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
• • 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
  • 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

• TITLE NEW BIOCONTROL AGENT AND ITS USE FOR THE CONTROL OF FUNGAL DISEASES OF PLANTS
• the present invention relates to a novel biocontrol agent and its use for the control of fungal diseases of plants.
• ⁇ leaf diseases eg powdery mildew, septoria, helminthosporiosis, yellow rust and brown rust
• ⁇ and ear diseases eg, fusarium head blight, ear powdery mildew and septoria.
• “Wheat septoria” refers to two diseases:
• Phaeosphaeria nodorum anamorph / asexual form: Stagonospora nodorum which attacks the leaves but also the ears and grains of wheat;
• Fusarium head blight is associated with a complex of fungal species belonging to the genera Fusarium and Microdochium.
• the genus Fusarium was first described in 1809 by Link and owes its name to the Latin "fusus" (spindle) in relation to the spindle-shaped shape of its spores. It belongs to the division of Ascomycetes, to the order of Hypocreales and to the family of Nectriaceae.
• F. graminearum is one of the most problematic species in France due to its production of mycotoxins including trichothecene deoxynivalenol (DON).
• Brown rust of wheat is a disease which usually appears quite late in the spring. It can cause significant damage if poorly controlled.
• the fungus overwinters mainly on the regrowth of cereals and early sowing crops in the form of uredospores. These uredospores germinate in the presence of free water and at temperatures between 15 and 25 ° C. The sporulation period is 5 to 25 days depending on the temperature and the variety. Under favorable conditions, more than 3000 uredospores can be produced per pustule, which explains the character explosive disease.
• the spores are dispersed mainly by the wind (Ali, S., Gladieux, P., Leconte, M., Gautier, A., Justesen, AF, Hovmoller, MS, Enjalbert, J., de Vallavieille-Pope, C. ( 2014). Origin, Migration Routes and Worldwide Population Genetic Structure of the Wheat Yellow Rust Pathogen Puccinia striiformis f.sp. tritici. PLoS Pathogens, 10 (1), el003903; De Vallavieille-Pope, C., Ali, S., & Leconte, M. (2012) Special Report Virulence Dynamics and Regional Structuring of Puccinia striiformis f sp. Tritici in France Between 1984 and 2009.).
• Yellow rust of wheat is a disease which can be very harmful in susceptible varieties if attacked early.
• the cool and humid conditions are favorable to its development.
• Yellow rust spends the winter as mycelium or active spores on regrowth of cereals or crops sown in early fall. This fungus is resistant to freezing temperatures and usually survives the winter on infected plants. In the spring, yellow rust begins to develop and produces active spores, especially since the weather is cool and humid.
• Optimal conditions for spore germination are met when temperatures are between 10 and 13 ° C with 100% relative humidity.
• the spores are dispersed mainly by the wind (Rossi, V., Racca, P., Giosue ', S., Pancaldi, D., & Alberti, F (1997).
• control agents biological or BCA can control target pathogens through one or more modes of action:
• ISR Induced Systemic Resistance
• Trichoderma comprises various species of fungi that may be biocontrol agents
• their mode of action has been the subject of much work. They report in particular a capacity 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 of natural defenses plants, etc. They colonize the roots of herbaceous and woody plants without any damage.
• this fungus can penetrate the roots and promote development, nutrition and disease resistance.
• the effects are uneven from one species to another or even from one strain to another because a good biological control agent must be a good colonizer and a good antagonist, but above all, it must also be compatible with the rhizosphere. and with plants.
• a first aim of the invention therefore consists in providing an effective strain, such as a biological control agent (or biocontrol agent) .
• a second aim of the invention is to provide phytosanitary compositions for the prevention and / or treatment of fungal diseases of plants.
• a third aim of the invention is also to provide phytosanitary coating compositions where, in association with a plant seed, the strain of the invention is used as an agent for accelerating the germination of said seed.
• Another object of the invention is to provide methods for manufacturing said compositions and for implementing the prevention and / or treatment of fungal diseases of plants, and the acceleration of germination.
• a first aspect of the invention relates to an isolated strain of Trichoderma atroviride in which the ech42 gene has a sequence identity of at least 98% with the sequence SEQ ID NO: 1 of the ech42 gene of the TAL-17 strain deposited on July 03. 2018 at the CNCM under number CNCM 1-5333.
• sequence identity of at least 98% with the sequence SEQ ID NO: 1 is meant a sequence identity of at least 98.5%, at least 99% or even at least 99, 5% with the sequence SEQ ID NO: 1 represented by the following nucleic acid sequence:
• sequence identity is measured by conventional tools for comparing sequences known to those skilled in the art such as the algorithms of the BLAST platform or the MatGat program (Campanella, Bitincka and Smalley, 2003) .
