WO2005068609A1 - Trichoderma harzianum clones, method for isolating and culturing and for the use thereof in the form of a phytosanitary product - Google Patents
Trichoderma harzianum clones, method for isolating and culturing and for the use thereof in the form of a phytosanitary product Download PDFInfo
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- WO2005068609A1 WO2005068609A1 PCT/FR2005/000008 FR2005000008W WO2005068609A1 WO 2005068609 A1 WO2005068609 A1 WO 2005068609A1 FR 2005000008 W FR2005000008 W FR 2005000008W WO 2005068609 A1 WO2005068609 A1 WO 2005068609A1
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- Prior art keywords
- clones
- trichoderma harzianum
- spores
- growth
- medium
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Links
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- LKPLKUMXSAEKID-UHFFFAOYSA-N pentachloronitrobenzene Chemical compound [O-][N+](=O)C1=C(Cl)C(Cl)=C(Cl)C(Cl)=C1Cl LKPLKUMXSAEKID-UHFFFAOYSA-N 0.000 description 1
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- AZJPTIGZZTZIDR-UHFFFAOYSA-L rose bengal Chemical compound [K+].[K+].[O-]C(=O)C1=C(Cl)C(Cl)=C(Cl)C(Cl)=C1C1=C2C=C(I)C(=O)C(I)=C2OC2=C(I)C([O-])=C(I)C=C21 AZJPTIGZZTZIDR-UHFFFAOYSA-L 0.000 description 1
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- JXKPEJDQGNYQSM-UHFFFAOYSA-M sodium propionate Chemical compound [Na+].CCC([O-])=O JXKPEJDQGNYQSM-UHFFFAOYSA-M 0.000 description 1
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Classifications
-
- 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
- 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
-
- 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
- C12N3/00—Spore forming or isolating processes
-
- 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
- the present invention essentially relates to clones of Trichoderma harzianum, a process for the isolation and culture of these clones and the application of these as phytosanitary products which can be used in the context of the biological control of the fungi responsible for diseases of the wood of the vine: Esca (Phaeoacremonium aleophilum, Phaeomoniella chiamydospora, Fomitiporia punctata, Phellinus igniarius, Phellinus viticola and Eutypa lata), Eutypiose (Eutypa lata and Botryospheria spp), Excoriosis (Phomopsis viticola bot and sherry berry and Phytospheria viticola bot sherry bot and sherry bot) obtusa), Black Dead Arm (Botryospheria dothidea), Transplant-welded symptoms (Botryospheria Stevensii).
- Trichoderma harzianum (Classification of Rifai), isolated by the depositor, were deposited with the National Collection of Cultures of Microorganisms (CNCM) of the Pasteur Institute on May 4, 1995 under number 1-1571 for the first and on December 26, 2003 under number 1-3151 for the second. It is recalled that the genus Trichoderma is an agronomically important group because it includes fungi "biocontrol agents" whose mode of action has been the subject of numerous studies.
- a sampling phase in an appropriate place that is to say known to contain populations of Trichoderma spp mushrooms and more specifically Trichoderma harzianum; - an isolation phase of Trichoderma spp fungi on selective media;
- the smooth-walled spores are oval in shape and green in color (approximately 3 ⁇ m in diameter).
- the cultural characters of the clones according to the invention therefore made it possible to confirm that they indeed belong to the species Trichoderma harzianum described according to the morphological classification of Rifai.
- Trichoderma harzianum The optimum growth temperature for Trichoderma harzianum is 20 to 25 ° C on conventional media such as malt-agar, PDA, beet pulp, oatmeal, with a pH between 4 and 7. Growth is aerobic, greater sporulation on a rich medium.
- the aim of the isolation of the clones and of confronting the pathogens is to obtain clones showing:
- the specific nutrient media are: - TME medium from Papavizas (1982): a mixture of glucose (1 g), agar (20 g) and distilled water (800 ml) is autoclave. 200 ml of V-8 are added, the pH must be between 3.8 and 4. In the still lukewarm medium, neomycin sulfate (100 mg), bacitracin (100 mg), penicillin G are then added (100 mg), chloroneb (100 mg), nystatin (20 mg), chlortetracycline HCI (25 mg), sodium propionate (500 mg).
- This medium consists of MgSO 4 , 7 H 2 O (0.2 g); K 2 HPO 4 (0.9 g); KCI (150 mg); NH 4 NO 3 (1 g); glucose (3 g); Chloramphenicol (250 mg); Fenaminosulf (300 mg); quintozene (200 mg); bengal rose (150 mg); agar (20 g); distilled water (1000 ml).
- the isolation technique used is that of direct incorporation of the soil.
- the selective isolation medium being poured and maintained in supercooling in Petri dishes (37 to 40 ° C), a small amount of earth is sprinkled and immediately dispersed in the medium by stirring until solidification.
- the cultures are then incubated for several days. We then proceed to the examination, the enumeration then the isolation of the colonies. Knowing the quantity of the soil sample before and after sowing, we can therefore know the mass of soil analyzed and, consequently, calculate the number of propagules present per gram of soil.
- the isolation technique used is that of dilution suspensions: After sieving and grinding in the presence of sterile water, different successive dilutions of the suspension obtained are incorporated into the isolation medium. To do this, 10 ml of each of the dilutions are poured into an Erlenmeyer flask containing 90 ml of selective isolation medium maintained in supercooling in a water bath (between 37 ° C and 40 ° C). After homogenization, the 100 ml are distributed in petri dishes placed under optimal conditions for isolation of Trichoderma. After an incubation time of 3 to 7 days, the dilution is identified having a sufficient number of colonies but without confluence.
- the colonies can be isolated. After growth of the colonies of Trichoderma population in a Petri dish at 24 ° C., in the light for 3 to 7 days, the isolations are purified, that is to say the monosporal isolation of each of the colonies. For this, for each colony, the green spores are removed, then suspended in sterile water in order to dissociate them. A separate culture is carried out for each dissociated spore on a rich agar medium such as PDA or oat flakes in a Petri dish. After 48 hours, all of the spores are resuspended and the titer is adjusted to 10 6 spores / milliliter.
- the objective is to select the clones capable of grow best in poor environments or at low temperatures of around 15 ° C.
- the selection process is characterized in that it comprises: - for growth in poor medium: a sample of each clone from the suspension obtained in point 3 which is inoculated into a poor agar medium, for example at l malt extract (20 g / 1) in Petri dish and incubated at 24 ° C.
- a rich agar medium for example PDA (40 g / 1) in a petri dish and incubated at 15 ° C.
- the present invention also relates to a method for culturing the Trichoderma clones thus isolated and selected and preferably the Trichoderma harzianum clones according to the invention.
- This process is characterized in that it consists in cultivating said clones and preferably the Trichoderma harzianum clones according to the invention by a surface culture method carried out in a disc fermenter with a capacity of 12 liters: fermentation on PDA agar medium ( dextrose [20 g / l]; potato infusion [200 g / l]; agar [15 g / l]; distilled water [qs 1000 ml]
- PDA agar medium dextrose [20 g / l]; potato infusion [200 g / l]; agar [15 g / l]; distilled water [qs 1000 ml]
- the method according to the invention is characterized in that it comprises the following stages: - Inoculation of the culture medium: the medium is directly sterilized in the reactor.
- the inoculation is carried out by means of 3. 10 8 spores / g of carbon substrate initially present. Rotation of the discs at low speed (10 rpm) allows the medium to be retained on the surface of the discs.
- - Incubation the fermenter is placed in an enclosure thermostatically controlled at 24 ° C. The incubation time is 7 days. Aeration of the culture is carried out by passing a stream of sterile air humidified by bubbling through the water. The aeration rate is 500 ml / min.
- the spores produced on the surface of the discs are harvested by 5 successive washes, by introducing into the reactor 2 liters of sterile distilled water added with 1% tween 80 and glycerol while maintaining a rapid rotation of the discs (500 rpm) for 30 minutes. The suspended spores are then counted on a Malassez cell by counting. A sterile process of placing in sealed ampoules is then initiated. The bulbs are kept cold.
- the spores can also be dried. A sterile filtration of the liquid containing the spores is then carried out. The filter "cake” is then dried and then finely ground and sieved. The final product is a fine green powder with a diameter of less than 200 ⁇ m and containing the viable spores of Trichoderma harzianum; this product can easily resuspend in water to be sprayed or brushed.
- These viable spore powders of Trichoderma harzianium referenced 1-3151 or I-1571 constitute the fundamental product according to the invention and can be declined in different formulations for phytosanitary application. These powders are characterized in that they contain at least 10 10 spores / gram.
- the products obtained according to the invention and derived from the clones of Trichoderma harzianium 1-3151 and 1-1571 have been found to be non-cellulolytic. Indeed, certain clones of Trichoderma, like many cellulolytic microorganisms produce cellulase which degrades the cellulose of the plant walls. This degradation involves a multi-component system.
- the pathogenic fungus Phaeomoniella chiamydospora is isolated from plants with Esca. For multiplication, it is subcultured from the storage tubes in a Petri dish containing a malt-agar nutrient medium (20 g / l agar, 15 g / l malt, per 1000 ml of water. The medium is sterilized at 120 ° C for 20 minutes).
- the technique of confrontation between the two fungi is as follows: Two mycelial implants of 6 mm in diameter of each of the two fungi Phaeomoniella chiamydospora and Trichoderma harzianum are taken from an actively growing colony on malt medium and placed in the petri dish of diametrically opposite. The distance between the two implants is 45 mm. Due to the slow growth of Phaeomoniella chiamydospora, its sowing is advanced by 15 days compared to the confronted Trichoderma harzianum. Incubation is carried out at 25 ° C for 1 to 21 days. Three repetitions are performed. The control corresponds to the growth of the fungus alone (without the presence of Trichoderma).
- the boxes are observed daily, noted and photographed, then weekly.
- the ratings consist in evaluating the inhibition of the growth of the mycelial colony of Phaeomoniella chiamydospora.
- the inhibition rate is calculated after 9 days.
- the results are also read by observing the confronting mycelia: presence of an inhibition zone, overlap of one mycelium by another, spatial competition, melanization zones, fruiting bodies ...
- each implant is put back on a malt-agar culture medium (20 g / l agar, 15 g / l malt, for 1000 ml of water.
- the medium is sterilized at 120 ° C for 20 minutes). The resumption or not of the colony is then noted.
- Botryosphaeria obtusa, Botryosphaeria dothidea and Botryosphaeria stevensii are isolated from vines either affected by dieback of Syrah, or showing symptoms of Black Dead Arm or even grafted-soldered. For multiplication, they are subcultured from the Petri dish storage tubes containing a malt-agar nutrient medium (20 g / l agar, 15 g / l malt, per 1000 ml of water. The medium is sterilized at 120 ° C for 20 minutes). The confrontation technique is identical to that used in Example 2.
- Phaeomoniella aleophilum Fomitiporia punctata, Eutypa lata, Botryosphaeria obtusa, Botryosphaeria dothidea, Botryosphaeria stevensii their seedings are advanced by 8 days compared to the two Trichoderma harzianum 1-3151 and 1-1571 confronted. Incubation is carried out at 25 ° C for 1 to 50 days. The inhibition rate is calculated after 4 to 12 days depending on the case.
- the plant protection product is obtained by suspending the sterile concentrate or the powder obtained according to Example 1 in water, so as to obtain a concentration of 10 8 viable spores / ml of solution.
- the concentrate or the powder is preferably suspended just before application by spraying or brushing on the vine stocks to protect them against the wood diseases mentioned.
- Example 5 Adaptation of the Trichoderma harzianum 1-3151 and 1-1571 clones to make them resistant to various fungicides and bactericides.
- Trichoderma clones In order to integrate the use of Trichoderma clones in a treatment program including either conventional fungicides or bactericides, it is necessary to make these clones resistant to various chemical agents, in particular wood disinfectants (copper, synthetic molecules , etc.) We therefore proceed to an adaptation of the clones of Trichoderma harzianum 1-3151 and 1-1571 by confronting them with increasing amounts of fungicides or chemical bactericides.
