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WO2010115802A1 - New actinomycetes strain compositions and their use for the prevention and/or the control of micro organism inducing plant diseases - Google Patents

New actinomycetes strain compositions and their use for the prevention and/or the control of micro organism inducing plant diseases Download PDF

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Publication number
WO2010115802A1
WO2010115802A1 PCT/EP2010/054299 EP2010054299W WO2010115802A1 WO 2010115802 A1 WO2010115802 A1 WO 2010115802A1 EP 2010054299 W EP2010054299 W EP 2010054299W WO 2010115802 A1 WO2010115802 A1 WO 2010115802A1
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cfu
actinomycetes
strain
plant
compounds
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PCT/EP2010/054299
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French (fr)
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Essaid Ait Barka
Christophe Clement
Jean-Hugues Renault
Jean-Marc Nuzillard
Alix Toribio
Souad Loqman
Yder Ouhdouch
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Universite De Reims Champagne-Ardenne
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    • 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
    • C12N1/20—Bacteria; Culture media therefor
    • 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
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial 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
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/28—Streptomyces
    • 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
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/465—Streptomyces

Definitions

  • the present invention relates to new actinomycetes strain compositions and their use for the prevention and/or the control of micro organism inducing plant diseases.
  • Botrytis cinerea The causal agent of grey mould, Botrytis cinerea, is a ubiquitous necrotrophic pathogen and can infect almost every plant part on a wide range of plants worldwide (Rosslenbroich and Stuebler, 2000, Botrytis cinerea - history of chemical control and novel fungicides for its management. Crop Prot. 19, 557-561).
  • the control of this and other fungal diseases of grapevine is mainly achieved by use of chemical fungicides. Widespread use of chemical fungicides has certainly decreased the incidence of fungal diseases, but at the same time has contributed to the appearance of fungicide resistant strains of the pathogens. Due to consumer resistance to chemical residues in food and public concern for environmental safety, there is an increasing demand to develop alternative methods for disease control.
  • microbe-based biological control of plant-pathogenic fungi has increased in agricultural importance as replacements or supplements for agrochemicals, which may pollute the environment.
  • Public concern about chemical pesticides has fostered an interest in application of bacteria for biological control to protect agricultural crops against pathogenic fungi.
  • Microbe- based biological control of plant diseases has increased in agricultural importance as replacements or supplements for agrochemicals, which may pollute the environment and affect non-target organisms (Gerhardson et al., 2002, Biological substitutes for pesticides. Trends in Biotechnology. 20(8) 338-343; Welbaum et al., 2004, Fertilizing soil microorganisms to improve productivity of agroecosystems. Crit. Rev. Plant Sci. 23, 175-193.).
  • Beneficial bacteria known as plant growth-promoting rhizobacteria (PGBR) (Kloepper et al., 1991, Plant promotion mediated by rhizosphere colonizers. In: Keister, D.L., Cregan, P.B. (Eds.), The Rhizosphere and Plant Growth. Kluwer Academic Publishers, Dordrecht, pp. 315- 326), colonize roots, enhance shoot emergence, and stimulate plant growth either directly, by producing plant hormones and improving nutrient uptake, or indirectly, by changing the microbial balance in the rhizosphere in the favor of the beneficial microorganisms (Nowak et al., 1988, Benefits of in vitro "biotization" of tissue cultures with microbial inoculants.
  • PGBR plant growth-promoting rhizobacteria
  • Actinomycetes especially Streptomyces spp., are among the most frightening microorganisms. Actinomycetes are well-known isolated from the rhizosphere and have been the focus for excellent biocontrol agents to soilborne plant diseases (Fabre et al., 1988, A simple screening method for insecticidal substances from actinomycetes. J. Antibiot. (Tokyo) 41, 12- 219; Tokala et al., 2002, Novel plant microbe rhizosphere interaction involving Streptomyces Lydicus WYEC108 and the pea plant (Pisum sativum). Appl. Environ. Microbiol.
  • the actinomycetes are important producers of bioactive compounds (Lange et al. 1993; Microbial Fungicides - the Natural choice. Pestic. Sci. 39:155-160) and constitute a potential as biocontrol agents (Kim et al. 2000, Structure elucidation and fungal activity of an anthracycline antibiotic, daunomycin, isolated from Actinomadura roseola. J. Agr. Food Chem. 48:1875-1881; Gomes et al. 2000, Chitinolytic activity of actinomycetes from a cerrado soil and their potential in biocontrol. Letters Appl.. Microbiol. 30:146-150; Ouhdouch et al.
  • Actinomycetes from Painless Plant Habitats: Screening for antifungal activites. Eur. J. soil Biol. 37:1-6). Actinomycetes represent a high proportion of soil microbial biomass, and appear to be of importance among the microbial flora of the rhizosphere (Sardi et al. 1992, Isolation of endophytic Streptomyces strains from surface-sterillized roots. Appl. Environ. Microbiol. 58:2691-2698). Associations between actinomycetes and plant organs could be deleterious or beneficial for the host. Some actinobacteria are also known to inhabit the tissues of healthy plants.
  • the actinomycetes residing in healthy plant tissues without causing symptoms of disease were defined as endophytic actinomycetes.
  • the first endophytic actinomycetes were studied from nonleguminous plants (Benson et al, 1993, Biology of Frankia strains actinomycete symbionts of actinorhizal plant. Microbiological Reviews 57, 293-319).
  • endophytic actinomycetes such as Streptomyces , Streptoverticillium, Nocardia, Micromonospora, Microbispora, and Streptosporangium strains were isolated surface-sterilized roots of different plant species in Italy (Sardi et al., 1992, Isolation of endophytic Streptomyces strains from surface-sterilized roots. Appl. Environ. Microbiol. 58, 2691-2693) and of maize in Brazil (Araujo et al, 2000, Diversity of endophytic bacterial populations and their interaction with Xylella fastidiosa in citrus plants. Appl. Environ. Microbiol. 68,4906-4914).