• one of the objects of the invention is the isolated strain of Trichoderma atroviride deposited on July 3, 2018 at the CNCM under the number CNCM 1-5333 and / or one of its mutants.
• isolated strain is meant the culture of a single microorganism which has been isolated from various microorganisms present on and / or in the tissues of a leaf fragment. of wheat grown in fields.
• one of its mutants is meant mutant strains obtained by mutations or genetic manipulations of a reference strain, which in the context of the invention is the isolated strain of Trichoderma atroviride TAL-17 filed on July 3, 2018 at the CNCM under the number CNCM 1-5333. These mutants, however, retain the same physiological properties as the reference strain or even may exhibit improved physiological properties (eg. Improvement in biocontrol capacities).
• one of the subjects of the invention also relates to an isolated strain of Trichoderma atroviride, the ech42 gene of which has a sequence identity of at least 98% with the sequence SEQ ID NO: 1 of the ech42 gene of the TAL-17 strain deposited on July 3, 2018 at the CNCM under the number CNCM 1-5333 and / or one of its mutants,
• said isolated strain of Trichoderma atroviride is the TAL-17 strain deposited on July 3, 2018 at the CNCM under the number CNCM 1-5333 and / or one of its mutants, and preferably, said isolated strain of Trichoderma atroviride is the strain TAL-17 filed on July 03, 2018 at the CNCM under number CNCM 1-5333.
• strains ie TAL-17 and its mutants
• qPCR real-time Polymerase Chain Reaction
• the inventors have developed specific tools thereof namely, a TaqMan ® probe GS285741-P1 and a pair of nucleotide primers associated GS285741-F1 and GS285741-R1 (see Table 1), which can amplify a sequence specific to the strains of the invention ⁇ cf. Table 2).
• FAM 6-carboxyfluorescein
• MGB minor groove binder
• Table 2 Target sequence of oligonucleotides and of the TaqMan ® probe in strain TAL-17 and / or its mutants
• one of the subjects of the invention relates to an isolated strain of Trichoderma atroviride, the ech42 gene of which has a sequence identity of at least 98% with the sequence SEQ ID NO: 1 of the ech42 gene of the TAL- strain. 17 filed on 03 July 2018 at the CNCM under number CNCM 1-5333,
• said isolated strain of Trichoderma atroviride is the TAL-17 strain deposited on July 3, 2018 at the CNCM under the number CNCM 1-5333.
• one of the objects of the invention is the isolated strain of Trichoderma atroviride deposited on July 3, 2018 at the CNCM under number CNCM 1-5333.
• another of the objects of the invention relates to the isolated strain of Trichoderma atroviride of the invention in the form of spores, produced by fermentation in solid or liquid medium, said spores possibly in purified form or in the matrix of production, said production matrix being solid or liquid.
• spores in the form of spores
• FMS solid medium
• liquid fermentation is meant any process allowing the development of the microorganism using a production matrix with the presence of free water.
• production matrix any natural and / or synthetic substrate, which allows the development of the microorganism and induces the production of biomass, and / or enzymes, and / or primary and / or secondary metabolites.
• families of enzymes or enzymes produced by the fungus are proteases, chitinases and beta-1,3-glucanase.
• sporestiti in purified form is meant the concentrated spores following the elimination of the major part of the remainder of the matrix after production which has not been used for the formation of the spores, with the help of 'a sieving process for example.
• sporestiti in the production matrix is meant the spores that remain in the production matrix at the end of the manufacturing process, without a purification step.
• Another object of the invention relates to the isolated strain of Trichoderma atroviride of the invention as a biological control agent.
• biological control agent is meant that a strain of Trichoderma atroviride according to the invention can interfere with the growth and / or the survival of pathogens, thus making it possible to control them, via one or more of the methods. action described above (eg via the development of its biomass and / or its production of enzymes and / or secondary metabolites).
• one of the means of demonstrating that a strain of Trichoderma atroviride according to the invention is a biological control agent is to carry out a Dual culture test on culture medium in a Petri dish such as those presented among the examples. below.
• Double culture test is meant a test on a Petri dish in the laboratory on culture medium, where the strain of the microorganism, biological control agent and the strain of the pathogen are cultured on the same dish in order to observe. the effect of one strain on another over time and vice versa.
• Another object of the invention relates to the isolated strain of Trichoderma atroviride of the invention, as an antifungal agent of plants.
• plant antifungal agent is meant that a strain of Trichoderma atroviride according to the invention is capable of combating phytopathogenic fungi.