- the adaptation process is characterized in that it comprises the following stages: Evaluation of the minimum inhibitory quantities of antifungals or bactericides: the appreciation of these quantities is made by observing, for each fungicide or for each bactericide, the development of the clones of Trichoderma cultured on an oatmeal agar medium to which increasing amounts of the fungicide or bactericide have been added. When the growth of the Trichoderma harzianum clones is stopped, the minimum fungicidal or bactericidal inhibitory amount is reached.
- an amount of fungicide or bactericide, less than the minimum inhibiting amount is added to an agar medium based on oat flakes and on which a suspension of spores of Trichoderma harzianum 1-3151 or 1-1571 is spread.
- the dishes are incubated for 5 days, at 24 ° C., under light. Following this incubation time, a few colonies of Trichoderma harzianum appear. These colonies are removed and then subcultured on an agar medium based on oat flakes containing a concentration of fungicide or bactericide greater than or equal to that previously used. This operation is repeated until colonies resistant to the amounts of fungicide or bactericide usually used in viticulture are obtained.
- the pathogenic fungus Eutypa lata is isolated from vines with Eutypiosis.
- the pathogenic fungi Botryosphaeria obtusa, Botryosphaeria dothidea and Botryosphaeria stevensii are isolated from vines either affected by dieback of Syrah, or showing symptoms of Black Dead Arm or even grafted-soldered.
- the potential effectiveness of the product is judged according to the behavior of the treated plants compared to that of control plants (and inoculated).
- the type of result expected is a decrease in frequency and / or intensity of leaf symptoms.
- Plant material Several batches of cuttings are exploited, inoculated and cultivated in the greenhouse. Three different batches of cuttings are thus used:
- the pathogen and Trichoderma isolates were cultivated in petri dishes on malt-agar medium (15 g / l and 20 g / l respectively) at a temperature of 20 to 25 ° C for 7 to 21 days.
- a volume of water of around 5mL is placed in each of the boxes.
- the mycelium is separated from the agar by scraping the surface with a rake glass or the sterile blade of a scalpel. It is then collected with a micropipette and a tip, the end of which has been cut with a scalpel to allow aspiration of the mycelial hyphae.
- the mycelium sample is then placed in a plastic tube. The volume is adjusted if necessary (suspension too dense) with sterile water. The tube is strongly agitated manually in order to homogenize the suspension. Inoculation of cuttings
- the rooted or non-rooted cuttings grown in the greenhouse were therefore inoculated as follows: the bark under the eye is disinfected with cotton wool soaked in alcohol, a hole of 2 mm in diameter is drilled to the marrow , about 2 cm below the eye, on the orthoptic axis of the cutting (vertical to the eye) using a small drill fitted with a flame sterilized drill bit.
- the mycelium of the pathogen suspended in water (about 20 ⁇ l) is injected using a pipette into the inoculation hole which is immediately covered with hot paraffin.
- the processing methods are:
- the processing methods are:
- the results are analyzed by a simple test of X 2 adapted to the comparison of two observed distributions. The result is a significant difference between the two distributions, thus authorizing the interpretation of the numbers in the different classes.
- the agronomic result is a regression of the damage due to these wastage resulting in a number of “vines showing a symptom less severe ”greater in the stocks treated and conversely a smaller number of“ stocks showing an equivalent or less severe symptom ”.
- the clones of Trichoderma harzianum 1-3151 and 1-1571 used in the treatment of wood diseases in viticulture are preferably the clones made resistant to fungicides and bactericides according to the process mentioned above.
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Abstract
The invention relates to Trichoderma harzianum clones and is characterised in that said Trichoderma harzianum clones are deposited in the National Collection of Microorganism Cultures (CNCM) of the Pasteur Institute under numbers I-3151 and I-1571. A method for isolating, selecting and culturing said clones and the use thereof in the form of a phytosanitary product usable for biologically controlling fungus causing tree diseases are also disclosed.
Description
CLONES DE TRICHODERMA HARZIANUM, PROCEDE D'ISOLEMENT ET DE CULTURE ET APPLICATION COMME PRODUIT PHYTOSANITAIRE DESCRIPTION CLONES OF TRICHODERMA HARZIANUM, METHOD OF ISOLATION AND CULTURE AND APPLICATION AS A PHYTOSANITARY PRODUCT DESCRIPTION
La présente invention concerne essentiellement des clones de Trichoderma harzianum, un procédé d'isolement et de culture de ces clones et l'application de ceux-ci en tant que produits phytosanitaires utilisables dans le cadre de la lutte biologique contre les champignons responsables des maladies du bois de la vigne : Esca (Phaeoacremonium aleophilum, Phaeomoniella chiamydospora, Fomitiporia punctata, Phellinus igniarius, Phellinus viticola et Eutypa lata), Eutypiose (Eutypa lata et Botryospheria spp), Excoriose (Phomopsis viticola et Botryosphaeria spp), dépérissement de la Syrah (Botryospheria obtusa), Black Dead Arm (Botryospheria dothidea), Symptômes de greffés-soudés (Botryospheria Stevensii). Les clones de Trichoderma harzianum (Classification de Rifai), isolés par le déposant, ont été déposés auprès de la Collection Nationale de Cultures de Micro- organismes (CNCM) de l'Institut Pasteur le 4 Mai 1995 sous le numéro 1-1571 pour le premier et le 26 Décembre 2003 sous le numéro 1-3151 pour le second. On rappelle que le genre Trichoderma est un groupe agronomiquement important car il comprend des champignons « agents de biocontrôle » dont le mode d'action a fait l'objet de nombreux travaux. Des études récentes font en effet état d'une capacité de ce champignon filamenteux à intervenir selon divers mécanismes : mycoparasitisme, antagonisme (compétition), antibiose (production d'antibiotiques), stimulation racinaire, stimulation de la croissance par solubilisation de minéraux fertilisants, stimulation des défenses naturelles des plantes, etc. Le procédé d'obtention des clones selon l'invention, prévoit :The present invention essentially relates to clones of Trichoderma harzianum, a process for the isolation and culture of these clones and the application of these as phytosanitary products which can be used in the context of the biological control of the fungi responsible for diseases of the wood of the vine: Esca (Phaeoacremonium aleophilum, Phaeomoniella chiamydospora, Fomitiporia punctata, Phellinus igniarius, Phellinus viticola and Eutypa lata), Eutypiose (Eutypa lata and Botryospheria spp), Excoriosis (Phomopsis viticola bot and sherry berry and Phytospheria viticola bot sherry bot and sherry bot) obtusa), Black Dead Arm (Botryospheria dothidea), Transplant-welded symptoms (Botryospheria Stevensii). The clones of Trichoderma harzianum (Classification of Rifai), isolated by the depositor, were deposited with the National Collection of Cultures of Microorganisms (CNCM) of the Pasteur Institute on May 4, 1995 under number 1-1571 for the first and on December 26, 2003 under number 1-3151 for the second. It is recalled that the genus Trichoderma is an agronomically important group because it includes fungi "biocontrol agents" whose mode of action has been the subject of numerous studies. Recent studies indeed state an ability of this filamentous fungus to intervene according to various mechanisms: mycoparasitism, antagonism (competition), antibiosis (production of antibiotics), root stimulation, growth stimulation by solubilization of fertilizing minerals, stimulation natural defenses of plants, etc. The process for obtaining the clones according to the invention provides:
- une phase de prélèvement dans un endroit approprié, c'est à dire connu pour renfermer des populations de champignons Trichoderma spp et plus spécifiquement le Trichoderma harzianum ; - une phase d'isolement des champignons Trichoderma spp sur milieux sélectifs ;- a sampling phase in an appropriate place, that is to say known to contain populations of Trichoderma spp mushrooms and more specifically Trichoderma harzianum; - an isolation phase of Trichoderma spp fungi on selective media;
- une phase d'isolement monoclonal (purification des isolements) de différents clones dans le but de générer une « mycothèque », laquelle sera par la suite confrontée, pour chacun de ses membres, aux clones des différents pathovars revendiqués par la présente invention ; - la sélection des clones les plus performants ;a phase of monoclonal isolation (purification of the isolations) of different clones with the aim of generating a "mycotheque", which will then be compared, for each of its members, with the clones of the different pathovars claimed by the present invention; - the selection of the most efficient clones;
- la méthode de culture et de développement de ces clones.- the method of culture and development of these clones.
Par la suite, l'aspect cultural de ces clones a été identifié. Il se caractérise par une colonie d'abord blanche puis devenant verte, d'aspect irrégulier et s'organisant en
agrégats plus ou moins condensés. Le revers est incolore, jaune ou brun. L'observation microscopique à faible grossissement révèle des arborescences terminées par des formations sphériques irrégulières (« têtes ») qui se différencient à partir d'un mycélium plus ou moins dense, étalé sur le milieu gélose. A plus fort grossissement, l'organisation des structures conidiogènes est telle que des ramifications se répartissent perpendiculairement et de façon opposée de part et d'autre d'un axe principal, le conidiophore. Les cellules conidiogènes (phialides) apparaissent ampulliformes. Les spores, à paroi lisse, sont de forme ovalaire et de couleur verte (environ 3 μm de diamètre). Les caractères culturaux des clones selon l'invention ont donc permis de confirmer qu'ils appartiennent bien à l'espèce Trichoderma harzianum décrite selon la classification morphologique de Rifai.Subsequently, the cultural aspect of these clones was identified. It is characterized by a colony initially white then becoming green, irregular in appearance and organized in more or less condensed aggregates. The reverse is colorless, yellow or brown. Microscopic observation at low magnification reveals trees ending in irregular spherical formations ("heads") which differentiate from a more or less dense mycelium, spread over the agar medium. At higher magnification, the organization of conidiogenic structures is such that ramifications are distributed perpendicularly and in opposite directions on either side of a main axis, the conidiophore. The conidiogenic cells (phialides) appear ampulliform. The smooth-walled spores are oval in shape and green in color (approximately 3 μm in diameter). The cultural characters of the clones according to the invention therefore made it possible to confirm that they indeed belong to the species Trichoderma harzianum described according to the morphological classification of Rifai.
La température de croissance optimale du Trichoderma harzianum est de 20 à 25°C sur milieux classiques comme le malt-agar, le PDA, pulpe de betterave, flocons d'avoine, avec un pH compris entre 4 et 7. La croissance est aérobie, la sporulation plus importante sur un milieu riche. Selon l'invention, le procédé d'isolement des clones et de confrontation aux pathogènes, a pour objectif d'obtenir des clones montrant :The optimum growth temperature for Trichoderma harzianum is 20 to 25 ° C on conventional media such as malt-agar, PDA, beet pulp, oatmeal, with a pH between 4 and 7. Growth is aerobic, greater sporulation on a rich medium. According to the invention, the aim of the isolation of the clones and of confronting the pathogens is to obtain clones showing:
- d'une part une croissance rapide afin qu'ils puissent être les plus efficaces vis à vis des pathogènes et présentant la plus importante production de spores par unité de milieu gélose;- On the one hand, rapid growth so that they can be the most effective with respect to pathogens and having the highest production of spores per unit of agar medium;
- d'autre part une production importante de composés antifongiques, production évaluée par comparaison des pouvoirs antagonistes à distance.- on the other hand, a significant production of antifungal compounds, production evaluated by comparison of the antagonistic powers at a distance.
Enfin, un pouvoir antagoniste puissant par contact. Les deux derniers points seront développés dans les exemples 1 et 2.Finally, a powerful antagonistic power by contact. The last two points will be developed in Examples 1 and 2.
Le procédé d'isolement selon l'invention est caractérisé en ce qu'il comprend les étapes suivantes :The isolation process according to the invention is characterized in that it comprises the following steps:
- Prélèvement des champignons du genre Trichoderma dans un milieu adéquat comme un sol humide (sols maraîchers, viticole ou arboricole, matières organiques en décomposition) ou dans des bois pourris issus de ceps de vigne ou de branches d'arbres fruitiers.- Collection of fungi of the genus Trichoderma in a suitable environment such as moist soil (market garden, wine or arboreal soil, decomposing organic matter) or in rotten wood from vine stocks or branches of fruit trees.
