-glucuronidase and alpha-fucosidase are not detected.
De acuerdo con la metodología usada, presenta los siguientes bandas de isoenzimas, obtenidas mediante electroforesis en geles de poliacrilamida al 7.5% (10% para la actividad catalasa): actividad fosfatasa alcalina (Rf 0.43-0.51); According to the methodology used, it presents the following isoenzyme bands, obtained by electrophoresis in 7.5% polyacrylamide gels (10% for catalase activity): alkaline phosphatase activity (Rf 0.43-0.51);
actividad catalasa (Rf 0.16-0.34); Catalan activity (Rf 0.16-0.34);
actividad fosfatasa acida (Rf 0.25-0.31) débil y variable,  weak and variable acid phosphatase activity (Rf 0.25-0.31),
actividad fosfatasa acida (Rf 0.32-0.43);  acid phosphatase activity (Rf 0.32-0.43);
actividad esterasa (Rf 0.53) variable,  esterase activity (Rf 0.53) variable,
actividad esterasa (Rf 0.57),  esterase activity (Rf 0.57),
actividad esterasa (Rf 0.63) débil y variable, actividad esterase (Rf 0.67) variable,  esterase activity (Rf 0.63) weak and variable, esterase activity (Rf 0.67) variable,
actividad esterasa (Rf 0.69) variable,  esterase activity (Rf 0.69) variable,
actividad esterasa (Rf 0.74) variable,  esterase activity (Rf 0.74) variable,
actividad esterasa (Rf 0.76),  esterase activity (Rf 0.76),
actividad esterasa (Rf 0.78) variable, y  esterase activity (Rf 0.78) variable, and
actividad esterasa (Rf 1.00).  esterase activity (Rf 1.00).
Tricboderma harzianua IMI 352941:  Tricboderma harzianua IMI 352941:
Posee los atributos morfológicos característicos de la especie Trichoderma harzianum Rifai, la conidiogénesis es enteroblástica fialídica, con células conidiógenas con una longitud superior a 10 mm; los conidios son subglobosos, con diámetro superior a 2,4 mm; y produce clamidosporas terminales con diámetro inferior a 10 mm.  It has the characteristic morphological attributes of the Trichoderma harzianum Rifai species, conidiogenesis is phialidic enteroblastic, with conidiogenic cells with a length greater than 10 mm; the conidia are subglobosous, with a diameter greater than 2.4 mm; and produces terminal clamidospores with a diameter of less than 10 mm.
De acuerdo con la metodología usada.  According to the methodology used.
crece muy bien en la superficie del medio, con esporulación variable, cuando se cultiva en glucosa 1% como fuente de carbono;  It grows very well on the surface of the medium, with variable sporulation, when grown in 1% glucose as a carbon source;
crece muy bien en la superficie del medio, con esporulación variable, cuando se cultiva en etanol 1% como fuente de carbono;  It grows very well on the surface of the medium, with variable sporulation, when grown in 1% ethanol as a carbon source;
crece en la superficie del medio, con buena esporulación, cuando se cultiva en ácido cítrico 1%;  grows on the surface of the medium, with good sporulation, when grown in 1% citric acid;
crece débilmente en la superficie del medio, y es capaz de esporular, cuando se cultiva en ácido láctico 1% como fuente de carbono;  it grows weakly on the surface of the medium, and is capable of sporulating, when grown in 1% lactic acid as a carbon source;
crece en la superficie del medio, con esporulación variable, cuando se cultiva en glicina 0.2% como fuente de nitrógeno; grows on the surface of the medium, with variable sporulation, when grown in 0.2% glycine as a source of nitrogen;
crece muy bien en la superficie del medio, con buena esporulación, cuando se cultiva en oxalato amónico 0.2% como fuente de nitrógeno;  It grows very well on the surface of the medium, with good sporulation, when grown in 0.2% ammonium oxalate as a source of nitrogen;
crece en la superficie del medio, con esporulación, cuando se cultiva en urea 0.2% como fuente de nitrógeno; no crece en la superficie del medio cuando se cultiva en creatina 0.2% como fuente de nitrógeno;  it grows on the surface of the medium, with sporulation, when it is grown in 0.2% urea as a source of nitrogen; it does not grow on the surface of the medium when grown in 0.2% creatine as a source of nitrogen;
crece a 4ºC;  grows at 4 ° C;
no crece en medios adicionados de cristal violeta a una concentración de 10mg/litro;  it does not grow in violet crystal added media at a concentration of 10mg / liter;
no esporula, pero crece, formando agrupaciones de hifas aéreas en forma de rosetón, cuando se cultiva en medios adicionados de selenito sódico a una concentración de 320mg/litro;  it does not sporulate, but it grows, forming groups of aerial hyphae in the form of a rosette, when grown in sodium selenite-added media at a concentration of 320mg / liter;
desarrolla colonias de diámetro inferior a 25mm en el medio agar extracto de malta adicionado de 200 mg/litro de sulfato de cobre II, después de 24 horas de incubación a 25°C;  develops colonies with a diameter of less than 25 mm in the agar medium added malt extract of 200 mg / liter of copper sulfate II, after 24 hours of incubation at 25 ° C;
hidroliza fuertemente la gelatina y esporula en ese mismo medio de cultivo;  strongly hydrolyzes the gelatin and sporula in that same culture medium;
crece y esporula bien en agar Czapek-amonio después de 10 días de incubación a 25ºC;  grows and sporulates well in Czapek-ammonium agar after 10 days of incubation at 25 ° C;
crece, con fuerte esporulación, en agar nitritosacarosa después de 10 días de incubación a 25⍜C;  it grows, with strong sporulation, on nitritic agarose agar after 10 days of incubation at 25⍜C;
las colonias no alcanzan un diámetro superior a 20mm en el medio agar glicerol-nitrato, después de 10 días de incubación a 25ºC.  the colonies do not reach a diameter greater than 20mm in the glycerol-nitrate agar medium, after 10 days of incubation at 25 ° C.