• one of the means of demonstrating that a strain of Trichoderma atroviride according to the invention is an antifungal agent for plants is to carry out a dual culture test such as those presented among the examples below.
• one of the objects of the invention relates to the isolated strain of Trichoderma atroviride of the invention as a biological control agent
• a second aspect of the invention relates to the use of the isolated strain of Trichoderma atroviride of the invention, in particular in the field of malting.
• the invention relates to the use of said isolated strain of Trichoderma atroviride in the prevention and / or treatment of fungal diseases of plants, in particular of plants belonging to the Poaceae family, in particular wheat and / or corn and / or barley.
• the invention relates to the use of said isolated strain of Trichoderma atroviride in the prevention and / or treatment of fungal diseases of wheat and / or corn and / or barley.
• the invention also relates to the use of said isolated strain of Trichoderma atroviride for promoting the germination of a seed of plants belonging to the Poaceae family, in particular wheat and / or corn and / or barley.
• the invention relates to the use of said isolated strain of Trichoderma atroviride to promote the germination of a seed of wheat and / or corn and / or barley.
• the germination aid advantageously and at the same time allows the prevention and / or treatment of fungal diseases at the time of sowing. The development of a disease after sowing is thus avoided and the yield is optimized via the biostimulant aspect of the strain of the invention.
• this aspect also makes it possible to avoid a significant slowdown in the development of the crop if a period of drought occurs some time after sowing (which can happen in spring) and therefore to avoid loss of yields at harvest.
• a third aspect of the invention relates to a phytosanitary composition
• a phytosanitary composition comprising: the isolated strain of Trichoderma atroviride of the invention as an active principle; or
• BCA biological control agent
• metabolites emitted during the production of the strain or “its metabolites emitted during its production” is meant any molecule produced by the strain of the invention during its development.
• metabolites can be enzymes produced by the fungus such as proteases, chitinases and beta-1,3-glucanase.
• biological control agent is meant a microorganism capable of interfering with the growth and / or survival of pathogens, thus making it possible to control them.
• the strain of the invention TAL-17 (deposited on July 3, 2018 at the CNCM under the number CNCM 1-5333) is an example, but within the meaning of the invention it is a question here of combining the strain of invention to at least one other biological control agent, which is different from the strain of the invention.
• at least one other biological control agent is meant that the plant protection composition of the invention can contain 2 biological control agents (that of the invention and another), just as it can contain 3, 4 or more. 5 (that of the invention and 2, 3 or 4 others).
• one of the objects of the invention relates to the phytosanitary composition described above, said phytosanitary composition being initially (on purchase) in the form of a solid or concentrated liquid composition.
• a solid or concentrated liquid composition can be, for example, obtained by means of a particular formulation of the spores of the isolated strain of Trichoderma atroviride of the invention produced by fermentation in solid or liquid medium, said spores possibly being in purified form or in the production matrix, said production matrix being solid or liquid.
• concentration composition is meant a composition in which the concentration of the active principle (ie the strain of the invention, which can be in the form of spores) is greater than an effective amount of 10 7 spores / g, i.e. 10 7 CFU / g.
• concentration of the active principle ie the strain of the invention, which can be in the form of spores
• concentration of the active principle ie the strain of the invention, which can be in the form of spores
• concentration of the active principle ie the strain of the invention, which can be in the form of spores
• concentration of the active principle ie the strain of the invention, which can be in the form of spores
• greater than an effective amount of 10 7 spores / g is also meant greater than an effective amount of 10 8 spores / g, greater than an effective amount 10 9 spores / g, greater than an effective amount 10 10 spores / g. g, greater than an effective amount of 10 11 spores / g or greater than an effective amount of 10 12 spores / g.
• the user of the aforesaid composition must then incorporate or suspend it in a solid (eg. Soil, etc.) or aqueous (eg water) medium to obtain a dilute composition comprising an effective amount of 10 4 to 10 12 spores / g of the Trichoderma atroviride strain of the invention, which may be in the form of spores.
• a solid eg. Soil, etc.
• aqueous (eg water) medium e.g.
• the resulting diluted solid or liquid composition can then be applied to plants for which prevention and / or treatment of a fungal plant disease is desired.
• the spreading of the aforesaid composition comprises in particular the conventional spreading techniques known to those skilled in the art, which are used to spread solid materials (eg sewage sludge, manure over an area to be treated). , etc.) or liquid (eg pesticides, etc.) of agronomic interest.
• solid materials eg sewage sludge, manure over an area to be treated. , etc.
• liquid eg pesticides, etc.