- Isolement de la population de champignons Trichoderma prélevée, sur des milieux spécifiques choisis. Les milieux nutritifs spécifiques sont :
- Milieu TME de Papavizas (1982) : un mélange de glucose (1g), gélose (20 g) et d'eau distillée (800 ml) est autoclave. On ajoute 200 ml de V-8, le pH doit être compris entre 3,8 et 4. Au milieu encore tiède, on ajoute alors du sulfate de néomycine (100 mg), de la bacitracine (100 mg), de la pénicilline G (100 mg), du chloronèbe (100 mg), de la nystatine (20 mg), de la chlortétracycline HCI (25 mg), du propionate de sodium (500 mg).- Isolation of the population of Trichoderma mushrooms collected, on selected specific media. The specific nutrient media are: - TME medium from Papavizas (1982): a mixture of glucose (1 g), agar (20 g) and distilled water (800 ml) is autoclave. 200 ml of V-8 are added, the pH must be between 3.8 and 4. In the still lukewarm medium, neomycin sulfate (100 mg), bacitracin (100 mg), penicillin G are then added (100 mg), chloroneb (100 mg), nystatin (20 mg), chlortetracycline HCI (25 mg), sodium propionate (500 mg).
- Milieu TSM dElad et al. (1981) : Ce milieu est constitué de MgSO4, 7 H2O (0,2 g); K2HPO4 (0,9 g); KCI (150 mg); NH4NO3 (1 g); glucose (3 g); Chloramphénicol (250 mg); Fénaminosulf (300 mg); quintozène (200 mg); rose bengale (150 mg); gélose (20 g); eau distillée (1000 ml).- TSM medium of Elad et al. (1981): This medium consists of MgSO 4 , 7 H 2 O (0.2 g); K 2 HPO 4 (0.9 g); KCI (150 mg); NH 4 NO 3 (1 g); glucose (3 g); Chloramphenicol (250 mg); Fenaminosulf (300 mg); quintozene (200 mg); bengal rose (150 mg); agar (20 g); distilled water (1000 ml).
- Milieu de Davet (1979) : Ca(NO3)2 (1g) ; CaCI2, 2H2O (1 g) ; KNO3 (250 mg) ; MgSO4, 7H2O (250 mg) ; KH2PO4 (125 mg) ; saccharose (2 g) ; acide citrique (50 mg) ; gélose (25 g); eau distillée (1000 ml). Après autoclavage, le pH est ajusté à 4,5 avec du HCI 1 N et on ajoute au milieu tiède du sulfate de streptomycine (30 mg), de la vinchlozoline (2,5 mg) et de l'alcool allylique (0,5 ml).- Davet's medium (1979): Ca (NO 3 ) 2 (1g); CaCl 2 , 2H 2 O (1 g); KNO 3 (250 mg); MgSO 4 , 7H 2 O (250 mg); KH 2 PO 4 (125 mg); sucrose (2 g); citric acid (50 mg); agar (25 g); distilled water (1000 ml). After autoclaving, the pH is adjusted to 4.5 with 1 N HCl and to the warm medium are added streptomycin sulfate (30 mg), vinchlozoline (2.5 mg) and allyl alcohol (0.5 ml).
Dans le cas d'un prélèvement de sol, la technique d'isolement utilisée est celle de l'incorporation directe du sol. Le milieu sélectif d'isolement étant coulé et maintenu en surfusion en boîtes de Pétri (37 à 40°C), une faible quantité de terre est saupoudrée et immédiatement dispersée dans le milieu par agitation jusqu'à solidification. Les cultures sont alors mises en incubation pendant plusieurs jours. On procède alors à l'examen, au dénombrement puis à l'isolement des colonies. Connaissant la quantité de l'échantillon de terre avant et après l'ensemencement, on peut donc connaître la masse de terre analysée et par voie de conséquence, calculer le nombre de propagules présentes par gramme de terre. Dans le cas d'un prélèvement de matières organiques en décomposition ou de bois pourris, la technique d'isolement utilisée est celle des suspensions dilutions : Après tamisage et broyage en présence d'eau stérile, différentes dilutions successives de la suspension obtenue sont incorporées dans le milieu d'isolement. Pour ce faire, 10 ml de chacune des dilutions sont versés dans un erlenmeyer contenant 90 ml de milieu sélectif d'isolement maintenu en surfusion au bain-marie (entre 37°C et 40°C). Après homogénéisation, les 100 ml sont répartis dans des boîtes de pétri placées dans les conditions optimales d'isolement des Trichoderma. Après un temps d'incubation de 3 à 7 jours, on repère la dilution présentant un nombre de colonies suffisant mais sans confluence. La dilution étant choisie, on peut procéder à l'isolement des colonies.
Après croissance des colonies de population de Trichoderma en boîte de Pétri à 24°C, à la lumière pendant 3 à 7 jours, on procède à la purification des isolements, c'est à dire à l'isolement monosporal de chacune des colonies. Pour cela, pour chaque colonie, les spores de couleur verte sont prélevées, puis mises en suspension dans l'eau stérile afin de les dissocier. On réalise une culture séparée pour chaque spore dissociée sur un milieu gélose riche tel le PDA ou flocons d'avoine en boîte de Pétri. Après 48 heures, l'ensemble des spores est remis en suspension et le titre est ajusté à 106 spores/millilitre. Dans le but d'obtenir les clones présentant la meilleure croissance et la meilleure production de spores, différents tests ont été mis en place pour évaluer la qualité des clones : en ce qui concerne la croissance, l'objectif est de sélectionner les clones capables de se développer le mieux sur milieu pauvre ou à des températures basses de l'ordre de 15°C. Le procédé de sélection est caractérisé en ce qu'il comprend : - pour la croissance en milieu pauvre : un prélèvement de chaque clone issu de la suspension obtenue dans le point 3 que l'on inocule dans un milieu gélose pauvre, par exemple à l'extrait de malt (20 g/1) en boîte de Pétri et incubé à 24°C.In the case of soil sampling, the isolation technique used is that of direct incorporation of the soil. The selective isolation medium being poured and maintained in supercooling in Petri dishes (37 to 40 ° C), a small amount of earth is sprinkled and immediately dispersed in the medium by stirring until solidification. The cultures are then incubated for several days. We then proceed to the examination, the enumeration then the isolation of the colonies. Knowing the quantity of the soil sample before and after sowing, we can therefore know the mass of soil analyzed and, consequently, calculate the number of propagules present per gram of soil. In the case of a sample of decomposing organic matter or rotten wood, the isolation technique used is that of dilution suspensions: After sieving and grinding in the presence of sterile water, different successive dilutions of the suspension obtained are incorporated into the isolation medium. To do this, 10 ml of each of the dilutions are poured into an Erlenmeyer flask containing 90 ml of selective isolation medium maintained in supercooling in a water bath (between 37 ° C and 40 ° C). After homogenization, the 100 ml are distributed in petri dishes placed under optimal conditions for isolation of Trichoderma. After an incubation time of 3 to 7 days, the dilution is identified having a sufficient number of colonies but without confluence. The dilution being chosen, the colonies can be isolated. After growth of the colonies of Trichoderma population in a Petri dish at 24 ° C., in the light for 3 to 7 days, the isolations are purified, that is to say the monosporal isolation of each of the colonies. For this, for each colony, the green spores are removed, then suspended in sterile water in order to dissociate them. A separate culture is carried out for each dissociated spore on a rich agar medium such as PDA or oat flakes in a Petri dish. After 48 hours, all of the spores are resuspended and the titer is adjusted to 10 6 spores / milliliter. In order to obtain the clones having the best growth and the best production of spores, various tests have been set up to assess the quality of the clones: with regard to growth, the objective is to select the clones capable of grow best in poor environments or at low temperatures of around 15 ° C. The selection process is characterized in that it comprises: - for growth in poor medium: a sample of each clone from the suspension obtained in point 3 which is inoculated into a poor agar medium, for example at l malt extract (20 g / 1) in Petri dish and incubated at 24 ° C.
- pour la croissance à basse température : un prélèvement de chaque clone issu de la suspension obtenue dans le point 3 que l'on inocule dans un milieu gélose riche, par exemple au PDA (40 g/1) en boîte de Pétri et incubé à 15°C.- for growth at low temperature: a sample of each clone from the suspension obtained in point 3 which is inoculated into a rich agar medium, for example PDA (40 g / 1) in a petri dish and incubated at 15 ° C.
- pour l'évaluation de la meilleure production de spores par unité de surface de milieu gélose : un prélèvement de chaque clone issu de la suspension obtenue dans le point 3 que l'on inocule dans un milieu gélose riche, par exemple au PDA (40 g/l) en boîte de Pétri et incubé à 24°C. Dans le but d'estimer le pouvoir antagoniste des clones vis à vis des champignons, Phaeoacremonium aleophilum, Phaeomoniella chiamydospora, Fomitiporia punctata, Phellinus igniarius, Phellinus viticola, Eutypa lata, Phomopsis viticola et Botryosphaeria sp, on réalise un prélèvement de chaque clone issu de la suspension obtenue dans le point 3. On inocule chacun des prélèvements dans une boîte de Pétri contenant du milieu nutritif malt - agar (20 g/l de gélose, 15 g/l de malt). Dans ces mêmes milieux, on place un implant prélevé dans une boîte de Pétri où l'on a réalisé préalablement la croissance du pathogène concerné. On mesure alors le pouvoir antagoniste des clones soit par contact, soit à distance par rapport à la croissance témoin de ce même pathogène. L'incubation est réalisée à 25°C pendant 1 à 50 jours.
Les clones qui ont démontré la plus grande performance et l'efficacité la plus importante vis à vis de l'ensemble de ces essais décrits aux points 4 et 5 sont les clones selon l'invention référencés 1-3151 et 1-1571. La présente invention concerne également un procédé de culture des clones de Trichoderma ainsi isolés et sélectionnés et de préférence les clones de Trichoderma harzianum selon l'invention. Ce procédé est caractérisé en ce qu'il consiste à cultiver lesdits clones et de préférence les clones de Trichoderma harzianum selon l'invention par une méthode de culture en surface réalisée dans un fermenteur à disques de capacité 12 litres : fermentation sur milieu gélose PDA (dextrose [20 g/l] ; infusion de pomme de terre [200 g/l] ; Agar [15 g/l] ; eau distillée [qsp 1000 ml]. Exemple 1 :- for the evaluation of the best production of spores per unit area of agar medium: a sample of each clone from the suspension obtained in point 3 which is inoculated into a rich agar medium, for example PDA (40 g / l) in Petri dish and incubated at 24 ° C. In order to estimate the antagonistic power of the clones with respect to the fungi, Phaeoacremonium aleophilum, Phaeomoniella chiamydospora, Fomitiporia punctata, Phellinus igniarius, Phellinus viticola, Eutypa lata, Phomopsis viticola and Botryosphaeria sp, a sample is taken from each clone the suspension obtained in point 3. Each of the samples is inoculated in a Petri dish containing malt-agar nutrient medium (20 g / l agar, 15 g / l malt). In these same media, an implant taken in a Petri dish is placed where the growth of the pathogen concerned has been carried out beforehand. The antagonistic power of the clones is then measured either by contact or at a distance from the control growth of this same pathogen. Incubation is carried out at 25 ° C for 1 to 50 days. The clones which have demonstrated the greatest performance and the most important efficiency with respect to all of these tests described in points 4 and 5 are the clones according to the invention referenced 1-3151 and 1-1571. The present invention also relates to a method for culturing the Trichoderma clones thus isolated and selected and preferably the Trichoderma harzianum clones according to the invention. This process is characterized in that it consists in cultivating said clones and preferably the Trichoderma harzianum clones according to the invention by a surface culture method carried out in a disc fermenter with a capacity of 12 liters: fermentation on PDA agar medium ( dextrose [20 g / l]; potato infusion [200 g / l]; agar [15 g / l]; distilled water [qs 1000 ml] Example 1:
Le procédé selon l'invention est caractérisé en ce qu'il comprend les étapes suivantes : - L'inoculation du milieu de culture : le milieu est directement stérilisé dans le réacteur.The method according to the invention is characterized in that it comprises the following stages: - Inoculation of the culture medium: the medium is directly sterilized in the reactor.
