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WO1997016974A1 - Formulation liquide a base de souches des champignons filamenteux trichoderma harzianum et trichoderma viride - Google Patents

Formulation liquide a base de souches des champignons filamenteux trichoderma harzianum et 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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WIPO (PCT)
Prior art keywords
trichoderma
harzianum
imi
trichoderma harzianum
grows
Prior art date
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Ceased
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PCT/ES1996/000206
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English (en)
Spanish (es)
Inventor
Enrique Monte Vazquez
Isabel Grondona España
Isabel Garcia Ancha
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Consejo Superior de Investigaciones Cientificas CSIC
Universidad de Salamanca
Original Assignee
Consejo Superior de Investigaciones Cientificas CSIC
Universidad de Salamanca
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Publication of WO1997016974A1 publication Critical patent/WO1997016974A1/fr
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Ceased legal-status Critical Current

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Classifications

    • 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

Formulation liquide à base de souches de champignons filamenteux Trichoderma harzianum et Trichoderma viride. La formulation comprend des sources de carbone, des sels minéraux et des traces d'éléments avec substances nutritives organiques et inorganiques, et des structures végétatives et reproductrices de souches du champignon filamenteux Trichoderma harzianum Rifai qui ont reçu l'identification Trichoderma harzianum IMI 352940, Trichoderma harzianum IMI 352941, et Trichoderma harzianum CECT 20179; et Trichoderma viride Pers.: Fr qui a reçu l'identification Trichoderma viride CECT 20178. La formulation est utile comme agent de régulation biologique pour combattre les maladies de végétaux et de matériau végétal ainsi que la biodétérioration produites ou transmises par des champignons filamenteux et autres ennemis des plantes, et comme agent de biolixiviation.
PCT/ES1996/000206 1995-11-07 1996-11-06 Formulation liquide a base de souches des champignons filamenteux trichoderma harzianum et trichoderma viride Ceased WO1997016974A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ES09502266A ES2109182B1 (es) 1995-11-07 1995-11-07 Formulacion liquida a base de cepas de los hongos filamentosos trichoderma harzianum y trichoderma viride.
ESP9502266 1995-11-07

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WO1997016974A1 true WO1997016974A1 (fr) 1997-05-15

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004054365A3 (fr) * 2002-12-13 2004-12-02 A M C Chemical S L Agent de lutte biologique et compositions
CN1302710C (zh) * 2004-01-18 2007-03-07 沈阳农业大学 多功能生防菌剂及其制备方法
WO2010009241A3 (fr) * 2008-07-17 2011-02-10 Bioworks, Inc. Contrôle de maladies de plante et amélioration de la croissance de plante utilisant une combinaison d'une espèce de trichoderma virens et d'une espèce de trichoderma harzianum ayant une compétence rhizosphérique
CN102726454A (zh) * 2012-07-16 2012-10-17 陕西省科学院酶工程研究所 用于枯萎病的生物防治剂及其制备和使用方法
US9578873B2 (en) 2011-10-11 2017-02-28 Universidad Nacional De Quilmes Methods for controlling leaf-cutting ants
WO2019067379A1 (fr) * 2017-09-28 2019-04-04 Locus Agriculture Ip Company, Llc Production à grande échelle de produits liquides et solides à base de trichoderma
WO2019103588A1 (fr) 2017-11-24 2019-05-31 Université Ibn Tofail Production, formulation et recyclage d' un produit biofongicide et biostimulant à base de 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

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ES2200602B1 (es) * 2000-04-28 2005-04-01 Universidad De Salamanca Composicion que comprende hongos del genero trichoderma util como agente de control biologico y sus aplicaciones.

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US4293333A (en) * 1980-02-12 1981-10-06 Engelhard Minerals & Chemicals Corporation Microbiological recovery of metals
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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 (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004054365A3 (fr) * 2002-12-13 2004-12-02 A M C Chemical S L Agent de lutte biologique et compositions
CN1302710C (zh) * 2004-01-18 2007-03-07 沈阳农业大学 多功能生防菌剂及其制备方法
WO2010009241A3 (fr) * 2008-07-17 2011-02-10 Bioworks, Inc. Contrôle de maladies de plante et amélioration de la croissance de plante utilisant une combinaison d'une espèce de trichoderma virens et d'une espèce de trichoderma harzianum ayant une compétence rhizosphérique
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 (zh) * 2012-07-16 2012-10-17 陕西省科学院酶工程研究所 用于枯萎病的生物防治剂及其制备和使用方法
US11825827B2 (en) 2016-11-16 2023-11-28 Locus Solutions Ipco, Llc Materials and methods for the control of nematodes
US12465041B2 (en) 2016-11-16 2025-11-11 Locus Solutions Ipco, Llc Materials and methods for the control of nematodes
US11172669B2 (en) 2016-11-16 2021-11-16 Locus Agriculture Ip Company, Llc Materials and methods for the control of nematodes
WO2019067379A1 (fr) * 2017-09-28 2019-04-04 Locus Agriculture Ip Company, Llc Production à grande échelle de produits liquides et solides à base de trichoderma
US11286456B2 (en) 2017-09-28 2022-03-29 Locus Agriculture Ip Company, Llc Large scale production of liquid and solid trichoderma products
WO2019103588A1 (fr) 2017-11-24 2019-05-31 Université Ibn Tofail Production, formulation et recyclage d' un produit biofongicide et biostimulant à base de 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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