• one of the subjects of the invention relates to the phytosanitary composition described above, comprising an effective amount of 10 4 to 10 12 spores / g, more particularly 10 7 to 10 9 spores / g,
• said phytosanitary composition optionally being obtained after a preparation step (e.g. dilution by suspending or mixing) making it ready for spreading.
• a preparation step e.g. dilution by suspending or mixing
• the expression "from 10 4 to 10 12 spores / g" can also mean from 10 4 to 10 5 spores / g, from 10 4 to 10 6 spores / g, from 10 4 to 10 7 spores / g, from 10 4 to 10 8 spores / g, from 10 4 to 10 9 spores / g, from 10 4 to 10 10 spores / g, from 10 4 to 10 11 spores / g, from 10 5 to 10 6 spores / g, from 10 5 to 10 7 spores / g, from 10 5 to 10 8 spores / g, from 10 5 to 10 9 spores / g, from 10 5 to 10 10 spores / g, from 10 5 to 10 11 spores / g, from 10 5 to 10 12 spores / g, from 10 6 to 10 7 spores / g, from 10 6 to 10 8 spor
• the latter also relates to a phytosanitary coating composition comprising the isolated strain of Trichoderma atroviride of the invention and optionally at least one fixing agent, said phytosanitary coating composition making it possible to produce a coated seed.
• the invention therefore also relates to a phytosanitary coating composition comprising the isolated strain of Trichoderma atroviride of the invention, said phytosanitary coating composition making it possible to produce a coated seed; a phytosanitary coating composition comprising the isolated strain of Trichoderma atroviride of the invention and at least one fixing agent, said phytosanitary coating composition making it possible to produce a coated seed.
• One of the objects of the invention therefore relates to a coated seed comprising a plant seed, said plant seed being coated with said phytosanitary coating composition and said plant belonging in particular to the Poaceae family, in particular wheat and / or corn and / or barley.
• coated seed is meant a seed selected for sowing, said seed having undergone a particular treatment which has enabled it to be coated in a phytosanitary coating composition.
• the invention relates to the coated seed as described above, said plant being wheat and / or corn and / or barley.
• the invention also relates to the coated seed as described above, said coated seed comprising an effective amount of 10 5 to 10 8 (or 10 6 to 10 7 ) spores / coated seed, preferably 10 6 ( or 10 7 ) coated spores / seed.
• a fourth aspect of the invention relates to the use of the composition of the invention. More particularly, the invention relates to the use of the composition of the invention in the prevention and / or treatment of fungal diseases of plants, in particular of plants belonging to the Poaceae family, in particular wheat and / or corn. and / or barley.
• the invention relates to the use of the composition of the invention in the prevention and / or treatment of fungal diseases of wheat and / or corn and / or barley.
• the invention also relates to the use of the composition of the invention for promoting the germination of a seed of plants belonging to the Poaceae family, in particular wheat and / or corn and / or barley.
• the invention relates to the use of the composition of the invention for promoting the germination of a seed of wheat and / or corn and / or barley.
• one of the subjects of the invention relates to the use of the isolated strain of Trichoderma atroviride of the invention or of the phytosanitary composition described above in the prevention and / or treatment of fungal diseases of plants, in particular of plants belonging to the Poaceae family, in particular wheat and / or corn and / or barley; and or
• a fifth aspect of the invention relates to a method of protecting and / or treating a plant against a disease caused by a pathogenic fungus for plants comprising the application of the composition of the invention to at least part of said plant or soil near said plant.
• the invention relates to said method of the invention, wherein said part of said plant is a leaf, a fruit, a seed.
• one of the objects of the invention relates to a method of protecting and / or treating a plant against a disease caused by a pathogenic fungus for plants comprising the application of the composition of the invention to at least part of said plant or of the soil near said plant,
• said part of said plant is a leaf, a fruit, a seed, and
• said plant belongs to the Poaceae family, in particular wheat and / or corn and / or barley.
• the invention relates to the method described above, in which said composition is applied
• 10 to 500 L / ha also means 10 to 400 L / ha, 10 to 300 L / ha, 10 to 200 L / ha, 10 to 100 L / ha , 100 to 500 L / ha, 200 to 500 L / ha, 300 to 500 L / ha, 400 to 500 L / ha, 50 to 500 L / ha, 50 to 250 L / ha or 250 to 500 L / ha.
• the expression "from 10 10 to 10 14 spores / ha” also means from 10 10 to 10 11 spores / ha, from 10 10 to 10 13 spores / ha, from 10 11 to 10 12 spores / ha, from 10 11 to 10 13 spores / ha, 10 11 to 10 14 spores / ha, 10 12 to 10 13 spores / ha, 10 12 to 10 14 spores / ha or 10 13 to 10 14 spores / ha.