Produce las siguientes actividades enzimáticas extracelulares, medidas con el sistema API ZYM (Biomérieux S.A., Marcy-1'Etoile, Francia): fosfatasa alcalina, esterasa, leucina arilaminidasa, fosfatasa acida, naftol-AS-BI-fosfato, alfa-galactosidasa, beta-galactosidasa, alfa-glucosidasa, beta-glucosidasa, y N-acetil-beta-glucosaminidasa con intensidad máxima; esterasa lipasa, lipasa, valina arilaminidasa, cistina arilamldasa, tripsina, alfa-quimiotripsina, beta-glucuronidasa y alfa-manosidasa, con intensidad moderada; y no se detecta alfa-fucosidasa. It produces the following extracellular enzymatic activities, measured with the API ZYM system (Biomérieux SA, Marcy-1'Etoile, France): alkaline phosphatase, esterase, arylaminidase leucine, acid phosphatase, naphthol-AS-BI-phosphate, alpha-galactosidase, beta -galactosidase, alpha-glucosidase, beta-glucosidase, and N-acetyl-beta-glucosaminidase with maximum intensity; esterase lipase, lipase, valine arylaminidase, cystine arylamldasa, trypsin, alpha-chymotrypsin, beta-glucuronidase and alpha-mannosidase, with moderate intensity; and alpha-fucosidase is not detected.
De acuerdo con la metodología usada, presenta los siguientes bandas de isoenzimas, obtenidas mediante electroforesis en geles de poliacrilamida al 7.5% (10% para la actividad catalasa):  According to the methodology used, it presents the following isoenzyme bands, obtained by electrophoresis in 7.5% polyacrylamide gels (10% for catalase activity):
actividad fosfatasa alcalina (Rf 0.40-0.48);  alkaline phosphatase activity (Rf 0.40-0.48);
actividad catalasa (Rf 0.16-0.34); Catalan activity (Rf 0.16-0.34);
actividad fosfatasa acida (Rf 0.32-0.43) variable; actividad celulasa Rf 0.10-0.13),  acid phosphatase activity (Rf 0.32-0.43) variable; cellulase activity Rf 0.10-0.13),
actividad celulasa Rf 0.15-0.18),  cellulase activity Rf 0.15-0.18),
actividad celulasa Rf 0.28-0.33),  cellulase activity Rf 0.28-0.33),
actividad celulasa Rf 0.45-0.48);  cellulase activity Rf 0.45-0.48);
actividad esterasa Rf 0.25),  esterase activity Rf 0.25),
actividad esterasa Rf 0.31) débil,  esterase activity Rf 0.31) weak,
actividad esterasa Rf 0.51),  esterase activity Rf 0.51),
actividad esterasa Rf 0.53),  esterase activity Rf 0.53),
actividad esterasa Rf 0.55),  esterase activity Rf 0.55),
actividad esterasa Rf 0.62),  esterase activity Rf 0.62),
actividad esterasa Rf 0.65),  esterase activity Rf 0.65),
actividad esterasa Rf 0.78),  esterase activity Rf 0.78),
actividad esterasa Rf 0.82-0.84), y  esterase activity Rf 0.82-0.84), and
actividad esterasa ,Rf 1.00).  esterase activity, Rf 1.00).
Trlchoderma harzianum CECT 20179:  Trlchoderma harzianum CECT 20179:
Posee los atributos morfológicos característicos de la especie Trichoderma harzianum Rifai, la conidiogénesis es enteroblástica fialidica, con células conidiógenas con una longitud superior a 10 mm y anchura algo mayor de 3 mm; los conidios son subglobosos, con diámetro superior a 2,4 mm; y produce clamidosporas terminales e intercalares con diámetro superior a 10 mm.  It has the characteristic morphological attributes of the Trichoderma harzianum Rifai species, the conidiogenesis is phialidic enteroblastic, with conidiogenic cells with a length greater than 10 mm and a width slightly greater than 3 mm; the conidia are subglobosous, with a diameter greater than 2.4 mm; and produces terminal and intercalar clamidospores with a diameter greater than 10 mm.