  • endophytic actinomycetes as biocontrol agents of soil-borne roots disease is of interest through their ability to colonize healthy plant tissue and produce antibiotics in situ (Kunoh et al, 2002, Endophytic actinomycetes: attractive biocontrol agents. Journal of General Plant Pathology 68, 249-252; Cao et al, 2004, Isolation and characterization of endophytic Streptomyces strains from surface-sterilized tomato (Lycopersicon esculentum) roots. Lett. Appl. Microbial. 39, 425-430; Conn et al, 2008, Endophytic actinobacteria induce defence pathways in Arabidopsis thaliana. MoI. Plant Microbe Interact. 21, 208-218).
  • One of the aims of the invention is to provide new compositions comprising actinomycetes strains or compounds thereof and their use for their stimulation and/or fungicide properties against micro organisms responsible of plant diseases without showing chemical fungicide drawbacks.
  • Another aim of the invention is to provide an extraction and purification process of compounds contained in said actinomycetes strains.
  • Another aim of the invention is to provide prevention and/or control process of micro organism inducing plant diseases.
  • the present invention relates to the use of a composition comprising one or more actinomycetes strains, said composition having stimulation properties of plant natural defences and/or fungistatic properties, for the prevention and/or the control of micro organism inducing plant diseases, provided that when said plant is soybean, and when only one strain of actinomycetes is used, said actinomycetes strain is different from Streptomyces melanosporofaciens (EF-76).
  • EF-76 Streptomyces melanosporofaciens
  • Actinobacteria or actinomycetes are a group of Gram-positive bacteria. They include some of the most common soil life, playing an important role in decomposition of organic materials, such as cellulose and chitin and thereby playing a vital part in organic matter turnover and carbon cycle.
  • Actinobacteria of medical or economic significance are in subclass Actinobacteridae, order Actinomycetales, and several genus are found, the most important of them being the following: Actinomyces, Arthrobacter, Corynebacterium, Frankia, Micrococcus, Micromonospora, Mycobacterium, Nocardia, Propionibacterium, Streptomyces .
  • Actinomycetes can be isolated from soil of various origin and in particular from theixie soil according to procedures well-known from a man skilled in the art, and for instance, according to example 1.
  • strain also refers to one of its variant or mutant.
  • a mutant of a strain contains at least one modification of the genotype of said strain provided that said mutant belongs to the same species of the strain.
  • a variant of a strain contains one or more modification of the genotype of said strain without altering the phenotype of said strain.
  • stimulation means: "something that causes someone or something to become more active or to develop or function”.
  • plant means living organisms belonging to the kingdom Plantae. They include familiar organisms such as trees, herbs, bushes, grasses, vines, ferns, mosses, and green algae.
  • plant natural defences is meant the implementation of one or more mechanism(s) of defence by the plant itself during an infection of said plant by a pathogen agent, such as a fungi, i.e. the implementation of mechanisms of control of the pathogen by the plant.
  • Said involved mechanisms of defence are chosen among the following: - the hypersensitive reaction (HR), i.e. a localised and induced cellular death of infected tissues, the "Local Acquired Resistance” (LAR), i.e. an induction phenomenon.
  • LAR is induced by the cells in a state of cellular death via the transmission of signal molecules such as jasmonic acid (JA), salicylic acid (SA) or ethylene (ET), - the "systemic acquired resistance” (SAR) or the "induced systemic resistance” (ISR).
  • stimulation properties of plant natural defences means that said actinomycetes strains are able to trigger said mechanisms of defence in order to control pathogens.
  • fungistatic refers to any agent that inhibits fungal growth without killing the fungus.
  • fungistatic properties means that said actinomycetes strains are able to inhibit fungal growth without killing the fungus.
  • micro organism induced disease is meant a disease caused by a micro organism or a microbe, i.e. by an organism that is microscopic (usually too small to be seen by the naked human eye).
  • Microorganisms are very diverse. They include bacteria, fungi, archaea, and protists; microscopic plants (called green algae); and animals such as plankton, the planarian and the amoeba.
  • Such diseases are various and can be for example, but without being limited to: the mildew (or blew spot, or downy mildew or blue mold) caused by fungi selected from the list consisting of phytophthora, plasmopara or peronospora, such as phytophthora infestans on potato or tomato, or Peronospora tabacina on tobacco, or Plasmora viticola on vine, the powdery mildew caused by a fungus of the family Erysiphaceae such as Erysiphe necator on vine, or a fungus of the genus Oidium distinguished by an abundant powdery conidia produced on the host, eutypiose disease of the vine caused by the fungus Eutypa lata after pruning, black dead arm (BDA) caused by Botrytosphaeria sp., grey mould of the vine caused by Botrytis cinerea, and other wood diseases or ESCA (Galet , 1977, Les Maladies et Les Parasite
  • compositions of the present invention are their capacity to develop or promote the natural defences of the plant and to inhibit or stop the growth of the fungi.
  • compositions of the invention have prevention properties or fungistatic properties but can also possess both properties.
  • the composition is, in one hand, able to prevent or to protect plants from a the aggression of a pathogen agent, i.e. to avoid a pathogen agent such as a micro organism from attacking said plant and further causing a disease but it is also able, on the other hand to control, i.e. to fight against said micro organism, i.e. to protect the plant against the micro organism leading to the development control of said micro organism without having harmful impact neither on the growth of said plant and thus on the biomass yield, nor on the environment.