- L'inoculation se réalise au moyen de 3. 108 spores/g de substrat carboné initialement présent. Une rotation des disques à faible vitesse (10trs/min) permet de retenir le milieu à la surface des disques. - L'incubation : le fermenteur est placé dans une enceinte thermostatée à 24°C. Le temps d'incubation est de 7 jours. L'aération de la culture est réalisée par passage d'un flux d'air stérile et humidifié par barbotage dans l'eau. Le débit d'aération est de 500 ml/min. - La récolte des spores : les spores produites à la surface des disques sont récoltées par 5 lavages successifs, en introduisant dans le réacteur 2 litres d'eau distillée stérile additionnée de tween 80 à 1% et de glycérol en maintenant une rotation rapide des disques (500 trs/min) pendant 30 minutes. Les spores mises en suspension sont ensuite dénombrées sur cellule de Malassez par comptage. Un procédé stérile de mise en ampoules scellées est alors engagé. Les ampoules sont conservées au froid.- The inoculation is carried out by means of 3. 10 8 spores / g of carbon substrate initially present. Rotation of the discs at low speed (10 rpm) allows the medium to be retained on the surface of the discs. - Incubation: the fermenter is placed in an enclosure thermostatically controlled at 24 ° C. The incubation time is 7 days. Aeration of the culture is carried out by passing a stream of sterile air humidified by bubbling through the water. The aeration rate is 500 ml / min. - Harvesting of spores: the spores produced on the surface of the discs are harvested by 5 successive washes, by introducing into the reactor 2 liters of sterile distilled water added with 1% tween 80 and glycerol while maintaining a rapid rotation of the discs (500 rpm) for 30 minutes. The suspended spores are then counted on a Malassez cell by counting. A sterile process of placing in sealed ampoules is then initiated. The bulbs are kept cold.
Les spores peuvent être aussi séchées. On procède alors à une filtration stérile du liquide contenant les spores. Le « gâteau » de filtre est alors séché puis finement broyé et tamisé. Le produit final est une poudre verte, fine présentant un diamètre inférieur à 200 μm et contenant les spores viables de Trichoderma harzianum ; ce
produit peut se remettre aisément en suspension dans l'eau afin d'être pulvérisé ou badigeonné.The spores can also be dried. A sterile filtration of the liquid containing the spores is then carried out. The filter "cake" is then dried and then finely ground and sieved. The final product is a fine green powder with a diameter of less than 200 μm and containing the viable spores of Trichoderma harzianum; this product can easily resuspend in water to be sprayed or brushed.
Ces poudres de spores viables de Trichoderma harzianium référencés 1-3151 ou I- 1571 constituent le produit fondamental selon l'invention et peuvent être déclinées en différentes formulations pour application phytosanitaire. Ces poudres sont caractérisées en ce qu'elles contiennent au moins 1010 spores / gramme. En outre, les produits obtenus selon l'invention et issu des clones de Trichoderma harzianium 1-3151 et 1-1571 se sont révélés non cellulolytiques. En effet, certains clones de Trichoderma, comme de nombreux micro-organismes cellulolytiques produisent de la cellulase qui dégrade la cellulose des parois végétale. Cette dégradation fait intervenir un système à composantes multiples. Trois éléments associés en un complexe enzymatique sont nécessaires pour cela : la Crcellulase, la Cχ-cellulase et la β-glucosidase. Or, les clones de Trichoderma harzianum 1-3151 et 1-1571 ne présentent aucune activité Ci-cellulase (test sur cellulose microcristalline [Avicel]).These viable spore powders of Trichoderma harzianium referenced 1-3151 or I-1571 constitute the fundamental product according to the invention and can be declined in different formulations for phytosanitary application. These powders are characterized in that they contain at least 10 10 spores / gram. In addition, the products obtained according to the invention and derived from the clones of Trichoderma harzianium 1-3151 and 1-1571 have been found to be non-cellulolytic. Indeed, certain clones of Trichoderma, like many cellulolytic microorganisms produce cellulase which degrades the cellulose of the plant walls. This degradation involves a multi-component system. Three elements associated in an enzymatic complex are necessary for this: C r cellulase, Cχ-cellulase and β-glucosidase. However, the clones of Trichoderma harzianum 1-3151 and 1-1571 show no Ci-cellulase activity (test on microcrystalline cellulose [Avicel]).
Il n'existe donc aucune dégradation possible de la cellulose, constituant principal de la paroi des végétaux. Par conséquent, les produits selon l'invention ne pourront en aucun cas être la cause de dommages sur les végétaux lors de son application comme produit phytosanitaire. Exemple 2There is therefore no possible degradation of cellulose, the main constituent of the wall of plants. Consequently, the products according to the invention can in no case be the cause of damage to plants during its application as a phytosanitary product. Example 2
Essai de pouvoir antagoniste (contact) des Trichoderma harzianum 1-3151 et 1-1571 vis à vis de Phaeomoniella chiamydosporaTest of antagonistic power (contact) of Trichoderma harzianum 1-3151 and 1-1571 against Phaeomoniella chiamydospora
Le champignon pathogène Phaeomoniella chiamydospora est isolé de plantes atteintes d'Esca . Pour la multiplication, il est repiqué à partir des tubes de conservation en boîte de Pétri contenant un milieu nutritif malt - agar (20 g/l de gélose, 15 g/l de malt, pour 1000 ml d'eau. Le milieu est stérilisé à 120°C pendant 20 minutes).The pathogenic fungus Phaeomoniella chiamydospora is isolated from plants with Esca. For multiplication, it is subcultured from the storage tubes in a Petri dish containing a malt-agar nutrient medium (20 g / l agar, 15 g / l malt, per 1000 ml of water. The medium is sterilized at 120 ° C for 20 minutes).
La technique de confrontation entre les deux champignons est la suivante : Deux implants mycéliens de 6 mm de diamètre de chacun des deux champignons Phaeomoniella chiamydospora et Trichoderma harzianum sont prélevés d'une colonie à croissance active sur milieu malt et déposés dans la boîte de Pétri de façon diamétralement opposée. La distance entre les deux implants est de 45 mm. Du fait de la croissance lente de Phaeomoniella chiamydospora, son ensemencement est avancé de 15 jours par rapport au Trichoderma harzianum mis en confrontation. L'incubation est réalisée à 25°C pendant 1 à 21 jours. Trois
répétitions sont effectuées. Le témoin correspond à la croissance du champignon seul (sans la présence de Trichoderma).The technique of confrontation between the two fungi is as follows: Two mycelial implants of 6 mm in diameter of each of the two fungi Phaeomoniella chiamydospora and Trichoderma harzianum are taken from an actively growing colony on malt medium and placed in the petri dish of diametrically opposite. The distance between the two implants is 45 mm. Due to the slow growth of Phaeomoniella chiamydospora, its sowing is advanced by 15 days compared to the confronted Trichoderma harzianum. Incubation is carried out at 25 ° C for 1 to 21 days. Three repetitions are performed. The control corresponds to the growth of the fungus alone (without the presence of Trichoderma).
Pendant la première semaine, les boîtes sont observées tous les jours, notées et photographiées, puis toutes les semaines. Les notations consistent à évaluer l'inhibition de la croissance de la colonie mycélienne de Phaeomoniella chiamydospora. Le taux d'inhibition I, exprimé en millimètres, correspond à la différence entre le rayon R1 de la colonie de la boîte témoin et le rayon R2 qui est situé sur la droite reliant le centre entre les deux protagonistes en confrontation (I = R1 - R2). Le taux d'inhibition est calculé au bout de 9 jours. La lecture des résultats se fait également par observation des mycéliums en confrontation : présence d'une zone d'inhibition, recouvrement d'un mycélium par un autre, compétition spatiale, zones de mélanisation, fructifications...During the first week, the boxes are observed daily, noted and photographed, then weekly. The ratings consist in evaluating the inhibition of the growth of the mycelial colony of Phaeomoniella chiamydospora. The inhibition rate I, expressed in millimeters, corresponds to the difference between the radius R1 of the colony of the control box and the radius R2 which is located on the right connecting the center between the two protagonists in confrontation (I = R1 - R2). The inhibition rate is calculated after 9 days. The results are also read by observing the confronting mycelia: presence of an inhibition zone, overlap of one mycelium by another, spatial competition, melanization zones, fruiting bodies ...
A la fin des confrontations, chaque implant est remis sur un milieu de culture malt - agar (20 g/l de gélose, 15 g/l de malt, pour 1000 ml d'eau. Le milieu est stérilisé à 120°C pendant 20 minutes). La reprise ou non de la colonie est alors notée.At the end of the confrontations, each implant is put back on a malt-agar culture medium (20 g / l agar, 15 g / l malt, for 1000 ml of water. The medium is sterilized at 120 ° C for 20 minutes). The resumption or not of the colony is then noted.
Deux isolats de Phaeomoniella chiamydospora ont été testés (Pc LR81 et PcLR47) à encontre de deux clones de Trichoderma harzianum 1-3151 et 1-1571.Two isolates of Phaeomoniella chiamydospora were tested (Pc LR81 and PcLR47) against two clones of Trichoderma harzianum 1-3151 and 1-1571.
Les résultats sont représentés dans le tableau 1.The results are shown in Table 1.
Tableau 1 : Taux d'inhibition (exprimé en mm) de la croissance mycélienne de deux isolats de Phaeomoniella chiamydospora face aux clones de Trichoderma harzianum 1-3151 et 1-1571 (mesuré au bout de 9 jours de confrontation)Table 1: Inhibition rate (expressed in mm) of the mycelial growth of two isolates of Phaeomoniella chiamydospora against the clones of Trichoderma harzianum 1-3151 and 1-1571 (measured after 9 days of confrontation)
Pc LR81 Pc LR47Pc LR81 Pc LR47
Taux d'inhibition par Trichoderma harzianum I- 4,2 3 3151Inhibition rate by Trichoderma harzianum I- 4.2 3 3151
Taux d'inhibition par Trichoderma harzianum I- 5,3 4 1571Inhibition rate by Trichoderma harzianum I- 5.3 4 1571
Rayon de la colonie témoin 16 14Radius of the control colony 16 14
L'étude du comportement des deux isolats de Phaeomoniella chiamydospora à l'égard des 2 clones de Trichoderma harzianum 1-3151 et 1-1571 a permis de montrer :
Un recouvrement des deux isolats par Trichoderma harzianum 1-3151 et 1-1571 L'existence de fructifications de Trichoderma harzianum 1-3151 et 1-1571 sur les deux isolats.The study of the behavior of the two Phaeomoniella chiamydospora isolates with regard to the 2 clones of Trichoderma harzianum 1-3151 and 1-1571 made it possible to show: A covering of the two isolates by Trichoderma harzianum 1-3151 and 1-1571 The existence of fruiting bodies of Trichoderma harzianum 1-3151 and 1-1571 on the two isolates.