De acuerdo con la metodología usada:  According to the methodology used:
crece muy bien en la superficie del medio, con esporulación variable, cuando se cultiva en glucosa 1% como fuente de carbono; grows very well on the surface of the medium, with variable sporulation, when grown in 1% glucose as a carbon source;
crece muy bien en la superficie del medio, con esporulación débil, cuando se cultiva en etanol 1% como fuente da carbono;  It grows very well on the surface of the medium, with weak sporulation, when grown in 1% ethanol as a carbon source;
crece en la superficie del medio, con buena esporulación, cuando se cultiva en ácido cítrico 1% como fuente de carbono;  it grows on the surface of the medium, with good sporulation, when grown in 1% citric acid as a carbon source;
crece débilmente en la superficie del medio, y es capaz de esporular, cuando se cultiva en ácido láctico 1% como fuente de carbono;  it grows weakly on the surface of the medium, and is capable of sporulating, when grown in 1% lactic acid as a carbon source;
crece muy bien en la superficie del medio, con esporulación variable, cuando se cultiva en glicina 0.2% como fuente de nitrógeno;  It grows very well on the surface of the medium, with variable sporulation, when grown in 0.2% glycine as a source of nitrogen;
crece muy bien en la superficie del medio, con buena esporulación, cuando se cultiva en oxalato amónico 0.2% como fuente de nitrógeno;  It grows very well on the surface of the medium, with good sporulation, when grown in 0.2% ammonium oxalate as a source of nitrogen;
crece en la superficie del medio, con esporulación débil, cuando se cultiva en urea 0.2% como fuente de nitrógeno;  it grows on the surface of the medium, with weak sporulation, when grown in 0.2% urea as a source of nitrogen;
no crece en la superficie del medio cuando se cultiva en creatina 0.2% como fuente de nitrógeno;  it does not grow on the surface of the medium when grown in 0.2% creatine as a source of nitrogen;
no crece a 4⍜C;  does not grow at 4⍜C;
no crece en medios adicionados de cristal violeta a una concentración de 10mg/litro;  it does not grow in violet crystal added media at a concentration of 10mg / liter;
no esporula, pero crece en medios adicionados de selenito sódico a una concentración de 320mg/litro;  it does not sporulate, but it grows in media added sodium selenite at a concentration of 320mg / liter;
desarrolla colonias de diámetro superior a 25mm y normalmente inferior a 31mm, en el medio agar extracto de malta adicionado de 200 mg/litro de sulfato de cobre II, después de 24 horas de incubación a 25⍜C;  develops colonies with a diameter greater than 25mm and normally less than 31mm, in the agar medium added malt extract of 200 mg / liter of copper sulfate II, after 24 hours of incubation at 25⍜C;
no hidroliza la gelatina, pero esporula en ese mismo medio de cultivo;  it does not hydrolyse gelatin, but sporulates in that same culture medium;
crece y esporula muy bien en agar Czapek-amonio después de 10 días de incubación a 25⍜C; crece muy bien, con fuerte esporulación, en agar nitrito-sacarosa después de 10 días de incubación a 25⍜C; las colonias no alcanzan un diámetro superior a 20mm en el medio agar glicerol-nitrato, después de 10 días de incubación a 25⍜C. grows and sporulates very well in Czapek-ammonium agar after 10 days of incubation at 25⍜C; it grows very well, with strong sporulation, in nitrite-sucrose agar after 10 days of incubation at 25⍜C; the colonies do not reach a diameter greater than 20mm in the glycerol-nitrate agar medium, after 10 days of incubation at 25⍜C.
Produce las siguientes actividades enzimáticas extracelulares, medidas con el sistema API ZYM (Biomérieux S.A., Marcy-1'Etoile, Francia): fosfatasa alcalina, leucina arilaminidasa y naftol-AS-BI-fosfatoesterasa con intensidad máxima; esterasa lipasa, lipasa, valina arilaminidasa, cistina arilamidasa, fosfatasa acida, alfa-galactosidasa, beta-galactosidasa, alfa-glucosidasa, beta-glucosidasa, N-acetil-beta-glucosaminidasa y alfa-manosidasa con intensidad moderada; y no se detectan esterasa, tripsina, alfa-quimiotripsina, beta-glucuronidasa y alfa-fucosidasa.  It produces the following extracellular enzymatic activities, measured with the API ZYM system (Biomérieux S.A., Marcy-1'Etoile, France): alkaline phosphatase, arylaminidase leucine and naphthol-AS-BI-phosphate esterase with maximum intensity; esterase lipase, lipase, arylaminidase valine, cystine arylamidase, acid phosphatase, alpha-galactosidase, beta-galactosidase, alpha-glucosidase, beta-glucosidase, N-acetyl-beta-glucosaminidase and alpha-mannosidase with moderate intensity; and esterase, trypsin, alpha-chymotrypsin, beta-glucuronidase and alpha-fucosidase are not detected.