  • the present invention relates to the use of a composition comprising one or more actinomycetes strains, said composition having stimulation properties of plant natural defences and/or fungistatic properties, for the prevention and/or the control of micro organism induced plant diseases, provided that when said plant is soybean, said actinomycetes strain is different from Streptomyces melanosporofaciens (EF-76).
  • EF-76 Streptomyces melanosporofaciens
  • said actinomycetes strains above defined are of Streptomyces or Micromonospora genus.
  • Streptomyces genus comprised more than 500 species, for example, but without being limited to the following species:
  • the Micromonospora genus comprised several species, for example, but without being limited to the following species:
  • Micromonospora aurantiaca Micromonospora carbonacea, Micromonospora chalcea, Micromonospora chersina, Micromonospora citrea, Micromonospora coerulea, Micromonospora echinaurantiaca, Micromonospora echinofusca, Micromonospora echinospora, Micromonospora fulviviridis, Micromonospora gallica, Micromonospora halophytica, Micromonospora inositola, Micromonospora inyonensis, Micromonospora nigra, Micromonospora olivasterospora, Micromonospora pallida, Micromonospora peucetia, Micromonospora purpureochromogenes, Micromonospora rosaria, Micromonospora sagamiensis, Micromonospora viridifaciens .
  • said Streptomyces genus strains defined above are chosen among Streptomyces spp., i.e. among the 500 species above defined, provided that when only one strain is used in the composition, the Streptomyces melanosporofaciens (EF-76) strain is excluded from the compositions of the present invention.
  • the micro organisms above defined are chosen among Botrytis sp., Fusarium sp., Pythium sp. or Candida tropicalis R2, in particular Botrytis cinerea.
  • Botrytis sp. is a genus of Ascomycete fungi causing several plant diseases. The genus contains 22 species and one hybrid.
  • Plant diseases caused by Botrytis species appear primarily as blossom blights and fruit rots but also as leaf spots and bulb rots in the field and in stored products.
  • the fungi induce host cell death resulting in progressive decay of infected plant tissue, when they take nutrients.
  • Fusarium sp. is a large genus of filamentous fungi widely distributed in soil and in association with plants. Most species are harmless saprobes and are relatively abundant members of the soil microbial community.
  • the genus includes a number of economically important plant pathogenic species.
  • Fusarium graminearum commonly infects barley under unstable weather conditions such as late rain. Fusarium contamination in barley can result in head blight and in extreme contaminations the barley can appear pink
  • Fusarium graminearum can also cause root rot and seedling blight.
  • Pythium sp. is a genus of micro organism classified among oomycetes, previously classified as fungi. Pythium root rot is a common crop disease caused by a genus of organisms called "Pythium”. These are commonly called water moulds.
  • Pythium damping off is a very common problem in fields and greenhouses, where the organism kills newly emerged seedlings.
  • Candida tropicalis R2 is an amphotericin B and nystatin resistant fungal strain.
  • Botrytis cinerea is a necrotrophic fungus that affects many plant species, such as tomato or sunflower and in particular wine grapes.
  • This fungi exists under four forms in function of its biological cycle: sclerotia compact mass of hardened mycelium stored with reserve food material), mycelia (vegetative part of a fungus, consisting of a mass of branching, thread-like hyphae), conidia, sometimes termed conidiospores that are asexual non-motile spores of a fungus and apothecia that are particular bowl-shape sexed formation.
  • the stimulation properties of plant natural defences and/or the fungistatic properties of the compositions of the invention allows to prevent and/or control the micro organism induced plant diseases by interacting with one, two, three or four of the four forms of Botrytis cinerea fungi.
  • said micro organisms are in particular fungi responsible of wood diseases or ESCA and include Phaemonacremonium sp., Phaemoniella sp., Phellinus sp, or Eutypa lata ...
  • the actinomycetes strain above defined is used at a concentration from about 10 cfu/ml to about 10 cfu/ml, preferably from about 10 7 cfu/ml to about 10 cfu/ml, more preferably from about 10 7 cfu/ml to about 10 cfu/ml, in particular 10 7 cfu/ml.
  • compositions of the invention comprising the actinomycetes strains defined above are liable to prevent and/or control the micro organism induced plant diseases.
  • the composition above defined comprises or is constituted of the culture supernatant isolated from a culture medium of said actinomycetes strain.
  • culture medium or “growth medium” is meant a liquid or gel designed to support the growth of microorganisms or cells.
  • the "culture medium” may be commercially available.
  • the most common growth media for microorganisms are nutrient broths (liquid nutrient medium) or Luria Bertani medium (LB medium or Lysogeny Broth).
  • Liquid media are often mixed with agar and poured into petri dishes to solidify. They may be also specifically designed for a particular micro organism.
  • compositions comprise or consist in either the actinomycetes strains or in the supernatant of the culture medium of said actinomycetes strains and are both liable to prevent and/or control the micro organism induced plant diseases.
  • the culture supernatant isolated from a culture medium of said actinomycetes strain above defined is at a concentration from about 10- M to about 10- M, preferably from about 10- M to about 10- M, more preferably from about 10- M to about 10 -1 M, in particular 10 -2 M.
  • the composition above defined comprises or is constituted of one or more chemical compounds extracted and purified from said culture supernatant, or synthesized by a chemical way.
  • the man skilled in the art is able to extract and purify more than six fungicidal compounds, in particular against B. cinerea, belonging to several different chemical families, in particular from two to more than three different chemical families, such as compounds IV, V, VI, VII, VIII, IX and X (see example 5).
  • the composition can comprise a mixture of said above seven defined compounds, or a mixture of six, five, four, three or two compounds selected from the chemical compound list above defined, or a single compound from said list. Therefore, another advantage of the invention is to provide a composition comprising several compounds from different chemical families allowing the prevention and/or the control of plant diseases induced by one or more micro organisms.