• the invention relates more particularly to the process described above, in which said plant belongs to the Poaceae family, in particular wheat and / or corn and / or barley.
• the invention relates to the process described above, in which said plant is wheat and / or corn and / or barley.
• Septoria tritici is meant the pathogen of septoria.
• Gabberella zeae F. graminearum
• Fusarium wilt the pathogen of Fusarium wilt.
• Prosinia striiformis is understood to mean the pathogen of yellow rust of wheat.
• the invention relates more particularly to the method described above, in which
• the protection of said plant comprises a step of coating the seed with said phytosanitary composition
• the protection of said plant comprises spraying said composition at the time of sowing or at the germination of said plant;
• the protection of said plant comprises either a preventive application at soil level before sowing said phytosanitary composition with the addition of organic amendment, or a preventive application at soil level before sowing said phytosanitary composition on the previous crop; and or
• the treatment of said plant comprises spraying said composition during different phenological stages of the plant to prevent a risk of infection of said pathogenic fungus of the whole or part of the plant.
• Figure 1 represents the visual observations of the Dual culture test against Fusarium graminearum, to the left of the Petri dishes, at 6 (A and B) and 10 (C and D) days of cultures for the TAL-17 strain (A and C) and the BCA C strain (B and D), to the right of the Petri dishes.
• FIG. 2 represents the microscopic observation (x100 magnification) with an inverted microscope of the mycelia of F. graminearum (black arrow) and of the TAL-17 strain (white arrow) on a Petri dish containing PDA medium (potato, dextrose, agar), after 8 days of growth.
• Figure 3 represents the occupied surface (in%) and the associated photographs of the front face of a Petri dish inoculated with a rake with three strains of microorganisms (F. graminearum, TAL-17 and BCA C alone or in mixtures after 7 growing days Average ⁇ sd Different letters indicate a significant difference between the modalities (1-way ANOVA, p ⁇ 0.05).
• FIG. 4 represents the change in the surface area occupied by the BCA TAL-17 and Fusarium graminearum microorganisms alone or as a mixture after depositing 30 ⁇ L in the center of a culture dish with PDA medium after 72 h (A) and after 96 h (B) of culture at 25 ° C under a light hood.
• Figure 5 shows the change in the percentage inhibition of Fusarium graminearum on a dish depending on the formulation used.
• Figure 6 shows the percentage of CIV tubes showing septoria spots as a function of treatment after 18 days and 26 days of disease inoculation.
• FIG. 7 represents the percentage of healthy grains observed at harvest during the tests in the phytotronic room.
• FIG. 8 represents the mass of grains per ear at harvest during the tests in a phytotronic room.
• FIG. 9 represents the percentage of visual symptoms of contamination on ears by Fusarium wilt during the tests in the phytotronic room.
• FIG. 10 represents the mass of grains per ear at harvest during the tests in a phytotronic room.
• FIG. 11 represents the percentage of fusarious grains at harvest during the phytotronic room tests carried out.
• FIG. 12 represents the germination of maize plants after 6 days of growth with a sowing treatment of a suspension of TAL-17 spores at 1.10 5 CFU spores / seed or 1.10 8 CFU spores / seed.
• An aerial treatment is carried out later during the 5-leaf stage and is not taken into account when measuring germination.
• Mean ⁇ sd Different letters indicate a significant difference between the different modalities (1-way ANOVA, p ⁇ 0.05).
• FIG. 13 represents the measurement of the height in height of corn plants after 25 days of growth with a sowing treatment of a suspension of TAL-17 spores at 1.10 5 UFC spores / seed or 1.10 8 UFC spores / seed.
• An aerial treatment is carried out later during the 5-leaf stage and is not taken into account when measuring germination.
• Mean ⁇ sd Different letters indicate a significant difference between the different modalities (1-way ANOVA, p ⁇ 0.05).
• FIG. 14 represents the percentage of germination of seeds of spring wheat (Calixo) in the presence or absence of suspended spores of Trichoderma atroviride TAL-17 at sowing. Culture in a phytotron chamber with photoperiod. Mean ⁇ s.d. Significant difference between the water modality and the TAL-17 modality (1-factor ANOVA, p ⁇ 0.05).
• Figure 15 includes photographs (A) and a graphic (B).
• Figure 16 includes photographs (A) and a graphic (B).