De acuerdo con la metodología usada, presenta las siguientes bandas de isoenzimas, obtenidas mediante electroforesis en geles de poliacrilamida al 7.5% (10% para la actividad catalasa):  According to the methodology used, it presents the following isoenzyme bands, obtained by electrophoresis in 7.5% polyacrylamide gels (10% for catalase activity):
actividad catalasa (Rf 0.20-0.30),  Catalan activity (Rf 0.20-0.30),
actividad catalasa (Rf 0.26-0.34);  Catalan activity (Rf 0.26-0.34);
actividad fosfatasa acida (Rf 0.10),  acid phosphatase activity (Rf 0.10),
actividad fosfatasa acida (Rf 0.28-0.43);  acid phosphatase activity (Rf 0.28-0.43);
actividad malato deshidrogenasa (Rf 0.01),  malate dehydrogenase activity (Rf 0.01),
actividad malato deshidrogenasa (Rf 0.16-0.17), actividad malato deshidrogenasa (Rf 0.20-0.24), actividad malato deshidrogenasa (Rf 0.30-0.40); actividad celulasa (Rf 0.28-0.33),  malate dehydrogenase activity (Rf 0.16-0.17), malate dehydrogenase activity (Rf 0.20-0.24), malate dehydrogenase activity (Rf 0.30-0.40); cellulase activity (Rf 0.28-0.33),
actividad celulasa (Rf 0.40) variable,  variable cellulase activity (Rf 0.40),
actividad celulasa (Rf 0.42-0.54) variable, actividad celulasa (Rf 0.69-0.82);  variable cellulase activity (Rf 0.42-0.54), cellulase activity (Rf 0.69-0.82);
actividad esterasa (Rf 0.44),  esterase activity (Rf 0.44),
actividad esterasa (Rf 0.50),  esterase activity (Rf 0.50),
actividad esterasa (Rf 0.72) débil, actividad esterasa (Rf 0.75) débil, weak esterase activity (Rf 0.72), weak esterase activity (Rf 0.75),
actividad esterasa (Rf 0.77),  esterase activity (Rf 0.77),
actividad esterase (Rf 0.82-0.84),  esterase activity (Rf 0.82-0.84),
actividad esterasa (Rf 0.87)  esterase activity (Rf 0.87)
actividad esterasa (Rf 0.92-0.95).  esterase activity (Rf 0.92-0.95).
Trichoderma viride CECT 20178:  Trichoderma viride CECT 20178:
Posee los atributos morfológicos característicos de la especie Trichoderma viride Pers.:Fr., la conidiogénesis es enteroblástica fialídica, con células conidiógenas con una longitud inferior a 10 mm y anchure inferior a 3 mm; los conidios son ovales, con superficie rugosa, con diámetro superior a 2,4 mm; y produce clamidosporas terminales e intercalares con diámetro superior a 10 mm.  It has the characteristic morphological attributes of the species Trichoderma viride Pers.:Fr., The conidiogenesis is phialidical enteroblastic, with conidiogenic cells with a length less than 10 mm and an brochure less than 3 mm; the conidia are oval, with a rough surface, with a diameter greater than 2.4 mm; and produces terminal and intercalar clamidospores with a diameter greater than 10 mm.
De acuerdo con la metodología usada:  According to the methodology used:
crece muy bien en la superficie del medio, con esporulación abundante, cuando se cultiva en glucosa 1% como fuente de carbono;  It grows very well on the surface of the medium, with abundant sporulation, when grown in 1% glucose as a carbon source;
crece muy bien en la superficie del medio, con esporulación muy abundante, cuando se cultiva en etanol 1% como fuente de carbono;  It grows very well on the surface of the medium, with very abundant sporulation, when grown in 1% ethanol as a carbon source;
crece en la superficie del medio, con esporulación variable, cuando se cultiva en ácido cítrico 1% como fuente de carbono;  it grows on the surface of the medium, with variable sporulation, when grown in 1% citric acid as a carbon source;
crece muy bien en la superficie del medio, y es capaz de esporular débilmente, cuando se cultiva en ácido láctico 1% como fuente de carbono;  It grows very well on the surface of the medium, and is able to sporulate weakly, when grown in 1% lactic acid as a carbon source;
crece en la superficie del medio, pero no esporula, cuando se cultiva en oxalato amónico 1% como fuente de carbono;  grows on the surface of the medium, but does not sporulate, when grown in 1% ammonium oxalate as a carbon source;
crece de forma variable en la superficie del medio, con esporulación también variable, cuando se cultiva en nitrito sódico 0.2% como fuente de nitrógeno;  it grows variably on the surface of the medium, with variable sporulation, when grown in 0.2% sodium nitrite as a source of nitrogen;
crece muy bien en la superficie del medio, con buena esporulación, cuando se cultiva en glicina 0.2% como fuente de nitrógeno; It grows very well on the surface of the medium, with good sporulation, when grown in 0.2% glycine as nitrogen source;
crece de forma variable en la superficie del medio, con buene esporuleción, cuendo se cultive en oxeleto amónico 0.2% como fuente tía nitrógeno;  it grows variably on the surface of the medium, with good sporuleation, when grown in 0.2% ammonium oxeleto as a source of nitrogen aunt;
crece en la superficie del medio, con buena esporulación, cuando se cultiva en urea 0.2% como fuente de nitrógeno;  it grows on the surface of the medium, with good sporulation, when it is grown in 0.2% urea as a source of nitrogen;