  • the composition above defined comprises or is constituted of one or more chemical compounds belonging to one of the following chemical families: tryptophan derivatives, 2-terpenyl pyridines, ionophoretic polyethers, and chlorinated lipid compounds, extracted and purified from said culture supernatant.
  • Trp Tryptophan
  • Trp is one of the 20 standard amino acids, as well as an essential amino acid in the human diet and has the following formula (I):
  • tryptophan derivatives represents every chemical compound of formula I-a bearing an indole moiety and an oxazole moiety that can be substituted by one to eight substituents:
  • Rl to R7 are selected independently from each other from the group consisting in: hydrogen, halogen group such as chlorine, bromine, fluorine or iodine, alkyl or heteroalkyl group in Cl to ClO, linear or branched, saturated or not saturated, substituted or not with an hydroxyl, alkoxy or acyl group, hydroxyl, alkoxy or acyl group, or primary, secondary or tertiary amine,
  • R8 being selected from the group consisting in: hydrogen, alkyl group in Cl to ClO, linear or branched, saturated or not saturated, substituted or not with an hydroxyl, alkoxy or acyl group,
  • 2-terpenyl pyridines refers to a molecule that comprises a pyridine moiety substituted with Rl to R4 and a terpene moiety being into brackets with m and having the following general formula II,
  • Rl to R4 are selected independently from each other from the group consisting in:
  • - halogen group such as chlorine, bromine, fluorine or iodine, - alkyle, alkoxy or acyl group, or
  • R5 to R9 being selected independently from each other from the group consisting in:
  • halogen group such as chlorine, bromine, fluorine or iodine
  • the dashed line means that a double bond is present or not.
  • ionophoric polyethers refers to chemical compounds comprising at least two cyclic polyethers with 5 or 6 atoms linked between them by a bond or by a common carbon atom having the property to form a complex with an ion, and transport said ion across the membrane, with the general formula III:.
  • Rl to R7 are any chemically acceptable substituent.
  • the chemical compounds above defined of this embodiment can be extracted and purified by methods known from a man skilled in the art, or according to example 5, from the supernatant of the micro organism culture medium, or synthesized by a chemical or other way.
  • the composition comprises a mixture of two or several compounds belonging to one or two or three chemical families.
  • said chemical compounds are at a concentration from about 10- M to about 10- M, preferably from about 10- M to about 10- M, more preferably from about 10 -5 M to about 10 -9 M, more preferably from about 10 -6 M to about 10 -9 M, more preferably from about 10 -7 M to about 10 -9 M, more preferably from about 10 -8 M to about 10 -9 M, more preferably from about 10- M to about 10- M, and more preferably from about 10- M to about 10 -7 M.
  • said actinomycetes strain comprised or constituted in the above defined composition, is the actinomycetes strain S37, as deposited at the BCCM on the 03 rd Mai 2007 under the number LMG P-24130.
  • actinomycetes strain can be named S37 or SS37 and refers to the same strain. It can be also named S37 or SS37 actinomycete isolate.
  • the S37 actinomycetes strain has been tested for taxonomical diversity using morphological, cultural, physiological and biochemical criteria as a well as others features (example 2a) as shown in the table I below of example 2.
  • said chemical compounds are chosen among 4-chloro-5-
  • Abierixin has the following structure (VI):
  • the composition can comprise one single compound selected from compounds list IV, V and VI or two compounds selected from said compound list, i.e. compounds IV and V, or compounds IV and VI, or compounds V and VI, or the three compounds IV, V and VI.
  • compounds of the invention are able to control B. cinerea in grapevine in vitro ( Figure 14) as well as in vivo, see for example compound IV, figure 15C).
  • said stimulation properties of plant natural defences with the composition above defined are obtained by the signalling pathway activation, in particular leading to a local and systemic induction of at least one of the following genes: VvGIuC, VvChi4C, VvPR ⁇ and VvStSy with an induction factor higher than about 3, except for the activation of the fiavonoid synthesis pathway, the induction factor of which being less than 3, in particular the gene inducing the phenylalanine ammonia lyase (PAL).
  • PAL phenylalanine ammonia lyase
  • stimulation properties of plant natural defences is meant that the plant synthesizes one or more defence compounds such as PR (pathogenesis related) protein (van
  • the « signalling pathway activation» means that genes implicated in the plant natural defences and causing the resistance of the plant near and around the pathogen infection site, have an increased expression, i.e. they are stimulated or inducted by means of signal molecules such as jasmonic acid (JA), salicylic acid (SA), ethylene, systemin or abscissic acid.
  • the expression "local and systemic induction” means that the induction, i.e. the activation of the gene implicated in the plant natural defences is carried out near the pathogen infection site and activated by interaction of the plant with the pathogen agent.
  • VvGIuC is a gene coding for a basic PR-2 ( ⁇ -1,3-glucanase).
  • VvChi4C is a gene coding for an acidic PR-3 (chitinase).
  • VvPR6 is a gene coding for a PR-6 (proteinase inhibitor).
  • VvStSy is a gene coding for a stilbene synthase.
  • VvChi4C et VvGIuC are induced or regulated by both SA and JA.
  • VvPR6 is induced by JA.
  • the activation of said gene can be measured by quantitative RT-PCR (Bezier et al. 2002, Study of defence-related gene expression in grapevine leaves and berries infected with Botrytis cinerea. European Journal of Plant Pathology 108: 111-120)
  • induction factor measures the activation of the gene induced by means of signal molecules to produce a defence compounds such as PR.
  • said induction factor is efficient for the activation of said gene coding for said protein.