Ce test révèle donc l'importance de l'antagonisme par contact des Trichoderma harzianum 1-3151 et 1-1571 vis à vis de Phaeomoniella chiamydospora : Les clones de Trichoderma harzianum 1-3151 et 1-1571 inhibent bien la croissance des deux isolats. Elle recouvre la colonie de Phaeomoniella chiamydospora et produit des fructifications à sa surface. La remise des implants de Phaeomoniella chiamydospora en culture montre que les deux isolats, Pc LR81 et PcLR47, ne se développent pas. Exemple 3This test therefore reveals the importance of the antagonism by contact of Trichoderma harzianum 1-3151 and 1-1571 with respect to Phaeomoniella chiamydospora: The clones of Trichoderma harzianum 1-3151 and 1-1571 well inhibit the growth of the two isolates. It covers the colony of Phaeomoniella chiamydospora and produces fruiting bodies on its surface. The delivery of Phaeomoniella chiamydospora implants in culture shows that the two isolates, Pc LR81 and PcLR47, do not develop. Example 3
Essai de pouvoir antagoniste (contact) de Trichoderma harzianum 1-3151 et 1-1571 vis à vis de Phaeomoniella aleophilum, Fomitiporia punctata, Eutypa lata, Botryosphae a obtusa, Botryosphaeria dothidea, Botryosphaeria stevensii Les champignons pathogènes Phaeomoniella aleophilum, Fomitiporia punctata sont isolés de plantes atteintes d'Esca . Le champignon pathogène Eutypa lata est isolé de ceps atteints d'Eutypiose. Les champignons pathogènes Botryosphaeria obtusa, Botryosphaeria dothidea et Botryosphaeria stevensii sont isolés de ceps soit atteints par le dépérissement de la Syrah, soit manifestant des symptômes de Black Dead Arm ou encore de greffés-soudés. Pour la multiplication, ils sont repiqués à partir des tubes de conservation boîte de Pétri contenant un milieu nutritif malt - agar (20 g/l de gélose, 15 g/l de malt, pour 1000 ml d'eau. Le milieu est stérilisé à 120°C pendant 20 minutes). La technique de confrontation est identique à celle utilisée dans l'exemple 2. Du fait de la croissance lente de Phaeomoniella aleophilum, Fomitiporia punctata, Eutypa lata, Botryosphaeria obtusa, Botryosphaeria dothidea, Botryosphaeria stevensii leurs ensemencements sont avancés de 8 jours par rapport aux deux Trichoderma harzianum 1-3151 et 1-1571 mis en confrontation. L'incubation est réalisée à 25°C pendant 1 à 50 jours. Le taux d'inhibition est calculé au bout de 4 à 12 jours selon les cas.Test of antagonistic power (contact) of Trichoderma harzianum 1-3151 and 1-1571 against Phaeomoniella aleophilum, Fomitiporia punctata, Eutypa lata, Botryosphae a obtusa, Botryosphaeria dothidea, Botryosphaeria stevensii The pathogenic fungi Phaeomoniella aleophilum, plants with Esca. The pathogenic fungus Eutypa lata is isolated from vines with Eutypiosis. The pathogenic fungi Botryosphaeria obtusa, Botryosphaeria dothidea and Botryosphaeria stevensii are isolated from vines either affected by dieback of Syrah, or showing symptoms of Black Dead Arm or even grafted-soldered. For multiplication, they are subcultured from the Petri dish storage tubes containing a malt-agar nutrient medium (20 g / l agar, 15 g / l malt, per 1000 ml of water. The medium is sterilized at 120 ° C for 20 minutes). The confrontation technique is identical to that used in Example 2. Due to the slow growth of Phaeomoniella aleophilum, Fomitiporia punctata, Eutypa lata, Botryosphaeria obtusa, Botryosphaeria dothidea, Botryosphaeria stevensii their seedings are advanced by 8 days compared to the two Trichoderma harzianum 1-3151 and 1-1571 confronted. Incubation is carried out at 25 ° C for 1 to 50 days. The inhibition rate is calculated after 4 to 12 days depending on the case.
Deux isolats de Phaeomoniella aleophilum ont été testés (Pa LR3 et Pa LR23). Deux isolats de Fomitiporia punctata ont été testés (Fp LR30 et Fp LR14). Deux isolats ύ'Eutypa lata ont été testés (Elcc 260 et El MT 247). Deux isolats de Botryosphaeria obtusa ont été testés (Bo F 98-1 et Bo F 99-8). Un isolât de Botryosphaeria dothidea a été testé (Bd 021).
Un isolât de Botryosphaeria stevensii a été testé (Bs 001 ). Les résultats sont représentés dans les tableaux suivants. Tableau 2 : Taux d'inhibition (exprimé en mm) de la croissance mycélienne de deux isolats de Phaeomoniella aleophilum face aux clones de Trichoderma harzianum I- 3151 et 1-1571 (mesuré au bout de 4 ou 12 jours de confrontation)Two isolates of Phaeomoniella aleophilum were tested (Pa LR3 and Pa LR23). Two isolates of Fomitiporia punctata were tested (Fp LR30 and Fp LR14). Two ut'Eutypa lata isolates were tested (Elcc 260 and El MT 247). Two Botryosphaeria obtusa isolates were tested (Bo F 98-1 and Bo F 99-8). A Botryosphaeria dothidea isolate was tested (Bd 021). A Botryosphaeria stevensii isolate was tested (Bs 001). The results are shown in the following tables. Table 2: Inhibition rate (expressed in mm) of the mycelial growth of two isolates of Phaeomoniella aleophilum against the clones of Trichoderma harzianum I-3151 and 1-1571 (measured after 4 or 12 days of confrontation)
Pa LR3 Pa LR23 Taux d'inhibition par Trichoderma harzianum I- 10,7 3,5 3151 Taux d'inhibition par Trichoderma harzianum I- 9,3 3,8 1571Pa LR3 Pa LR23 Inhibition rate by Trichoderma harzianum I- 10.7 3.5 3151 Inhibition rate by Trichoderma harzianum I- 9.3 3.8 1571
Rayon de la colonie témoin 17,5 15,2Radius of the control colony 17.5 15.2
Tableau 3 : Taux d'inhibition (exprimé en mm) de la croissance mycélienne de deux isolats de Fomitiporia punctata face aux clones de Trichoderma harzianum 1-3151 et 1-1571 (mesuré au bout de 7 jours de confrontation)Table 3: Inhibition rate (expressed in mm) of the mycelial growth of two isolates of Fomitiporia punctata against the clones of Trichoderma harzianum 1-3151 and 1-1571 (measured after 7 days of confrontation)
Fp LR30 Fp LR14 Taux d'inhibition par Trichoderma harzianum I- 10,7 7,7 3151 Taux d'inhibition par Trichoderma harzianum I- 8,7 10,3 1571 Rayon de la colonie témoin 26,7 22,7Fp LR30 Fp LR14 Inhibition rate by Trichoderma harzianum I- 10.7 7.7 3151 Inhibition rate by Trichoderma harzianum I- 8.7 10.3 1571 Radius of the control colony 26.7 22.7
Tableau 4 : Taux d'inhibition (exprimé en mm) de la croissance mycélienne de deux isolats û'Eutypa lata face aux clones de Trichoderma harzianum 1-3151 et 1-1571 (mesuré au bout de 7 jours de confrontation)
Taux d'inhibition parTable 4: Inhibition rate (expressed in mm) of the mycelial growth of two isolates û'Eutypa lata against clones of Trichoderma harzianum 1-3151 and 1-1571 (measured after 7 days of confrontation) Inhibition rate by
Trichoderma harzianum 1- 9 12Trichoderma harzianum 1- 9 12
31513151
Taux d'inhibition parInhibition rate by
Trichoderma harzianum I- 11 ,1 13,6Trichoderma harzianum I- 11, 1 13.6
15711571
Rayon de la colonie témoin 36 38,7Radius of the control colony 36 38.7
Tableau 5 : Taux d'inhibition (exprimé en mm) de la croissance mycélienne de deux isolats de Botryosphaeria obtusa face aux clones de Trichoderma harzianum 1-3151 et 1-1571 (mesuré au bout de 5 jours de confrontation)Table 5: Rate of inhibition (expressed in mm) of the mycelial growth of two isolates of Botryosphaeria obtusa against the clones of Trichoderma harzianum 1-3151 and 1-1571 (measured after 5 days of confrontation)
Bo F 98-1 Bo F 99-8Bo F 98-1 Bo F 99-8
Taux d'inhibition par Trichoderma harzianum I- 27,3 24,6 3151Inhibition rate with Trichoderma harzianum I- 27.3 24.6 3,151
Taux d'inhibition par Trichoderma harzianum I- 28,2 24,6 1571Inhibition rate by Trichoderma harzianum I- 28.2 24.6 1571
Rayon de la colonie témoin 48,2 43,9Radius of the control colony 48.2 43.9
Tableau 6 : Taux d'inhibition (exprimé en mm) de la croissance mycélienne de l'isolât de Botryosphaeria dothidea face aux clones de Trichoderma harzianum I- 3151 et 1-1571 (mesuré au bout de 4 jours de confrontation)Table 6: Inhibition rate (expressed in mm) of the mycelial growth of the Botryosphaeria dothidea isolate against the clones of Trichoderma harzianum I-3151 and 1-1571 (measured after 4 days of confrontation)
Bd 021 Taux d'inhibition par Trichoderma harzianum I- 21 ,7 3151 Taux d'inhibition par Trichoderma harzianum I- 31 ,7 1571Bd 021 Inhibition rate by Trichoderma harzianum I- 21, 7 3151 Inhibition rate by Trichoderma harzianum I- 31, 7 1571
Rayon de la colonie témoin 48,6
Tableau 7 : Taux d'inhibition (exprimé en mm) de la croissance mycélienne de l'isolât de Botryosphaeria stevensii face aux clones de Trichoderma harzianum I- 3151 et 1-1571 (mesuré au bout de 5 jours de confrontation)Radius of the control colony 48.6 Table 7: Inhibition rate (expressed in mm) of the mycelial growth of the Botryosphaeria stevensii isolate against the clones of Trichoderma harzianum I-3151 and 1-1571 (measured after 5 days of confrontation)
Bs 001 Taux d'inhibition par Trichoderma harzianum I- 32,7 3151 Taux d'inhibition par Trichoderma harzianum I- 38.1 1571 Rayon de la colonie témoin 54,1Bs 001 Inhibition rate by Trichoderma harzianum I- 32.7 3151 Inhibition rate by Trichoderma harzianum I- 38.1 1571 Radius of the control colony 54.1
L'étude du comportement des deux isolats de Phaeomoniella aleophilum, Fomitiporia punctata, Eutypa lata, Botryosphaeria obtusa, et de l'isolât de Botryosphaeria dothidea, et Botryosphaeria stevensii à l'égard des clones de Trichoderma harzianum 1-3151 et 1-1571 a permis de montrer :Study of the behavior of the two isolates of Phaeomoniella aleophilum, Fomitiporia punctata, Eutypa lata, Botryosphaeria obtusa, and of the isolate of Botryosphaeria dothidea, and Botryosphaeria stevensii with regard to the clones of Trichoderma harzianum 1-3151 and 1-1571 a license to show:
La présence d'une zone d'inhibition entre chacun des deux protagonistes,The presence of an inhibition zone between each of the two protagonists,
Un recouvrement des isolats de chaque espèce par Trichoderma harzianum 1-3151 et 1-1571Coverage of isolates of each species by Trichoderma harzianum 1-3151 and 1-1571
L'existence de fructifications de Trichoderma harzianum 1-3151 et 1-1571 sur chacun des isolats.The existence of fruiting bodies of Trichoderma harzianum 1-3151 and 1-1571 on each of the isolates.
Ce test révèle donc l'importance de l'antagonisme par contact de Trichoderma harzianum 1-3151 et 1-1571 vis à vis de Phaeomoniella aleophilum, Fomitiporia punctata, Eutypa lata, Botryosphaeria obtusa, Botryosphaeria dothidea, Botryosphaeria stevensii. Les clones de Trichoderma harzianum 1-3151 et 1-1571 inhibent bien la croissance des isolats de chaque espèce. Ils recouvrent les colonies de Phaeomoniella aleophilum, Fomitiporia punctata, Eutypa lata, Botryosphaeria obtusa, Botryosphaeria dothidea, Botryosphaeria stevensii et produisent des fructifications à leur surface. La remise des implants de Phaeomoniella aleophilum Fomitiporia punctata, Eutypa lata, Botryosphaeria obtusa, Botryosphaeria dothidea, Botryosphaeria stevensii en culture montre que celles-ci ne se redéveloppent pas ou que très légèrement. Les clones de Trichoderma harzianum 1-3151 et 1-1571
limitent fortement le développement de la croissance mycélienne des isolats de chaque espèce.This test therefore reveals the importance of the antagonism by contact of Trichoderma harzianum 1-3151 and 1-1571 with respect to Phaeomoniella aleophilum, Fomitiporia punctata, Eutypa lata, Botryosphaeria obtusa, Botryosphaeria dothidea, Botryosphaeria stevensii. Clones of Trichoderma harzianum 1-3151 and 1-1571 well inhibit the growth of isolates from each species. They cover the colonies of Phaeomoniella aleophilum, Fomitiporia punctata, Eutypa lata, Botryosphaeria obtusa, Botryosphaeria dothidea, Botryosphaeria stevensii and produce fruiting bodies on their surface. The delivery of implants of Phaeomoniella aleophilum Fomitiporia punctata, Eutypa lata, Botryosphaeria obtusa, Botryosphaeria dothidea, Botryosphaeria stevensii in culture shows that they do not redevelop or only very slightly. The clones of Trichoderma harzianum 1-3151 and 1-1571 strongly limit the development of mycelial growth of isolates from each species.