crece en la superficie del medio y esporula cuando se cultiva en creatina 0.2% como fuente de nitrógeno; no crece a 4⍜C;  it grows on the surface of the medium and sporula when 0.2% creatine is grown as a source of nitrogen; does not grow at 4⍜C;
crece bien en medios adicionados de cristal violeta e une concentración de 10mg/litro, y débilmente en medios adicionados de cristal violeta a una concentración de 50mg/litro;  it grows well in violet crystal added media and a concentration of 10mg / liter, and weakly in violet crystal added media at a concentration of 50mg / liter;
no esporula, pero crece, formando agrupaciones de hifes eérees en forma de rosetón, en medios adicionados de selenito sódico a una concentración de 320mg/litro; desarrolla colonias con un diámetro 21% inferior al del testigo sin agente inhibidor, en el medio agar extracto de malta adicionado de 200 mg/litro de sulfato de cobre II, después de 24 horas de incubación a 25⍜C; desarrolle colonies con un diámetro superior a 65 mm, en el medio agar extracto de malta adicionado de 200 mg/litro de sulfato de cobre II, después de 14 días de incubación a 25⍜C;  it does not sporulate, but it grows, forming groups of erectheifes in the form of a rosette, in added media of sodium selenite at a concentration of 320mg / liter; develops colonies with a diameter 21% smaller than the control without inhibitory agent, in the agar medium added malt extract of 200 mg / liter of copper sulfate II, after 24 hours of incubation at 25 aC; develop colonies with a diameter greater than 65 mm, in the agar medium added malt extract of 200 mg / liter of copper sulfate II, after 14 days of incubation at 25⍜C;
hidroliza le geletina, con esporulación abundante en ese mismo medio de cultivo;  hydrolyzes geletine, with abundant sporulation in the same culture medium;
crece, con micelio aéreo abundente, y esporula muy bien en ager Czapek-amonio después de 10 días de incubación a 25⍜C;  it grows, with abundant aerial mycelium, and sporulates very well in Czapek-ammonium ager after 10 days of incubation at 25⍜C;
crece bien y esporula en agar nitrito-sacarosa después de 10 días de incubación a 25⍜C;  It grows well and sporulates in nitrite-sucrose agar after 10 days of incubation at 25⍜C;
crece bien en el medio agar glicerol-nitrato, después de 10 días de incubación a 25⍜C.  It grows well in the glycerol-nitrate agar medium, after 10 days of incubation at 25⍜C.
Produce las siguientes actividades enzimáticas extracelulares, medidos con el sisteme API ZYM (Biomérieux S.A., Marcy-1'Etoile, Francia): fosfatasa alcalina, esterasa lipasa, valina arilaminidase y alfa-glucosidasa, con intensided máxime; leucine erilaminidasa, naftol-AS-BI-fosfetoesterasa, cistine erilamidase, fosfetese acida, elfa-gelactosidase, beta-glucosidasa, N-acetil-beta-glucosaminidasa y alfa- menosidasa con intensidad moderede; y no se detecten lipese, tripsina, alfa-quimiotripsine, beta-gelactosidasa, bete-glucuronidase y elfa-fucosidasa. b) Característices moleculares: It produces the following enzymatic activities extracellular, measured with the API ZYM system (Biomérieux SA, Marcy-1'Etoile, France): alkaline phosphatase, esterase lipase, arylaminidase valine and alpha-glucosidase, with an intensified maximum; leucine erilaminidase, naphthol-AS-BI-phosphetosterase, cistine erilamidase, acid phosphetese, elfa-gelactosidase, beta-glucosidase, N-acetyl-beta-glucosaminidase and alpha-lessidase with moderate intensity; and no lipese, trypsin, alpha-chymotrypsin, beta-gelactosidase, bete-glucuronidase and elfa-fucosidase are detected. b) Molecular characteristics:
Los microorganismos utilizados en le formulación objeto de pétente son cepas sin ediciones ni deleciones de DNA, que hen sido eislados de la Naturaleze y su genome no he sido modificedo. El hecho de que sean cepas natureles dificulte su cerecterizeción al no existir DNA exógeno que sirve de mercador de cada una de ellas.  The microorganisms used in the formulation object of patent are strains without editions or deletions of DNA, which have been isolated from Naturaleze and its genome has not been modified. The fact that they are natural strains hinders their cerecterization because there is no exogenous DNA that serves as a merchant for each of them.
Dado que se trate de microorganismos fenotípicamente muy parecidos, y que sus atributos morfológicos, fisiológicos y bioquímicos pueden ser difíciles de valorar, hemos recurrido a le búsquede, dentro del genome de estos microorganismos, de marcedores moleculares que fueran estables, a nivel de especie y de cepa, pare poder cerecterizer e identificer los eislemientos utilizedos en le formulación.  Given that they are phenotypically similar microorganisms, and that their morphological, physiological and biochemical attributes may be difficult to assess, we have resorted to the search, within the genome of these microorganisms, of molecular markers that were stable, at the species level and strain, to be able to cerecterizer and identify the uses used in the formulation.