  • the composition comprising either one or more actinomycetes strain, or the culture supernatant isolated from a culture medium of said actinomycetes strain or one or more different chemical molecules is thus able to induce one of the genes selected from the list above defined or two genes such as VvGIuC and VvCi4C, or VvGIuC and VvPR6, or VvGIuC and VvStSy , or VvCM4C and VvPR6, or VvCM4C and VvStSy, or VvPR6 and VvStSy, or three genes such as VvGIuC, VvChi4C and VvPR6, or VvGIuC, VvPR6 and VvStSy, or VvChi4C, VvPR ⁇ and VvStSy, or the four genes of said list, leading thus to an improved resistance of the plants toward one or more micro organism induced plant diseases and thus to the prevention of one or more micro organism induced plant diseases and/or an
  • compound of the invention in particular compounds IV, V and VI are able to induce different genes, in particular STS, Chit4c, PR6 or Glue with different induction factors (figure 16 and 17).
  • the ratio corresponding to the activation of the flavonoid synthesis pathway is:
  • isoflavone before stimulation is lower than 0.45 and in particular the isoflavone quantity being present after stimulation of the natural defences is lower than 680 ⁇ g/g of dry matter.
  • Said ratio and said isoflavone quantity correspond to the activation of the expression of phenylalanine ammonia lyase (PAL).
  • PAL phenylalanine ammonia lyase
  • said composition used has also fungicidal properties and/or growth promoting properties.
  • Fungicidal properties can be evaluated according to Bauer et al. 1966, (Antibiotic susceptibility testing by standardized single disk method.Am J Clin Pathol 45 :493-497)
  • fungicidal refers to any agent that inhibits fungal growth and kills the fungus.
  • the actinomycete strain of example 1 (SS37) is able to inhibit the development of Botrytis cinerea when it is introduced on potato dextrose agar medium (PDA, figure 1) previously inoculated with Botrytis cinerea or the compounds of example 5 are able to inhibit the development of Botrytis cinerea when they are introduced on PDA previously inoculated with Botrytis cinerea (see example 5 and figure 8).
  • Table II (example 4b) shows the fungicidal activity against B. cinerea.
  • the compound X alone has fungicidal properties on B. Cinerea as shown by a local effect on B. Cinerea (figure 13) and compounds IV, V, VI, and to a lesser extent, compounds VII and VIII have a distance effect on the growth of B. cinerea, while compound X has no distance effect.
  • composition comprising a mixture of chemical compounds above defined is to exhibit a synergistic effect against one or more fungi compared to the effect of each compound taken alone against the same fungus or fungi (figure 13).
  • growth promoting properties refers to an agent able to increase the development of a plant.
  • the length of the principal root was boosted when the plantlets were inoculated with SS37.
  • the composition have plant natural defences and/or fungistatic properties as well as fungicidal properties and/or growth promoting properties and thus have two, three or four of the properties list above defined.
  • said composition above defined allows the implementation of a systemic acquired resistance (SAR, Durrant W.E et al. 2004, Systemic acquired resistance. Annual Review of Phytopathology 42: 185-209) and/or the implementation of an induced systemic resistance (ISR, Van Loon et al., 1998, Systemic resistance induced by rhizosphere bacteria. Annual Review of Phytopathology 36: 453-483, Pieterse, CMJ. et al., 2001,
  • Rhizobacteria-mediated induced systemic resistance triggering, signalling, and expression. European Journal of Plant Pathology 107: 51-61) in said plants.
  • the "systemic acquired resistance (SAR)” is a resistance mechanism activated by a first interaction with a pathogen leading to long time resistance of the plant (several weeks) against the pathogen infections caused by various pathogen micro organisms.
  • the ISR is induced following the plant interaction with beneficial bacteria.
  • the presence of actinomycete in the root tips (Fig. 1 IA), after inoculation, supports the possibility of entry of actinomycetes strain of the invention, in particular SS37, via root tips and the continuous presence of actinomycetes in the plant even in the second or third generation, allowing thus the plant to resist to other micro-organism attacks.
  • vitroplants of Chardonnay (grapevine), that are susceptible to fungal attack, inoculated with B. cinerea produced characteristic gray mold symptoms within 7 days (Fig. 12A and B).
  • endophyte actinomycete bacterized vitroplants appeared healthy (Fig. 12C and D) showing thus not only the stimulation properties of plant natural defences with SS37 but also the implementation of an induced systemic resistance (ISR) of said plant toward the disease and thus the prevention of said disease.
  • ISR induced systemic resistance
  • said plants are chosen among grapevine, or cereals such as wheat or rape.
  • the present invention relates to a composition comprising the actinomycetes strain S37 as deposited at the BCCM on the 03 rd Mai 2007 under the number LMG P-24130, as defined above, or a culture supernatant of a culture medium of said actinomycetes strain.
  • said strain comprised in the composition above defined is at a concentration from about 10 6 cfu/ml to about 10 9 cfu/ml, preferably from about 10 7 cfu/ml to about 10 cfu/ml, from about 10 cfu/ml to about 10 cfu/ml, in particular 10 cfu/ml.
  • said supernatant of the composition above defined is at a concentration from about 10 -4 M to about 10 -1 M, preferably from about 10 -3 M to about 10 -1 M, more preferably from about 10 -2 M to about 10 -1 M, in particular 10 -2 M.
  • the present invention relates to a composition
  • a composition comprising or being constituted of one or more chemical compounds belonging to one of the following chemical families: tryptophan derivatives, 2-terpenyl pyridines, ionophoretic polyethers, and chlorinated lipid compounds such as defined above, extracted and purified from the culture supernatant isolated from an actinomycetes strain culture medium, and when only one chemical compound is present in the composition, the following compounds 4-chloro-5-(3'-indolyle) oxazole, piericidine and abierixine are excluded.