• Line 1 corresponds to BCA alone at the dose of 1.10 4 CFU / mL
• line 2 corresponds to the BCA / Fusa ratio 2/1 mixture (BCA at 1.10 4 CFU / mL and Fusa at 5.10 3 CFU / mL)
• line 3 corresponds to the BCA / Fusa ratio 1/1 mixture (BCA at 1.10 4 UFC / mL and Fusa at 1.10 4 UFC / mL)
• line 4 corresponds to the BCA / Fusa ratio 1/2 mixture (BCA at 1.10 4 CFU / mL and Fusa at 2.10 4 CFU / mL).
• the bottom panel corresponds to the culture of Fusarium graminearum alone, from left to right, at a dose of 2.10 4 , 1.10 4 and
• Figure 17 includes photographs (A) and a graphic (B).
• Line 1 corresponds to BCA alone at a dose of 1.10 4 CFU / mL
• line 2 corresponds to the BCA / Fusa ratio 2/1 mixture (BCA at 1.10 4 UFC / mL and Fusa at 5.10 3 UFC / mL)
• line 3 corresponding to the BCA / Fusa ratio 1/1 mixture (BCA at 1.10 4 UFC / mL and Fusa at 1.10 4 UFC / mL)
• line 4 corresponds to the BCA / Fusa ratio 1/2 mixture (BCA at 1.10 4 UFC / mL and Fusa at 2.10 4 UFC / mL).
• the bottom panel corresponds to the culture of Fusarium graminearum alone, from left to right, at a dose of 2.10 4 , 1.10 4
• the leaf sample from the wheat crop was ground and then brought into contact in an infinitely mixed with a PDA (Potato Dextrose Agar) agar at 50 ° C which contains spores of Fusarium graminearum.
• the dish was cultured at 25 ° C. under a light hood. Thus only the strains being able to reproduce in the presence of Fusarium graminearum developed on the dish.
• TAL-17 was present.
• a specific sample from an area of interest in the box and several successive subcultures made it possible to isolate the TAL-17 strain, which was then deposited on July 3, 2018 at the CNCM under number CNCM 1-5333.
• strains TAL-17 and BCA C both exhibited an ability to occupy space, whereas strain TAL-17 unlike strain BCA C is capable of growing on the mycelium of Fusarium graminearum ( Figure 1).
• the TAL-17 strain therefore presents a much greater mycoparasitism potential than the other reference strain BCA C.
• this mixture also contains 0.5% of the Appyphyt formulation, which is an emulsion of vegetable oil esters (Vegetable oil esters : 60- 80%; Biobased emulsifier; 1-5%; Qsp Water).
• the mixture was inoculated after 2 h of contact.
• the Petri dishes were incubated at 25 ° C. under artificial light (24 hours a day) (fluorescent tube F36WT8 / 2084 Britegro). Measurements of the growth of F.
• endophyte comes from two Greek words “endon” and "phuton” which means respectively "inside” and "plant”.
• the definition of endophyte has changed very often over time.
• the definition concerned any organism that could live inside a plant.
• endophytic organisms are organisms having a way of life with the plant known as “endophism”. That is, endophytic species are able to colonize the internal structures of plants asymptomatically (Hardoim, PR, van Overbeek, LS, Berg, G., Pirttilà, AM, Compant, S., Campisano, A ., ... Sessitsch, A. (2015).
• Endophytes can be microorganisms like fungi. They can colonize the plant at different levels depending on the microorganism.
• the organs concerned can be, depending on the case: the stem, the roots, the leaf segments, the fruits, the buds, the seeds but also in the dead and hollow hyaline cells of the plants (Stçpniewska, Z., & Kuzniar, A. (2013). Endophytic microorganisms— promising applications in bioremediation of greenhouse gases. Applied Microbiology and Biotechnology, 97 (22), 9589-96.).
• Cotton blue (LACP-00D-100 Labkem ® ) with lactophenol was used in this study as a dye for the detection of fungi. Samples of 1 x 0.5 cm wheat leaf and 2.5 x 1.5 cm corn were taken for each modality studied. The procedure is as follows for the treatment of sections:
• the substrate used is a mixture of universal soil and expanded vermiculite (0.5-10 mm) (2: 1 v / v).
• the substrate was moistened 24 hours before sowing with reverse osmosis water.
• the seeds were moistened the day before sowing for 24 h at 4 ° C.
• the pots were placed under a light hood at 25 ° C (16/8 hour photoperiod) and were watered three times a week.
• Fungus mycelium was observed in the leaves and roots of the seedling treated plant in contrast to the untreated control plant.
• the substrate used is a mixture of universal soil and expanded vermiculite (0.5-10 mm) (2: 1 v / v).
• the substrate was moistened 24 hours before sowing with reverse osmosis water.