Nos fijamos en la región ITS1-ITS4, que engloba el gen que codifica el RNAr 5.8 S, y sintetizamos cebadores para amplificarla mediante reacción en cadene de le polimerase ( PCR).  We look at the ITS1-ITS4 region, which encompasses the gene encoding the 5.8 S RNAr, and synthesize primers to amplify it by means of a polymerase chain reaction (PCR).
Se emplificó el gen 5.8S rRNA con les regiones adyacentes al mismo, usando los cebadores ITS1 e ITS 4, mediante PCR, obteniéndose fragmentos de aproximedemente 0,6 kbp, pare les cepes de T. harzianum IMI 352939, IMI 352940 e IMI 352941, según se observó mediante electroforesis en gel de agerose, al 1%. El gen 5.8S rRNA (nucleótidos del 212 al 358) posea une gran semejanze con otros hongos filementosos, y, por tento, entre les 3 cepes de T. harzianum . Las diferencias se encontraron en las regiones adyacentes al gen, fundamentalmente en las secuencias comprendidas entre los nucleótidos 100 y 200, y entre los nucleótidos 408 y 440. The 5.8S rRNA gene was used with the regions adjacent to it, using primers ITS1 and ITS 4, by PCR, obtaining fragments of approximately 0.6 kbp, for cepes of T. harzianum IMI 352939, IMI 352940 and IMI 352941, as observed by agerose gel electrophoresis, 1%. The 5.8S rRNA gene (nucleotides from 212 to 358) has a great similarity with other filementous fungi, and, by temptation, among the 3 strains of T. harzianum. The differences were found in the regions adjacent to the gene, mainly in the sequences between nucleotides 100 and 200, and between nucleotides 408 and 440.
Las secuencias característices obtenidas para las tres cepas de T. harzianum se indican a continuación:  The characteristic sequences obtained for the three strains of T. harzianum are indicated below:
Tr±choderma harzianvm IMI 352939 (región ITS1-ITS4 del gen 5.8S rRNA ) :  Tr ± choderma harzianvm IMI 352939 (ITS1-ITS4 region of the 5.8S rRNA gene):
568 nucleótidos:  568 nucleotides:
Figure imgf000028_0002
Figure imgf000028_0002
Trichodenaa harzianum IMI 352940 ( región ITS1 - ITS4 del gen 5. 8S rRNA ) : Trichodenaa harzianum IMI 352940 (ITS1 - ITS4 region of gene 5. 8S rRNA):
582 nucleótidos :  582 nucleotides:
Figure imgf000028_0001
Tríchoderma harzianum IMI 352941 (región ITS1-ITS4 del gen 5.8S rRNA ) :
Figure imgf000028_0001
Tríchoderma harzianum IMI 352941 (ITS1-ITS4 region of the 5.8S rRNA gene):
568 nucleótidos:  568 nucleotides:
Figure imgf000029_0001
Figure imgf000029_0001
Las secuencias correspondientes a las cepas Trichoderma harzianum IMI 352939 y Trichoderma harzianum IMI 352941, han resultado ser idénticas para este gen. Por este motivo no se han secuenciado las regiones ITS1-ITS4 del gen 5.8S rRNA de las cepas Trichoderma harzianum CECT 20179 y Trichoderma viride CECT 20178. No obstante, se están buscando nuevos atributos moleculares que permitan identificar cada una de estas cepas sin la necesidad de recurrir a marcadores exógenos.  The sequences corresponding to the Trichoderma harzianum IMI 352939 and Trichoderma harzianum IMI 352941 strains have been identical for this gene. For this reason, the ITS1-ITS4 regions of the 5.8S rRNA gene of the Trichoderma harzianum CECT 20179 and Trichoderma viride CECT 20178 strains have not been sequenced. However, new molecular attributes are being sought to identify each of these strains without the need of resorting to exogenous markers.

Claims

REIVINDICACIONES
1.- Una formulación liquida a base de cepas de los hongos filamentosos Trichoderma harzianum y Trichoderma viride, caracterizada porque presenta la siguiente composición cuentitativa en % p/v:  1.- A liquid formulation based on strains of filamentous fungi Trichoderma harzianum and Trichoderma viride, characterized in that it has the following quantitative composition in% w / v:
Figure imgf000030_0001
Figure imgf000030_0001
Componentes biológicos: al menos una cepa elegida del grupo consistente en:  Biological components: at least one strain chosen from the group consisting of:
Trichoderma harzianum IMI 352939 c.s.p. 3×105-10×107 conidios/ml, Trichoderma harzianum IMI 352939 csp 3 × 10 5 -10 × 10 7 conidia / ml,
Trichoderma harzianum IMI 352940 c.s.p. 3×105-10×107 conidios/ml, Trichoderma harzianum IMI 352940 csp 3 × 10 5 -10 × 10 7 conidia / ml,
Trichoderma harzianum IMI 352941 c.s.p. 3×105-10×107 conidios/ml Trichoderma harzianum IMI 352941 csp 3 × 10 5 -10 × 10 7 conidia / ml
Trichoderma harzianum CECT 20179  Trichoderma harzianum CECT 20179
Trichoderma viride CECT 20178 y  Trichoderma viride CECT 20178 and
mezclas de tales cepas.  mixtures of such strains.