  • the composition is composed of chemical compounds from an extracted and purified origin, or synthesized by a chemical way.
  • said composition comprising or being constituted of one or more of said chemical compounds does not comprise 4-chloro-5-(3'-indolyle) oxazole, piericidine and abierixine.
  • said chemical compounds of the compositions above defined are at a concentration from about 10- M to about 10- M, preferably from about 10- M to about 10- M, more preferably from about 10- M to about 10- M, more preferably from about 10- 6 M to about 10 -9 M, more preferably from about 10 -7 M to about 10 -9 M, more preferably from about 10- M to about 10- M, more preferably from about 10 -7 M to about 10- M, and more preferably from about 10- M to about 10 -7 M.
  • the present invention relates to an actinomycetes stain S37 as deposited at the BCCM on the 03 rd Mai 2007 under the number LMG P-24130 such as defined in claim 10.
  • the present invention relates to culture medium allowing to cultivate the strain defined above, comprising or constituted from glucose, peptone (Difco) 2 g/1, yeast extract (Difco) 1 g/1, and meat extract (Difco) 1 g/1.
  • Glucose is commercially available and can be obtained for example from Merck (Merck Chemicals Limited, Padge Road, Nottingham, NG9 2JR) and is at a concentration comprised from about 1 g/1 to about 100 g/1, preferably from about 1 g/1 to about 50 g/1, more preferably from about 1 g/1 to about 20 g/1, in particular about 10 g/1.
  • Peptone, yeast extract and meat extract are commercially available and can be obtained for example from Difco (USA).
  • Peptone is used at a concentration from about 0.1 g/1 to about 10 g/1, preferably from about 0.1 g/1 to about 5 g/1, more preferably from about 0.5 g/1 to about 1 g/1, in particular about 2 g/1.
  • Yeast extract is used at a concentration from about 0.1 g/1 to about 10 g/1, preferably from about 0.1 g/1 to about 5 g/1, more preferably from about 0.5 g/1 to about 2 g/1, in particular about 1 g/1.
  • Meat extract is used at a concentration from about 0.1 g/1 to about 10 g/1, preferably from about 0.1 g/1 to about 5 g/1, more preferably from about 0.5 g/1 to about 2 g/1, in particular about
  • the pH is adjusted from about 7.0 to about 7.5, in particular 7.2 before sterilization at about 100oC to 120oC, in particular 110oC for about 10 to about 60 minutes, preferably about 10 to about 50 minutes, more preferably about 20 to about 40 minutes, in particular about 30 minutes.
  • the present invention relates to an extraction and purification process of chemical compounds, such as defined above, from a culture supernatant isolated from a culture medium of one or more actinomycetes strains such as defined above, comprising a chromatography step, in particular a centrifugal partition chromatography of one or several fractions of chemical compounds obtained by liquid liquid pre- fractionating of a culture supernatant solvent extract of said culture medium of one or more actinomycetes strain, said fractions presenting fungicidal properties, to obtain one or more said pure chemical compounds and, optionally, a mixture of other said chemical compounds.
  • a chromatography step in particular a centrifugal partition chromatography of one or several fractions of chemical compounds obtained by liquid liquid pre- fractionating of a culture supernatant solvent extract of said culture medium of one or more actinomycetes strain, said fractions presenting fungicidal properties, to obtain one or more said pure chemical compounds and, optionally, a mixture of other said chemical compounds.
  • the chromatography is a well known technique for a man skilled in the art.
  • the expression "centrifugal partition chromatography” is liquid-liquid chromatography without “solid-support”, requiring two immiscible solvent phases.
  • liquid liquid pre-fractionating refers to the use of a biphasic or triphasic system composed of organic solvents or water, the solvent system comprising at least 2 immiscible solvents.
  • Organic solvents are chosen among liquid alkanes (hexane, cyclohexane, heptane, petroleum ethers,...) aromatic hydrocarbons (toluene) ketones (acetone, methyl-isobutyl ketone, methyl-ethyl ketone, etc alcohols (methanol, ethanol, butanols) - esters (ethyl acetate, butyl acetate,%) halogenated solvents (chloroform, dichloro methane,%) ethers (methyl terbutyl ether, ethyl terbutyl ether, ...) acetonitrile
  • pure chemical compounds refers to a compound with more than about
  • the fungicidal properties of said fractions can be determined by techniques well known from the man skilled in the art.
  • the fungicidal properties of said fraction(s) of chemical compounds of the extraction and purification process defined above are determined by bioautography.
  • the bioautography technique is detailed in Petersen, M.; Simmonds, M. Rosmarinic acid. Phyto chemistry 2003, 62, 121-125) and allows to determine the activities of the products adsorbed on plate by chromatography on thin layer.
  • the extraction and purification process defined above comprises a second chromatography, in particular a centrifugal partition chromatography of said mixture of other chemical compounds, different from the previous centrifugal partition chromatography.
  • the first chromatography does not allow to separate all the compounds produced by the actinomycetes strains.
  • a second chromatography in particular a centrifugal partition chromatography, with a solvent system different from the one used in the first chromatography, of the collected fractions containing a mixture of chemical compounds not separated must be carried out to obtain all the compounds in a pure state.
  • pure state refers to a compound with more than about 95% of purity.
  • said actinomycetes extract being subjected to the extraction and purification process above defined, has been obtained by the culture supernatant extraction of culture medium of one or more actinomycetes strain by means of a solvent, in particular 1-butanol, then evaporating said solvent containing said strain extract.