• the pots were watered three times a week with an increasing volume of RO water as the plant developed.
• Fungus mycelium was observed in the leaves and roots of the seedling treated plant in contrast to the untreated control plant.
• Strain TAL-17 resulted in a 63% reduction in the development of Fusarium graminearum in a Petri dish at 72 h and 78% at 96 h. This reduction rose to 77% at 72 h and to 85% at 96 h when combined with the Appyphyt formulation ( Figure 5). It should be noted that at 96 h the Appyphyt formula alone did not show any inhibition on the growth of Fusarium graminearum.
• the in vitro culture was developed to perform a screening of the selected BCAs potentials against the septoria blight of the wheat Septoria tritici.
• the standard in vitro culture medium has been modified by removing the sucrose to avoid possible contamination.
• the disease and BCA were applied simultaneously by spraying under conditions as close to the field as possible.
• FIG. 6 The percentages of plants exhibiting septoria spots according to the methods are presented in FIG. 6. Two Trichoderma atroviride were tested, including TAL-17. The latter has proved to be the most interesting against this disease. Note the quantity of BCA is in number of viable spores (equivalent to 5.10 11 viable spores / hectare).
• the phytotronic room has the possibility of increasing to 90% RH humidity during infection at the time of flowering of the ears.
• the tray allows you to test 30 planters of 15 plants. The tests were carried out with “Calixo” spring wheat.
• a first test showed an interest for the TAL-17 strain ( Figures 7 and 8). This interest was confirmed thanks to a new test ( Figures 9, 10 and 11). The latter demonstrated an interest in the use of TAL-17 (equivalent to 10 11 UFC spores / ha) with the Appyphyt formulation (equivalent to lL / ha) on wheat for the visual symptoms of the disease and on the mass of grains per ear which are then both statistically different from the contaminated modality to Fusarium graminearum alone. However, the mass of grains per ear when the wheat was treated with TAL-17 and the Appyphyt formula does not reach the mass of uncontaminated control wheat (ie 0.831 g / ears for Figure 8 and 0.77 g / ears for figure 10).
• the TAL-17 strain showed an interest for an application at the flowering stage of wheat, but in view of its endophytic character, a much earlier application is of interest in the fight against fusarium head blight.
• the substrate used is a mixture of universal soil and expanded vermiculite (0.5-10 mm) (2: 1 v / v).
• the substrate was moistened 24 hours before sowing with reverse osmosis water.
• the pots were watered three times a week with an increasing volume of RO water as the plant developed.
• witness (A) did not receive any treatment; the sowing treatment (B and C) was carried out with a TAL-17 solution of 1.10 5 or 1.10 8 spores / seed (sp / seed); the spraying treatment at the 5-leaf stage (D and E), ie 21 days of growth, was carried out with a TAL-17 solution of 1.10 11 or 5.10 11 spores / ha.
• the germination rate tends towards 97% for the modalities having received the sowing treatment at 1.10 5 spores / seed and for those who have not yet received the aerial treatment.
• the control mode without treatment, increases up to a germination percentage of 93%.
• the results of the size-in-height measurements of the 25-day-old plants represented are presented in FIG. 13, and demonstrated a statistical difference between the sowing treatment of TAL-17 compared with the aerial spray treatment and the control.
• the sowing treatments at 1.10 5 and 1.10 8 spores / seed (viable spores) allow plants to grow in the order of 105 cm, while the aerial treatments, carried out 4 days earlier and the control, only allow growth ranging from 97 cm for the control, to 100.5 cm for the aerial treatment at 1.10 11 spores / ha and 101 cm for the treatment at 5.10 11 spores / ha. These values are found in the same statistical group.
• the substrate used is a mixture of universal soil and expanded vermiculite (0.5-10 mm) (2: 1 v / v). The substrate was moistened 24 hours before sowing with reverse osmosis water.
• the pots were placed in the phytotronic room (16 / 8h photoperiod) and were watered twice a week.
• the germination percentage was observed at 4 and 11 days depending on the modalities.
• the experimental protocol was the same as that described previously.
• the Dual culture test was performed with 30 m ⁇ of BCA or F. graminearum deposited at the ends of the Petri dish. After 6 and 10 days of culture at 25 ° C on PDA under a light hood with 16 h photoperiod, the dishes were observed ( Figure 15 A) and the length of the pathogen coverage by BCA was measured ( Figure 15B).
• the TAL-17 strain exhibited the particularity of growing on the mycelium of the pathogen over time, unlike the BCA D.