2.- El uso de Trichoderma herzienum IMI 352939, Trichoderme harzianum IMI 352940 y Trichoderma harzienum IMI 352941, Trichoderme harzianum CECT 20179 y Trichoderma viride CECT 20178 solos o mezclados, como agentes de control biológico de los enemigos de las plantas y material vegetal, y entidades biológicas causantes de biodeterioro, así como agentes de biolixiviación. 2.- The use of Trichoderma herzienum IMI 352939, Trichoderme harzianum IMI 352940 and Trichoderma harzienum IMI 352941, Trichoderme harzianum CECT 20179 and Trichoderma viride CECT 20178 alone or mixed, as biological control agents of the enemies of plants and plant material, and biological entities causing biodeterioration, as well as bioleaching agents.
3.- El uso de Trichoderma harzianum IMI 352939,3.- The use of Trichoderma harzianum IMI 352939,
Trichoderma harzianum IMI 352940 y Trichoderma harzianum IMI 352941, Trichoderma harzianum CECT 20179 c.s.p.Trichoderma harzianum IMI 352940 and Trichoderma harzianum IMI 352941, Trichoderma harzianum CECT 20179 c.s.p.
S×105-10×107 conidios/ml y Trichoderme viride CECT 20178 c.s.p. 3×105-10×107 conidios/ml y solos o mezclados, como genoma receptor de genes que incrementen su ectivided como agentes de control biológico de los enemigos de les plantas y material vegetal, y entidades biológicas causantes de biodeterioro, así como agentes de biolixiviación. S × 10 5 -10 × 10 7 conidia / ml and Trichoderme viride CECT 20178 csp 3 × 10 5 -10 × 10 7 conidia / ml and alone or mixed, as a genome receiving genes that increase its ectivided as biological control agents of the enemies of plants and plant material, and biological entities causing biodeterioration, as well as bioleaching agents.
PCT/ES1996/000206 1995-11-07 1996-11-06 Liquid formulation based on strains of the stringy fungi trichoderma harzianum and trichoderma viride WO1997016974A1 (en)

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004054365A2 (en) * 2002-12-13 2004-07-01 A.M.C. Chemical S.L. Biological control agent and formulations
CN1302710C (en) * 2004-01-18 2007-03-07 沈阳农业大学 Multifunctional biological control bacteria agent and its preparation method
WO2010009241A2 (en) * 2008-07-17 2010-01-21 Bioworks, Inc. Control of plant diseases and enhancing plant growth using a combination of a trichoderma virens species and a rhizosphere competent trichoderma harzianum species
CN102726454A (en) * 2012-07-16 2012-10-17 陕西省科学院酶工程研究所 Biological control agent for fusarium wilt and preparation and use method of biological control agent
US9578873B2 (en) 2011-10-11 2017-02-28 Universidad Nacional De Quilmes Methods for controlling leaf-cutting ants
WO2019067379A1 (en) * 2017-09-28 2019-04-04 Locus Agriculture Ip Company, Llc Large scale production of liquid and solid trichoderma products
WO2019103588A1 (en) 2017-11-24 2019-05-31 Université Ibn Tofail Production, formulation and recycling of a biofungicidal and biostimulant product based on trichoderma asperellum
US11172669B2 (en) 2016-11-16 2021-11-16 Locus Agriculture Ip Company, Llc Materials and methods for the control of nematodes
US11447430B2 (en) 2018-05-08 2022-09-20 Locus Agriculture Ip Company, Llc Microbe-based products for enhancing plant root and immune health
US11758924B2 (en) 2019-04-12 2023-09-19 Locus Solutions Ipco, Llc Pasture treatments for enhanced carbon sequestration and reduction in livestock-produced greenhouse gas emissions
US11963528B2 (en) 2018-02-26 2024-04-23 Locus Solutions Ipco, Llc Materials and methods for control of insect pests using entomopathogenic fungi

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2200602B1 (en) * 2000-04-28 2005-04-01 Universidad De Salamanca COMPOSITION THAT INCLUDES FUNDS OF THE GENUINE USEFUL TRICHODERMA AS A BIOLOGICAL CONTROL AGENT AND ITS APPLICATIONS.