  • the extraction and purification process defined above comprises the following steps: a. culture supernatant extraction of a culture medium of one or more actinomycetes strain, previously cultivated, by means of a solvent, in particular 1-butanol, to obtain a solution containing a culture supernatant extract of a culture medium of one or more actinomycetes strain, b. liquid liquid pre- fractionation of said culture supernatant extract by means of a triphasic or biphasic extraction system, after evaporation of said solvent containing extract, to obtain one or more fraction(s) containing chemical compounds from said culture supernatant extract, c.
  • the first step a allows the extraction of a mixture of compounds contained in the culture medium of the strain.
  • This step can be performed with different organic solvents such as butanol, ethyl acetate, hexane or derivatives, dichloromethane, methanol, ethanol, propanol, chloroform...., and in particular with 1-butanol that leads to the best results considering the detection of the compounds having a biological activity, i.e. the fungicidal activity, by bioautography.
  • the second step b.) allows to obtain one more fractions containing one or more chemical compound in order to evaluate the biological activity of each fraction.
  • This step corresponds to the partial purification of the butanolic extract obtained in step a.
  • the step c.) corresponds to the fungicidal evaluation of each fraction obtained in step b.
  • the step d.) allows to obtain pure chemical compounds from each biological active fraction determined in step c.
  • step d.) of the process does not lead always to the purification of all the biological obtained fractions of step c. and some of the chemical compounds contained in the biological active fractions are therefore not separated.
  • the process defined above comprises a further step of centrifugal partition chromatography of said mixture of other said chemical compounds above obtained, by means of a biphasic mixture different from the one used for the first centrifugal partition chromatography carried out in step d).
  • the chemical compounds contained in the biological active fractions that have not been separated in step d. of the previous defined process are collected, then evaporated and subjected to a further centrifugal partition chromatography using a biphasic mixture different from the previous one in order to separate the different chemical compounds of said biological active fractions that have not been separated in step d.
  • said strain being subjected to the process above defined is the actinomycetes strain S37 as deposited at the BCCM on the 03 rd Mai 2007 under the number LMG P-24130.
  • the present invention relates to products obtained by process as defined above, except for the compounds 4-chloro-5-(3'-indolyle) oxazole, piericidine and abierixine.
  • the present invention relates to a prevention and/or control process of plant diseases induced by microorganisms such as Botrytis sp., Fusarium sp., Pythium sp.
  • Candida tropicalis R2 in particular Botrytis cinerea induced diseases, comprising a spraying step of the aerial parts and/or an inoculation step of the underground parts of said plants with a composition comprising one or more actinomycetes strain such as defined above, preferably at a concentration from about 10 cfu/ml to about 10 cfu/ml, preferably from about 10 cfu/ml to about 10 9 cfu/ml, from about 10 7 cfu/ml to about 10 8 cfu/ml, in particular 10 7 cfu/ml.
  • a composition comprising one or more actinomycetes strain such as defined above, preferably at a concentration from about 10 cfu/ml to about 10 cfu/ml, preferably from about 10 cfu/ml to about 10 9 cfu/ml, from about 10 7 cfu/ml to about 10 8 cfu/ml, in particular 10 7 cf
  • the strains are the same as defined above provided that the Streptomyces melanosporofaciens is excluded from the species that can be used in the compositions of the present invention.
  • the spraying step of the aerial part allows to stop the development of the fungi, either by fungistatic activity of the composition (see figure 6), either by killing the fungi (see figure 8). Said spraying allows also (figure 3-5) to prevent the disease and develop a resistance by pre- treatment with the compositions of the invention
  • the inoculation of the underground parts allows to prevent the disease and develop a resistance by pre-treatment with the compositions of the invention (see figure 11, 12).
  • said spraying step of the aerial parts and/or an inoculation step of the underground parts of said plants in the process above defined is carried out with a composition comprising or constituted of the culture supernatant isolated from a culture medium of one or more actinomycetes strains, preferably at a concentration from about 10 ⁇ 4 M to about 10 -1 M, preferably from about 10 ⁇ 3 M to about 10 -1 M, more preferably from about 10 ⁇ 2 M to about 10 -1 M, in particular 10 ⁇ 2 M.
  • said spraying step of the aerial parts and/or an inoculation step of the underground parts of said plants of the process above defined is carried out with one or more chemical compounds belonging to one of the following chemical families: tryptophan derivatives, 2-terpenyl pyridines, ionophoretic polyethers, and chlorinated lipid compounds such as defined above, and preferably at a concentration from about 10- M to about 10- M, preferably from about 10- M to about 10- M, more preferably from about 10- M to about 10 -9 M, more preferably from about 10 -6 M to about 10 -9 M, more preferably from about 10 -7 M to about 10 -9 M, more preferably from about 10 -8 M to about 10 -9 M, more preferably from about 10- M to about 10- M, and more preferably from about 10- M to about 10- M.
  • tryptophan derivatives preferably from about 10- M to about 10- M
  • ionophoretic polyethers ionophoretic polyethers
  • chlorinated lipid compounds such as defined above
  • Figure 13 and 14 shows the in vitro fungicidal effect of the compounds of the invention.
  • Figures 11, 12, 15 shows that the inoculation with the compounds of the invention allows to protect the plant and to prevent the disease and develop a resistance against said disease.
  • the present invention relates to a promoting growth process of plant, said plants being chosen among grapevine, or cereals such as wheat or rape, comprising a spraying step of the aerial parts and/or an inoculation step of the underground parts of said plants with a composition comprising or constituted of strain above defined, or of culture supernatant isolated from a culture medium of one or more actinomycetes strains above defined, or of one or more chemical compounds belonging to one of the following chemical families: tryptophan derivatives, 2-terpenyl pyridines, ionophoretic polyethers, and chlorinated lipid compounds above defined.