• VII probe specificity Assay TaqMan ® GS285741-P1 and the torque associated GS285741 primer-F1-R1 and GS285741 vis-à-vis the strain of the invention (tal- 17 and / or a mutant thereof )
• An internal control standard is also produced with genomic DNA extracted from the strain of the invention, which has been serially diluted. All qPCR assays were performed using a CFX 96 touch thermal cycler from Biorad ® . The amplification program used is that recommended by the supplier with a temperature hybridization at 60 ° C. For the TaqMan ® method, the Luna ® Universal Probe qPCR Master Mix (NEB ® ) reaction mixture was used. The amplification program is 95 ° C 2 min followed by 40X 95 ° C 15 ”and 60 ° C 30”.
• SCI strain isolated Trichoderma atroviride present in the Vintec ® product.
• a Dual culture test was carried out on which 100 ⁇ L of a F. graminearum / T. atroviride (Fusa / BCA) was spread on PDA medium in a Petri dish.
• BCA / Fusa mixture (ratio 1/2): BCA at 1.10 4 CFU / mL and Fusa at 2.10 4 CFU / mL (lmL mixture of BCA at 2.10 4 CFU / mL + 1mL of fusa at 4.10 4 CFU / mL).
• BCA / Fusa mixture (ratio 2/1): BCA at 1.10 4 CFU / mL and Fusa at 5.10 3 CFU / mL (lmL mixture of BCA at 2.10 4 CFU / mL + 1mL of fusa at 1.10 4 CFU / mL).
• the strain of the invention TAL-17 at the same dose combats Fusarium graminearum more. This is clearly visible for the ratio of 2 CFU of Fusa / 1 CFU of BCA. In fact, under these conditions, the SCI strain stalls unlike the TAL-17 strain.
• the sown plot was divided into 4 micro-plots of the order of 20 m 2 per modality (see Table 4) and the actual test was able to start.
• two treatments were carried out at the 'ears 6 to 8' (T1) and 'early flowering' (T2) stage with conventional treatments (Onnel ® , Faxer ® , Librax ® ) in the usual dose (0.6 L / ha) or in half-dose (0.3 L / ha) in combination or not with a mixture of BCA (strain TAL-17 [BCA 1] or another strain of T. atroviride [BCA 2]) at a dose of 1.10 11 CFU / ha and Appyphyt at a dose of 1 kg / ha (see Table 4).
• the statistical analysis was carried out using StatBox ® analysis and statistical processing software. It is paired with Excel ® software and enriched its functions. It made it possible to carry out the ANOVAs which will be presented in the results section. The comparisons of means are carried out according to the Newman-Keuls test at the 5% threshold. Homogeneous statistical groups are determined, the modalities associated with the same letter are not statistically different.
• BCA 1 ie. Strain TAL-17
• BCA 2 another strain of Trichoderma atroviride
• This difference in efficacy is statistically different when comparing modality No. 9 to modality No. 7 of the test and looking at the notes on brown rust.
• BCA was applied alone to the first treatment and was also applied to the second treatment with a half-dose of conventional Librax ® fungicide.
• the TAL-17 strain showed in this test a statistical interest on the physical symptoms of brown rust.
• the TAL 17 strain when it is applied (cf. modality No. 9), it allowed to obtain a brown rust score of the same statistical group as the best modality (cf. modality No. 4) for the brown rust notation.

Landscapes

• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Health & Medical Sciences (AREA)
• Zoology (AREA)
• General Health & Medical Sciences (AREA)
• Microbiology (AREA)
• Biotechnology (AREA)
• Chemical & Material Sciences (AREA)
• Wood Science & Technology (AREA)
• Mycology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Organic Chemistry (AREA)
• Genetics & Genomics (AREA)
• Virology (AREA)
• Botany (AREA)
• Medicinal Chemistry (AREA)
• Biochemistry (AREA)
• General Engineering & Computer Science (AREA)
• Tropical Medicine & Parasitology (AREA)
• Biomedical Technology (AREA)
• Agronomy & Crop Science (AREA)
• Pest Control & Pesticides (AREA)
• Plant Pathology (AREA)
• Dentistry (AREA)
• Environmental Sciences (AREA)
• Agricultural Chemicals And Associated Chemicals (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=68806854

Family Applications (1)

Country Status (3)

Families Citing this family (3)

Family Cites Families (2)

• 2019
  • 2019-06-18 FR FR1906554A patent/FR3097557A1/fr active Pending
• 2020
  • 2020-06-18 WO PCT/EP2020/067034 patent/WO2020254533A1/fr unknown
  • 2020-06-18 EP EP20732637.2A patent/EP3987068A1/de active Pending

Also Published As

Similar Documents

Legal Events