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2345921A1 (en) * 1976-03-30 1977-10-28 Seppic Sa Use of Trichoderma viride suspensions - to treat vines and mushrooms infested with parasitic fungi
US4293333A (en) * 1980-02-12 1981-10-06 Engelhard Minerals & Chemicals Corporation Microbiological recovery of metals
EP0124388A1 (en) * 1983-04-28 1984-11-07 Les Produits Organiques Du Santerre Orsan Strain of Trichoderma harzianum, process for its isolation, process for its culture, peptides or compounds produced by this strain and application of this strain and these peptides or the product produced by the culture process as a means for biological control in the form of a phytosanitary product
EP0285987A2 (en) * 1987-04-03 1988-10-12 Cornell Research Foundation, Inc. Fused biocontrol agents
US4915944A (en) * 1983-03-15 1990-04-10 Yissum Research Development Corp. Of The Hebrew University Of Jerusalem Novel isolate of trichoderma, fungicidal compositions containing said isolate and use thereof
US5266316A (en) * 1990-07-12 1993-11-30 Peri Development Applications (1985) Ltd. Isolate of Trichoderma harzianum I-952 fungicidal compositions containing said isolate and use against B. cinerea and S. sclerotiorum
US5288634A (en) * 1990-08-03 1994-02-22 Cornell Research Foundation, Inc. Method of increasing the percentage of viable dried spores of a fungus

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2345921A1 (en) * 1976-03-30 1977-10-28 Seppic Sa Use of Trichoderma viride suspensions - to treat vines and mushrooms infested with parasitic fungi
US4293333A (en) * 1980-02-12 1981-10-06 Engelhard Minerals & Chemicals Corporation Microbiological recovery of metals
US4915944A (en) * 1983-03-15 1990-04-10 Yissum Research Development Corp. Of The Hebrew University Of Jerusalem Novel isolate of trichoderma, fungicidal compositions containing said isolate and use thereof
EP0124388A1 (en) * 1983-04-28 1984-11-07 Les Produits Organiques Du Santerre Orsan Strain of Trichoderma harzianum, process for its isolation, process for its culture, peptides or compounds produced by this strain and application of this strain and these peptides or the product produced by the culture process as a means for biological control in the form of a phytosanitary product
EP0285987A2 (en) * 1987-04-03 1988-10-12 Cornell Research Foundation, Inc. Fused biocontrol agents
US5266316A (en) * 1990-07-12 1993-11-30 Peri Development Applications (1985) Ltd. Isolate of Trichoderma harzianum I-952 fungicidal compositions containing said isolate and use against B. cinerea and S. sclerotiorum
US5288634A (en) * 1990-08-03 1994-02-22 Cornell Research Foundation, Inc. Method of increasing the percentage of viable dried spores of a fungus

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
G.C. PAPAVIZAS ET AL.: "Effect of Gliocladium and Trichoderma on damping -off and blight of snapbean caused by Sclerotium rolfsii in the greenhouse.", PLANT PATHOLOGY, vol. 38, 1989, pages 277 - 286, XP002024059 *
L MIHUTA-GRIMM ET AL.: "Trichoderma spp. as biocontrol agents of rhizoctonia damping-off radish in organic soil and comparison of four delivery systems.", PHYTOPATHOLOGY, vol. 76, no. 3, 1986, pages 306 - 312, XP002024058 *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004054365A3 (en) * 2002-12-13 2004-12-02 A M C Chemical S L Biological control agent and formulations
WO2004054365A2 (en) * 2002-12-13 2004-07-01 A.M.C. Chemical S.L. Biological control agent and formulations
CN1302710C (en) * 2004-01-18 2007-03-07 沈阳农业大学 Multifunctional biological control bacteria agent and its preparation method
WO2010009241A2 (en) * 2008-07-17 2010-01-21 Bioworks, Inc. Control of plant diseases and enhancing plant growth using a combination of a trichoderma virens species and a rhizosphere competent trichoderma harzianum species
WO2010009241A3 (en) * 2008-07-17 2011-02-10 Bioworks, Inc. Control of plant diseases and enhancing plant growth using a combination of a trichoderma virens species and a rhizosphere competent trichoderma harzianum species
US9681668B2 (en) 2008-07-17 2017-06-20 Bioworks, Inc. Control of plant diseases and enhancing plant growth using a combination of a Trichoderma virens species and a rhizosphere competent Trichoderma harzianum species
US9578873B2 (en) 2011-10-11 2017-02-28 Universidad Nacional De Quilmes Methods for controlling leaf-cutting ants
CN102726454A (en) * 2012-07-16 2012-10-17 陕西省科学院酶工程研究所 Biological control agent for fusarium wilt and preparation and use method of biological control agent
US11172669B2 (en) 2016-11-16 2021-11-16 Locus Agriculture Ip Company, Llc Materials and methods for the control of nematodes
US11825827B2 (en) 2016-11-16 2023-11-28 Locus Solutions Ipco, Llc Materials and methods for the control of nematodes
WO2019067379A1 (en) * 2017-09-28 2019-04-04 Locus Agriculture Ip Company, Llc Large scale production of liquid and solid trichoderma products
US11286456B2 (en) 2017-09-28 2022-03-29 Locus Agriculture Ip Company, Llc Large scale production of liquid and solid trichoderma products
WO2019103588A1 (en) 2017-11-24 2019-05-31 Université Ibn Tofail Production, formulation and recycling of a biofungicidal and biostimulant product based on trichoderma asperellum
US11963528B2 (en) 2018-02-26 2024-04-23 Locus Solutions Ipco, Llc Materials and methods for control of insect pests using entomopathogenic fungi
US11447430B2 (en) 2018-05-08 2022-09-20 Locus Agriculture Ip Company, Llc Microbe-based products for enhancing plant root and immune health
US11758924B2 (en) 2019-04-12 2023-09-19 Locus Solutions Ipco, Llc Pasture treatments for enhanced carbon sequestration and reduction in livestock-produced greenhouse gas emissions

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