Un test identique est réalisé pour démontrer le pouvoir antagoniste par contact vis à vis d'autres champignons pathogènes tels que Phellinus igniarius, Phellinus viticola, Phomopsis viticola. Dans tous les cas, les tests ont montré des résultats similaires à ceux obtenus avec les tests de confrontation cités en exemples. L'ensemble de ces tests montre bien l'intérêt des clones de Trichoderma harzianum 1-3151 et 1-1571 pour contrôler le développement de la majorité des champignons du bois. Exemple 4An identical test is carried out to demonstrate the antagonistic power by contact with respect to other pathogenic fungi such as Phellinus igniarius, Phellinus viticola, Phomopsis viticola. In all cases, the tests showed results similar to those obtained with the confrontation tests cited in examples. All of these tests clearly show the advantage of the clones of Trichoderma harzianum 1-3151 and 1-1571 in controlling the development of the majority of wood fungi. Example 4
Préparation de formulation phytosanitairePreparation of phytosanitary formulation
Le produit phytosanitaire est obtenu en mettant en suspension dans l'eau le concentré stérile ou la poudre obtenue selon l'exemple 1 , de façon à obtenir une concentration de 108 spores viables / ml de solution. Selon l'invention, le concentré ou la poudre sont mis en suspension de préférence juste avant l'application par pulvérisation ou badigeonnage sur les ceps de vigne pour les protéger contre les maladies du bois citées. Exemple 5 Adaptation des clones de Trichoderma harzianum 1-3151 et 1-1571 pour les rendre résistants à différents fongicides et bactéricides.The plant protection product is obtained by suspending the sterile concentrate or the powder obtained according to Example 1 in water, so as to obtain a concentration of 10 8 viable spores / ml of solution. According to the invention, the concentrate or the powder is preferably suspended just before application by spraying or brushing on the vine stocks to protect them against the wood diseases mentioned. Example 5 Adaptation of the Trichoderma harzianum 1-3151 and 1-1571 clones to make them resistant to various fungicides and bactericides.
Afin de pouvoir intégrer l'utilisation des clones de Trichoderma dans un programme de traitement incluant soit des fongicides classiques soit des bactéricides, il est nécessaire de rendre ces clones résistants à divers agents chimiques, en particulier les désinfectants des bois (cuivres, molécules de synthèses, etc..) On procède donc à une adaptation des clones de Trichoderma harzianum 1-3151 et 1-1571 en les confrontant à des quantités croissantes de fongicides ou de bactéricides chimiques.In order to integrate the use of Trichoderma clones in a treatment program including either conventional fungicides or bactericides, it is necessary to make these clones resistant to various chemical agents, in particular wood disinfectants (copper, synthetic molecules , etc.) We therefore proceed to an adaptation of the clones of Trichoderma harzianum 1-3151 and 1-1571 by confronting them with increasing amounts of fungicides or chemical bactericides.
Le procédé d'adaptation est caractérisé en ce qu'il comprend les étapes suivantes : Evaluation des quantités minimales inhibitrices des antifongiques ou bactéricides : l'appréciation de ces quantités se fait en observant, pour chaque fongicide ou pour chaque bactéricide, le développement des clones de Trichoderma mis en culture sur milieu gélose à base de flocon d'avoine auquel on a ajouté des quantités croissantes du fongicide ou du bactéricide. Lorsqu'il y a arrêt de la croissance des clones de Trichoderma harzianum, la quantité minimale inhibitrice du fongicide ou bactéricide est atteinte.
une quantité de fongicide ou bactéricide, inférieure à la quantité minimale inhibitrice est ajouté à un milieu gélose à base de flocons d'avoine et sur lequel on étend une suspension de spores de Trichoderma harzianum 1-3151 ou 1-1571. Les boîtes sont mises à incuber pendant 5 jours, à 24°C, sous lumière. Suite à ce temps d'incubation, on voit apparaître quelques colonies de Trichoderma harzianum. Ces colonies sont prélevées puis repiquées sur un milieu gélose à base de flocons d'avoine contenant une concentration de fongicide ou de bactéricide supérieure ou égale à celle précédemment utilisée. Cette opération est répétée jusqu'à l'obtention de colonies résistantes aux quantités du fongicide ou du bactéricide habituellement utilisé en viticulture. Exemple 6The adaptation process is characterized in that it comprises the following stages: Evaluation of the minimum inhibitory quantities of antifungals or bactericides: the appreciation of these quantities is made by observing, for each fungicide or for each bactericide, the development of the clones of Trichoderma cultured on an oatmeal agar medium to which increasing amounts of the fungicide or bactericide have been added. When the growth of the Trichoderma harzianum clones is stopped, the minimum fungicidal or bactericidal inhibitory amount is reached. an amount of fungicide or bactericide, less than the minimum inhibiting amount, is added to an agar medium based on oat flakes and on which a suspension of spores of Trichoderma harzianum 1-3151 or 1-1571 is spread. The dishes are incubated for 5 days, at 24 ° C., under light. Following this incubation time, a few colonies of Trichoderma harzianum appear. These colonies are removed and then subcultured on an agar medium based on oat flakes containing a concentration of fungicide or bactericide greater than or equal to that previously used. This operation is repeated until colonies resistant to the amounts of fungicide or bactericide usually used in viticulture are obtained. Example 6
Tests d'efficacité de Trichoderma harzianum 1-3151 et 1-1571 vis à vis de Phaeomoniella aleophilum, Fomitiporia punctata, Eutypa lata, Botryosphaeria obtusa, Botryosphaeria dothidea, Botryosphaeria stevensii Différents types d'essais d'efficacité des clones de Trichoderma à encontre des pathogènes des bois directement dans les plants de vigne ont été testés. Les premiers concernent les capacités des Trichoderma à coloniser les porte-greffes, les seconds à constater les performances anti-pathogéniques. Sur des jeunes plants de vigne, on applique le processus suivant : traitement au cryptonol puis greffage et chauffage optionnel, puis stratification en caisse dans une eau variant entre 28°C et 30°C pendant 3 semaines. C'est dans cette eau de trempage que sont introduits les divers micro-organismes antagonistes ou pathogènes, a) Comportement de Trichoderma dans les plants après divers traitements Les modalités testées concernent : eau courante; eau + chauffage ; eau + Trichoderma 107spores par ml de produit ; eau + chauffage + Trichoderma 107spores par ml de produit ; eau courante + sulfate de cuivre à 10g par 1001 d' eau ; eau courante + sulfate de cuivre + Trichoderma 107spores par ml de produit. On observe alors la colonisation par les Trichoderma après différentes périodes de 15 jours, en réalisant des coupes dans les bois, en observant au microscope et en mettant en milieu de culture ces dernières. On pratique quatre niveaux de coupes : 1 = Base; 2 = Oeil; 3 = sous la greffe; 4 = au-dessus de la greffe. On observe aussi les taux de reprise exprimés par la création du cal, du système racinaire et foliaire.
Matériel végétalEfficacy tests of Trichoderma harzianum 1-3151 and 1-1571 against Phaeomoniella aleophilum, Fomitiporia punctata, Eutypa lata, Botryosphaeria obtusa, Botryosphaeria dothidea, Botryosphaeria stevensii Different types of efficacy tests of Trichoderma clones against wood pathogens directly in vine plants have been tested. The first concerns the capacities of Trichoderma to colonize rootstocks, the second to observe anti-pathogenic performances. On young vine plants, the following process is applied: treatment with cryptonol then grafting and optional heating, then stratification in boxes in water varying between 28 ° C and 30 ° C for 3 weeks. It is in this soaking water that the various antagonistic or pathogenic microorganisms are introduced, a) Behavior of Trichoderma in the plants after various treatments The methods tested relate to: running water; water + heating; water + Trichoderma 10 7 spores per ml of product; water + heating + Trichoderma 10 7 spores per ml of product; running water + copper sulphate at 10g per 1001 of water; running water + copper sulphate + Trichoderma 10 7 spores per ml of product. Colonization by Trichoderma is then observed after different periods of 15 days, by making cuts in the woods, observing under a microscope and putting them in a culture medium. We practice four levels of cuts: 1 = Basic; 2 = Eye; 3 = under the graft; 4 = above the graft. We also observe the recovery rates expressed by the creation of callus, the root and leaf systems. Plant material
* Lots de 30 plants par modalité soit 180 plants par porte-greffe* Lots of 30 plants per modality, i.e. 180 plants per rootstock
* Essai sur 3 porte-greffes types Richter 10 ,504,3309* Test on 3 Richter 10 rootstocks, 504.3309
* Un seul greffon étudié. Par la suite les boutures ont été plantées en terre et en pots et des observations ont été faites deux fois par an, en vue de voir les effets de l'inoculation de Trichoderma sur le comportement de ces plants en milieu réel. Ces observations ont consisté aux prélèvements de 20 coursons ( de longueur 10 cm aux moins ) pour chaque modalité. Des tests biologiques ont été effectués au laboratoire pour connaître les aptitudes de Trichoderma à s'installer dans les tissus et à créer une barrière protectrice dans le bois. b) Efficacité du Trichoderma contre la maladie en pépinières Le principe de cette étude consiste à inoculer des boutures, racinées ou non, avec les champignons pathogènes respectifs. Les champignons pathogènes Phaeomoniella aleophilum, Fomitiporia punctata sont isolés de plantes atteintes d'Esca. Le champignon pathogène Eutypa lata est isolé de ceps atteints d'Eutypiose. Les champignons pathogènes Botryosphaeria obtusa, Botryosphaeria dothidea et Botryosphaeria stevensii sont isolés de ceps soit atteints par le dépérissement de la Syrah, soit manifestant des symptômes de Black Dead Arm ou encore de greffés-soudés. L'efficacité potentielle du produit est jugée en fonction du comportement des plantes traitées par rapport à celui de plantes témoins (et inoculées). Le type de résultat attendu est une diminution en fréquence et/ou en intensité des symptômes foliaires. Matériel végétal Plusieurs lots de boutures sont exploités, inoculés et cultivés en serre. Trois lots différents de boutures sont ainsi utilisés :* Only one graft studied. Subsequently the cuttings were planted in the ground and in pots and observations were made twice a year, in order to see the effects of the inoculation of Trichoderma on the behavior of these plants in real environment. These observations consisted of taking 20 coursons (at least 10 cm long) for each modality. Biological tests were carried out in the laboratory to determine the ability of Trichoderma to settle in the tissues and to create a protective barrier in the wood. b) Effectiveness of Trichoderma against disease in nurseries The principle of this study consists in inoculating cuttings, rooted or not, with the respective pathogenic fungi. The pathogenic fungi Phaeomoniella aleophilum, Fomitiporia punctata are isolated from plants with Esca. The pathogenic fungus Eutypa lata is isolated from vines with Eutypiosis. The pathogenic fungi Botryosphaeria obtusa, Botryosphaeria dothidea and Botryosphaeria stevensii are isolated from vines either affected by dieback of Syrah, or showing symptoms of Black Dead Arm or even grafted-soldered. The potential effectiveness of the product is judged according to the behavior of the treated plants compared to that of control plants (and inoculated). The type of result expected is a decrease in frequency and / or intensity of leaf symptoms. Plant material Several batches of cuttings are exploited, inoculated and cultivated in the greenhouse. Three different batches of cuttings are thus used:
- boutures fructifères âgées d'un an,- fruiting cuttings one year old,
- boutures non racinées « courtes », à un seul mérithalle d'une longueur de 8 à 10 cm, - boutures foliaires à deux ou trois mérithalles, longues de 25 cm à 30cm. Production de mycélium- "short" unrooted cuttings, with a single merithalle 8 to 10 cm long, - leaf cuttings with two or three merithalles, long from 25 cm to 30cm. Mycelium production
Les isolats de pathogènes et de Trichoderma ont été cultivés en boîtes de Pétri sur du milieu malt-agar (respectivement 15g/l et 20 g/l) à la température de 20 à 25°C durant 7 à 21 jours. Un volume d'eau d'environ 5mL est déposé dans chacune des boîtes. Le mycélium est séparé de la gélose en grattant la surface avec un râteau
en verre ou la lame stérile d'un scalpel. Il est ensuite récupéré avec une micropipette et un embout dont l'extrémité a été coupée au scalpel pour permettre l'aspiration des hyphes mycéliens. Le prélèvement de mycélium est alors placé dans un tube plastique. Le volume est ajusté si nécessaire (suspension trop dense) avec de l'eau stérile. Le tube est fortement agité manuellement afin d'homogénéiser la suspension. Inoculation des bouturesThe pathogen and Trichoderma isolates were cultivated in petri dishes on malt-agar medium (15 g / l and 20 g / l respectively) at a temperature of 20 to 25 ° C for 7 to 21 days. A volume of water of around 5mL is placed in each of the boxes. The mycelium is separated from the agar by scraping the surface with a rake glass or the sterile blade of a scalpel. It is then collected with a micropipette and a tip, the end of which has been cut with a scalpel to allow aspiration of the mycelial hyphae. The mycelium sample is then placed in a plastic tube. The volume is adjusted if necessary (suspension too dense) with sterile water. The tube is strongly agitated manually in order to homogenize the suspension. Inoculation of cuttings
La technique d'inoculation utilisée est celle précédemment exploitée avec succès par Laurence Chapuis (1995) pour reproduire des nécroses dans le bois. J.P. Péros a lui aussi de son côté mis au point en 1995 un autre modèle de reproduction des symptômes foliaires avec des boutures mises à raciner dans un support artificiel (pouzzolane) : ce modèle avait permis de comparer la sensibilité de différents cépages et l'agressivité de différents isolats en quelques mois seulement. Les boutures racinées ou non racinées et cultivées en serre ont donc été inoculées de la façon suivante : l'écorce sous l'œil est désinfectée avec un coton imbibé d'alcool, un trou de 2 mm de diamètre est percé jusqu'à la moelle, environ 2 cm en dessous de l'œil, sur l'axe orthoptique de la bouture (à la verticale de l'œil) à l'aide d'une petite perceuse munie d'une mèche stérilisée à la flamme. Le mycélium du pathogène en suspension dans l'eau (environ 20 μl) est injecté à l'aide d'une pipette dans le trou d'inoculation qui est immédiatement recouvert par de la paraffine chaude. Les modalités de traitement sont :The inoculation technique used is that previously successfully exploited by Laurence Chapuis (1995) to reproduce necrosis in wood. JP Péros, for its part, also developed in 1995 another model for reproducing leaf symptoms with cuttings rooted in an artificial support (pozzolana): this model had made it possible to compare the sensitivity of different grape varieties and the aggressiveness different isolates in just a few months. The rooted or non-rooted cuttings grown in the greenhouse were therefore inoculated as follows: the bark under the eye is disinfected with cotton wool soaked in alcohol, a hole of 2 mm in diameter is drilled to the marrow , about 2 cm below the eye, on the orthoptic axis of the cutting (vertical to the eye) using a small drill fitted with a flame sterilized drill bit. The mycelium of the pathogen suspended in water (about 20 μl) is injected using a pipette into the inoculation hole which is immediately covered with hot paraffin. The processing methods are:
- une seule dose de champignons antagonistes ( Trichoderma à 107spores par ml de produit), - une dose d'inoculum du champignon pathogène seul,- a single dose of antagonistic fungi (Trichoderma with 10 7 spores per ml of product), - a dose of inoculum of the pathogenic fungus alone,
- une dose d'inoculum du champignon pathogène suivit 24 heures après d'une dose de champignons antagonistes,- a dose of inoculum of the pathogenic fungus followed 24 hours after a dose of antagonistic fungi,
- un témoin non traité. Analyses On observe alors la colonisation par les micro-organismes après différentes périodes de 15 jours, en réalisant des coupes dans les bois, en observant au microscope et en mettant en milieu de culture ces dernières. On pratique quatre niveaux de coupes : 1 = Base; 2 = Oeil; 3 = sous la greffe; 4 = au-dessus de la greffe. On observe aussi les taux de reprise exprimés par la création du cal, du système racinaire et foliaire. Par la suite les boutures ont été plantées en terre et en
pots en vue de voir les effets de l'inoculation de Trichoderma sur le comportement de ces plants en milieu réel. L'efficacité est évaluée par au moins deux observations par an en fonction de l'amélioration constatée sur chacun des ceps (rémission des symptômes ou stabilisation) et par comparaison du comportement des ceps traités par rapport à celui des témoins. c) Efficacité de Trichoderma contre Phaeomoniella chiamydospora (Esca) et Eutypa lata (Eutypiose et Esca)- an untreated witness. Analyzes Colonization by microorganisms is then observed after different periods of 15 days, by making cuts in the woods, observing under a microscope and putting them in a culture medium. We practice four levels of cuts: 1 = Basic; 2 = Eye; 3 = under the graft; 4 = above the graft. We also observe the recovery rates expressed by the creation of callus, the root and leaf systems. Subsequently the cuttings were planted in the ground and in pots in order to see the effects of the inoculation of Trichoderma on the behavior of these plants in real environment. Efficacy is evaluated by at least two observations per year depending on the improvement observed on each of the stocks (remission of symptoms or stabilization) and by comparison of the behavior of the treated stocks compared to that of the controls. c) Effectiveness of Trichoderma against Phaeomoniella chiamydospora (Esca) and Eutypa lata (Eutypiosis and Esca)
Le principe de ces études a consisté :The principle of these studies consisted of:
- dans un premier temps, à identifier et repérer des souches malades isolées (mise en évidence de l'effet curatif) ou des souches apparemment saines (mise en évidence de l'effet préventif) dans des parcelles de vignobles atteints d'Esca,- firstly, to identify and locate isolated sick strains (highlighting the curative effect) or apparently healthy strains (highlighting the preventive effect) in plots of vineyards affected with Esca,
- puis à appliquer le produit,- then apply the product,
- pour observer ensuite pendant deux années consécutives au moins l'évolution de la symptomatologie dans les zones traitées ou non. Les modalités de traitement sont :- to then observe, for at least two consecutive years, the evolution of the symptomatology in the areas treated or not. The processing methods are:
- une seule dose de champignons antagonistes (107 spores par ml de produit),- a single dose of antagonistic fungi (10 7 spores per ml of product),
- deux doses d'inoculum (1 et 1/3) du champignon pathogène ; soit pour Eutypa lata : 150 et 200/3 spores, et pour Phaeomoniella chiamydospora : 500 et 500/3 spores, - une seule date d'inoculation : 24 heures après l'application des produits. Méthode d'évaluation de l'efficacité- two doses of inoculum (1 and 1/3) of the pathogenic fungus; either for Eutypa lata: 150 and 200/3 spores, and for Phaeomoniella chiamydospora: 500 and 500/3 spores, - a single date of inoculation: 24 hours after application of the products. Efficacy evaluation method
Chaque cep a fait l'objet au moins d'une observation par saison. L'efficacité a été évaluée en fonction de l'amélioration constatée sur chacun des ceps (rémission des symptômes ou stabilisation) et par comparaison du comportement des ceps traités par rapport à celui des témoins. Cette méthode a conduit, pour les besoins de l'analyse statistique, à répartir les ceps en trois classes :Each vine was subject to at least one observation per season. The effectiveness was evaluated according to the improvement observed on each of the stocks (remission of symptoms or stabilization) and by comparison of the behavior of the treated stocks compared to that of the controls. For the purposes of statistical analysis, this method led to dividing the stocks into three classes:
- cep montrant un symptôme plus grave que lors de la première notation,- vine showing a symptom more serious than at the first scoring,
- cep montrant un symptôme équivalent (stabilisation),- vine showing an equivalent symptom (stabilization),
- cep montrant un symptôme moins grave. Les résultats sont analysés par un simple test de X2 adapté à la comparaison de deux distributions observées. Le résultat est une différence significative entre les deux distributions autorisant ensuite l'interprétation des effectifs dans les différentes classes. Le résultat agronomique est une régression des dégâts dus à ces dépérissements se traduisant par un nombre de « ceps montrant un symptôme
moins grave » plus grand chez les ceps traités et inversement un nombre plus petit de « ceps montrant un symptôme équivalent ou moins grave ». Selon l'invention, les clones de Trichoderma harzianum 1-3151 et 1-1571 utilisés dans le traitement des maladies du bois en viticulture sont de préférence les clones rendus résistants aux fongicides et bactéricides suivant le procédé cité ci- dessus.
- vine showing a less severe symptom. The results are analyzed by a simple test of X 2 adapted to the comparison of two observed distributions. The result is a significant difference between the two distributions, thus authorizing the interpretation of the numbers in the different classes. The agronomic result is a regression of the damage due to these wastage resulting in a number of “vines showing a symptom less severe ”greater in the stocks treated and conversely a smaller number of“ stocks showing an equivalent or less severe symptom ”. According to the invention, the clones of Trichoderma harzianum 1-3151 and 1-1571 used in the treatment of wood diseases in viticulture are preferably the clones made resistant to fungicides and bactericides according to the process mentioned above.
Claims
REVENDICATIONS
1- Clones de Trichoderma harzianum caractérisés en ce qu'il s'agit des clones de Trichoderma harzianum déposés à la Collection Nationale de Cultures de Micro-organismes (CNCM) de l'Institut Pasteur sous les numéros 1-3151 et 1-1571. 2- Clones de Trichoderma harzianum, selon la revendication 1 , caractérisés en ce qu'ils possèdent les aspects culturaux suivants :1- Clones of Trichoderma harzianum characterized in that they are clones of Trichoderma harzianum deposited in the National Collection of Cultures of Microorganisms (CNCM) of the Pasteur Institute under the numbers 1-3151 and 1-1571. 2- Trichoderma harzianum clones, according to claim 1, characterized in that they have the following cultural aspects:
- des colonies d'abord blanches puis devenant vertes, d'aspect irrégulier et s'organisant en agrégats plus ou moins condensés ;- colonies that are initially white and then become green, irregular in appearance and organized into more or less condensed aggregates;
- des arborescences terminées par des formations sphériques irrégulières qui se différencient à partir d'un mycélium plus ou moins dense, étalé sur le milieu gélose, - l'organisation des structures conidiogènes est telle que des ramifications se répartissent perpendiculairement et de façon opposée de part et d'autre d'un axe principal, le conidiophore ;- trees ending in irregular spherical formations which differentiate from a more or less dense mycelium, spread over the agar medium, - the organization of the conidiogenic structures is such that the branches are distributed perpendicularly and in opposite ways from one side and on the other side of a main axis, the conidiophore;
- des cellules conidiogènes (phialides) apparaissent ampulliformes ;- conidiogenic cells (phialides) appear ampulliform;
- des spores, à paroi lisse, sont de forme ovalaire et de couleur verte (environ 3 μm de diamètre) ;- spores, with smooth walls, are oval in shape and green in color (approximately 3 μm in diameter);
- une température de croissance optimale de 20 à 25°C sur milieux classiques comme le malt-agar, le PDA, pulpe de betterave, flocons d'avoine, avec un pH compris entre 4 et 7 ;- an optimal growth temperature of 20 to 25 ° C on conventional media such as malt-agar, PDA, beet pulp, oat flakes, with a pH between 4 and 7;
- une croissance aérobie, la sporulation plus importante sur un milieu riche. 3- Procédé d'isolement des clones de Trichoderma harzianum, selon la revendication 1 ou 2, caractérisé en ce qu'il comprend les étapes suivantes : a) prélèvement de champignons Trichoderma harzianum dans un milieu adéquat comme un sol humide (sols maraîchers, viticole ou arboricole, matières organiques en décomposition) ou dans des bois pourris issus de ceps de vigne ou de branches d'arbres fruitiers ; b) isolement des populations de champignons Trichoderma harzianum prélevées, sur des milieux spécifiques choisis, de préférence le Milieu TME de Papavizas (1982), le Milieu TSM d'Elad et al. (1981) ou encore le Milieu de Davet (1979) ; c) croissance des colonies de populations de Trichoderma en boîte de Pétri à 24°C, à la lumière pendant 3 à 7 jours ; d) isolements monosporaux de chacune des colonies : les spores de couleur verte sont prélevées, puis mises en suspension dans l'eau stérile afin de les dissocier ; e) réalisation d'une culture séparée pour chaque spore dissociée sur un milieu gélose riche tel le PDA ou flocons d'avoine en boîte de Pétri ;
f) après 48 heures, l'ensemble des spores est remis en suspension et le titre est ajusté à 106 spores/millilitre. 4- Procédé de sélection des clones de Trichoderma harzianum obtenus selon le procédé de la revendication 3, présentant la meilleure croissance, la meilleure production de spores et le pouvoir antagonisme le plus important vis à vis des champignons responsables des maladies du bois de la vigne, caractérisé en ce qu'il comprend : a) une croissance des clones de Trichoderma harzianum en milieu pauvre par un prélèvement de chaque clone issu de la suspension obtenue dans la revendication 3, que l'on inocule dans un milieu gélose pauvre, notamment à l'extrait de malt (20 g/l) en boîte de Pétri et incubé à 24°C avec observation de la croissance des clones toutes les 24 heures et évaluation de la qualité de cette croissance pour les différents clones ; b) une croissance des clones de Trichoderma harzianum à basse température par un prélèvement de chaque clone issu de la suspension obtenue dans la revendication 3, que l'on inocule dans un milieu gélose riche, par exemple au PDA (40 g/l) en boîte de Pétri et incubé à 15°C avec observation de la croissance des clones toutes les 24 heures et évaluation de la qualité de cette croissance pour les différents clones. ; c) une évaluation de la meilleure production de spores par unité de surface de milieu gélose par un prélèvement de chaque clone issu de la suspension selon la revendication 3, que l'on inocule dans un milieu gélose riche, par exemple au PDA (40 g/l) en boîte de Pétri et incubé à 24°C ; d) une estimation du pouvoir antagoniste des clones vis à vis des champignons Phaeoacremonium aleophilum, Phaeomoniella chiamydospora, Fomitiporia punctata, Phellinus igniarius, Phellinus viticola, Eutypa lata, Phomopsis viticola et Botryosphaeria sp, par un prélèvement de chaque clone issu de la suspension de spores, que l'on inocule dans une boîte de Pétri contenant du milieu nutritif malt - agar préalablement ensemencé par le pathogène concerné avec incubation réalisée à 24°C pendant 5 jours et mesure du pouvoir antagoniste des clones soit par contact, soit à distance vis à vis du pathogène. 5- Procédé de culture des clones de Trichoderma harzianum obtenus selon le procédé de la revendication 4, caractérisé en ce qu'il consiste à cultiver lesdits clones par une méthode de culture en surface réalisée dans un fermenteur à disques (fermentation sur milieu gélose PDA) comprenant les étapes suivantes :
a) inoculation du milieu de culture effectuée dans le réacteur au moyen de 3. 108 spores/g de substrat carboné initialement présent ; b) incubation réalisée en plaçant le fermenteur dans une enceinte thermostatée à 24°C pendant 7 jours ; c) récolte des spores produites à la surface des disques par lavages successifs à l'eau distillée stérile additionnée de tween 80 à 1 % et de glycérol ; d) comptage des spores en suspension sur cellule de Malassez ; e) stérilisation de leur mise en ampoules scellées, puis conservation à températures négatives. 6- Procédé d'obtention d'un produit à base de clones de Trichoderma harzianum obtenus selon le procédé de la revendication 5, caractérisé en ce qu'il consiste : a) à filtrer stérilement le liquide contenant les spores ; b) à broyer et tamiser finement le filtrat préalablement séché. 7- Utilisation de clones de Trichoderma harzianum selon la revendication 1 ou 2, ou obtenus selon le procédé de la revendication 3, 4 ou 5 ou d'un produit obtenu selon le procédé de la revendication 6, comme produits phytosanitaires en tant que moyen de lutte biologique. 8- Utilisation de produits obtenus à partir de clones selon la revendication 1 ou 2, ou obtenus selon le procédé de la revendication 3, 4, 5 ou d'un produit obtenu selon le procédé de la revendication 6, comme moyen de lutte biologique et comme produit phytosanitaire, par pulvérisation ou badigeonnage des ceps de vignes, ou autres plantes pérennes. 9- Application de clones de Trichoderma harzianum selon la revendication 1 ou 2, ou obtenus selon le procédé de la revendication 3, 4, 5 ou d'un produit obtenu selon le procédé de la revendication 6, en tant que produit phytosanitaire pour lutter contre les champignons responsables des maladies du bois de la vigne et en particulier : Phaeoacremonium aleophilum, Phaeomoniella chiamydospora, Fomitiporia punctata, Phellinus igniarius, Phellinus viticola, Eutypa lata, Phomopsis viticola et Botryosphaeria sp , mais aussi contre les maladies des bois des arbres fruitiers, d'ornement.
- aerobic growth, greater sporulation on a rich medium. 3- A method of isolating the clones of Trichoderma harzianum, according to claim 1 or 2, characterized in that it comprises the following stages: a) sampling of Trichoderma harzianum mushrooms in an adequate medium such as moist soil (market garden soil, wine-growing or arboreal, decaying organic matter) or in rotten wood from vine stocks or fruit tree branches; b) isolation of populations of Trichoderma harzianum fungi taken from selected specific media, preferably TME medium from Papavizas (1982), TSM medium from Elad et al. (1981) or even the Middle of Davet (1979); c) growth of the colonies of Trichoderma populations in a petri dish at 24 ° C., in the light for 3 to 7 days; d) monosporal isolates from each of the colonies: the green spores are taken, then suspended in sterile water in order to dissociate them; e) carrying out a separate culture for each dissociated spore on a rich agar medium such as PDA or oat flakes in a Petri dish; f) after 48 hours, all of the spores are resuspended and the titer is adjusted to 10 6 spores / milliliter. 4- A method for selecting the clones of Trichoderma harzianum obtained according to the method of claim 3, having the best growth, the best production of spores and the greatest antagonism with respect to the fungi responsible for diseases of the wood of the vine, characterized in that it comprises: a) a growth of the clones of Trichoderma harzianum in poor medium by a sample of each clone resulting from the suspension obtained in claim 3, which is inoculated in a poor agar medium, in particular at l 'malt extract (20 g / l) in Petri dish and incubated at 24 ° C with observation of the growth of the clones every 24 hours and evaluation of the quality of this growth for the different clones; b) growth of the Trichoderma harzianum clones at low temperature by taking a sample from each clone obtained from the suspension obtained in claim 3, which is inoculated into a rich agar medium, for example PDA (40 g / l) in Petri dish and incubated at 15 ° C with observation of the growth of the clones every 24 hours and evaluation of the quality of this growth for the different clones. ; c) an evaluation of the best production of spores per unit area of agar medium by sampling each clone from the suspension according to claim 3, which is inoculated into a rich agar medium, for example PDA (40 g / l) in Petri dish and incubated at 24 ° C; d) an estimate of the antagonistic power of the clones with respect to the fungi Phaeoacremonium aleophilum, Phaeomoniella chiamydospora, Fomitiporia punctata, Phellinus igniarius, Phellinus viticola, Eutypa lata, Phomopsis viticola and Botryosphaeria sp, by taking a sample of each clone from the suspension , which is inoculated into a Petri dish containing malt-agar nutrient medium previously seeded with the pathogen concerned with incubation carried out at 24 ° C. for 5 days and measurement of the antagonistic power of the clones either by contact or remotely screw of the pathogen. 5- A method of culturing Trichoderma harzianum clones obtained according to the method of claim 4, characterized in that it consists in cultivating said clones by a surface culture method carried out in a disc fermenter (fermentation on PDA agar medium) including the following steps: a) inoculation of the culture medium carried out in the reactor using 3. 10 8 spores / g of carbon substrate initially present; b) incubation carried out by placing the fermenter in a chamber thermostatically controlled at 24 ° C for 7 days; c) harvesting of the spores produced on the surface of the discs by successive washes with sterile distilled water added with 1% tween 80 and glycerol; d) counting of spores in suspension on a Malassez cell; e) sterilization of their placing in sealed ampoules, then storage at negative temperatures. 6- A process for obtaining a product based on Trichoderma harzianum clones obtained according to the process of claim 5, characterized in that it consists of: a) sterile filtering the liquid containing the spores; b) grinding and finely sieving the previously dried filtrate. 7- Use of Trichoderma harzianum clones according to claim 1 or 2, or obtained according to the process of claim 3, 4 or 5 or of a product obtained according to the process of claim 6, as phytosanitary products as a means of biological control. 8- Use of products obtained from clones according to claim 1 or 2, or obtained according to the process of claim 3, 4, 5 or of a product obtained according to the process of claim 6, as a means of biological control and as a phytosanitary product, by spraying or brushing vine stocks, or other perennial plants. 9- Application of Trichoderma harzianum clones according to claim 1 or 2, or obtained according to the process of claim 3, 4, 5 or of a product obtained according to the process of claim 6, as a phytosanitary product for combating the fungi responsible for vine wood diseases and in particular: Phaeoacremonium aleophilum, Phaeomoniella chiamydospora, Fomitiporia punctata, Phellinus igniarius, Phellinus viticola, Eutypa lata, Phomopsis viticola and Botryosphaeria sp, but also against diseases of the woods of fruit trees, 'ornament.
Applications Claiming Priority (2)
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|---|---|---|---|
| FR0400076 | 2004-01-07 | ||
| FR0400076A FR2864832B1 (en) | 2004-01-07 | 2004-01-07 | CLICHES OF TRICHODERMA HERZIANUM, METHODS OF ISOLATION AND CULTURE AND APPLICATION AS PHYTOSANITARY PRODUCT |
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| PCT/FR2005/000008 WO2005068609A1 (en) | 2004-01-07 | 2005-01-05 | Trichoderma harzianum clones, method for isolating and culturing and for the use thereof in the form of a phytosanitary product |
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| FR (1) | FR2864832B1 (en) |
| WO (1) | WO2005068609A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019142009A1 (en) * | 2018-01-16 | 2019-07-25 | Debreceni Egyetem | Method for the treatment of grapevine diseases |
| WO2023084126A1 (en) * | 2021-11-15 | 2023-05-19 | Bes, Claude | Method for sampling, isolating, selecting, propagating and inoculating indigenous microbial populations of soils |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007110686A2 (en) * | 2006-03-28 | 2007-10-04 | Council Of Scientific And Industrial Research | A synergistic composition useful as bioinoculant |
| FR2915999B1 (en) | 2007-05-09 | 2013-01-18 | Agrolor | ATROVIRID TRICHODERMA STRAIN AND ITS USE AS A PHYTOSANITARY PRODUCT FOR VINE DISEASES. |
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| US4748021A (en) * | 1983-07-28 | 1988-05-31 | Yissum Research And Development Company Of The Hebrew University Of Jerusalem | Antifungal compositions containing trichoderma active against fusarium |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019142009A1 (en) * | 2018-01-16 | 2019-07-25 | Debreceni Egyetem | Method for the treatment of grapevine diseases |
| WO2023084126A1 (en) * | 2021-11-15 | 2023-05-19 | Bes, Claude | Method for sampling, isolating, selecting, propagating and inoculating indigenous microbial populations of soils |
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| Publication number | Publication date |
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| FR2864832B1 (en) | 2006-02-24 |
| FR2864832A1 (en) | 2005-07-08 |
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