  • said chemical compounds of the process above defined are chosen among 4-chloro-5-(3'-indolyle) oxazole, piericidine or abierixine.
  • said strain of the process above defined is the actinomycetes strain S37 as deposited at the BCCM on the 03 rd Mai 2007 under the number LMG P-24130.
  • Figure 1 presents the interaction between selected SS37 actinomycete isolate and B. cinerea when inoculated together on PDA (distance confrontation).
  • the fungus has been placed in the centre of the Petri dish. This figure shows the significant zone of inhibition around the fungus inoculums due to the effect of SS37 strain against B. cinerea.
  • the SS37 strain was used at 10 7 CFU/ml.
  • the fungus was used at 10 5 CFU/ml.
  • Figure 2 presents the hyphal mycelium from the zone of interaction between B. cinerea and SS37 actinomycete isolate.
  • Part a normal hypha of B. cinerea.
  • Parts b, c, d mycelium taken in the zone of interaction between B. cinerea and isolate
  • Actinomycete isolates were effective in restricting radial growth of B. cinerea.
  • Figure 3, part a) leaves from control plantlets;
  • Figure 3, part b) leaves inoculated with actinomycetes (isolate SS37);
  • Figure 3, part c leaves inoculated with B. cinerea and exhibiting the symptoms of gray mold disease;
  • the fungus was used at 10 5 CFU/ml.
  • Figure 5 presents the light micrographs of samples from grapevine leaves.
  • FIG 5, part c leaves inoculated with B. cinerea and exhibiting the symptoms of gray mold disease;
  • the hyphae of the pathogen abundantly colonize the epidermis and the parenchyma. Fungal growth occurs both intracellularly and intercellularly.
  • Control leaves show unaltered tissues (Fig. 5, part a).
  • Treatment of leaves with isolates has no incidence on leaf structure (Fig. 5, part b), while the parenchyma of pathogen-inoculated leaves showed an intensive colonization by B. cinerea (Fig. 5, part c).
  • Pathogen ingress toward the inner tissues coincided with extensive plant cell disorganization such as host cell wall alteration (Fig. 5, part c). In massively invaded areas cell walls are no longer discernible in extreme cases (Fig. 5, part c).
  • leaves pre -treated with actinomycetes isolates before their inoculation with B. cinerea showed that pathogen growth was stopped at the leaf surface (Fig. 5, part d).
  • the SS37 strain was used at 10 7 CFU
  • Figure 6 presents the effect of SS37 Actinomycete isolate on hyphal mycelium growth of B. cinerea when inoculated together on grapevine tissue extract agar medium.
  • the SS37 strain was used at 10 7 CFU/ml.
  • the fungus was used at 10 5 CFU/ml.
  • Figure 7 presents the chromatograms obtained from CPC experiments A (Fig.7, partA) and B (Fig.7, part B).
  • Fig. 7, part A shows the compounds IV, VIII and IX obtained by experiment A.
  • Compound X can be obtained by dual mode (see example 5)
  • part B shows the compounds V, VI and VII obtained by experiment B after collecting the fraction containing these compounds in experiment A.
  • Figure 8 presents the fungicidal activity against B. cinerea of compounds control, IV, V, VI, VII, VIII and X.
  • Fig.8 part A shows the fungicidal activity against B. cinerea of compounds control, VI, VII, and X.
  • a mother solution was prepared (45.76 mM in MeOH), then 1 ml of this solution was added in 99 ml sterile distilled water to obtain a dilute solution and l ⁇ l of said diluted solution was deposited on the sides of the Petri dish.
  • the fungus was used at 10 5 CFU/ml.
  • Figure 9 presents the different types of PR protein according to Van Loon et al. (2006).
  • Figure 10 presents the in vitro responses of grapevine plantlets co-cultured with SS37 strain; the bacterisation process of plantlets was accomplished according to Ait Barka et al, 2000, Enhancement of in vitro growth and resistance to gray mould of Vitis vinifera L. co- cultured with plant growth-promoting rhizobacteria. FEMS Microbiol. Lett. 186, 91-95) Figure 10, part A: control
  • part B grapevine plantlets co-cultured with SS37 strain
  • This figure shows that SS37 promotes the growth of treated grapevine plantlets with SS37.
  • the SS37 strain was used at 10 7 CFU/ml.
  • Figure 11 presents the microscopic observation showing emergence of SS37 mycelia from disinfected root cuttings (figure 11, part A) and a test of the presence of actinomycete colonies in leaf tissues sampled from bacterized plantlets after four days of incubation on solid Bennett medium (figure 11, part B): (1) First generation, (2) Second generation, (3) Third generation, (T) control plantlets.
  • FIG. 11, part A This figure shows that the presence of actinomycete in the root tips (Fig. 11, part A) after inoculation supports the possibility of entry of SS37 via root tips and that the actinomycetes is still present in the plant even in the second or third generation allowing thus the plant to resist to an micro-organism attack.
  • the SS37 strain was used at 10 7 CFU/ml.
  • Figure 12 presents the effect of inoculation with B. cinerea on grapevine plantlets bacterized or nonbacterized with SS37.
  • This figure shows the stimulation properties of plant natural defences with SS37 but also the implementation of a resistance of said plant toward the disease and thus the prevention of said disease.
  • FIG. 13 presents the effect of chemical compounds IV, V, VI, VII, VII, and X on the growth of B. Cinerea (chemical compounds were deposited simultaneously with the fungus at the centre of the Petri dish). Fig.13, part A: compounds IV, V, VI, VII, VIII and B. Cinerea.
  • a mother solution was prepared (45.76 mM in MeOH), then 1 ml of this solution was added in 99 ml sterile distilled water to obtain a diluted solution and l ⁇ l of said diluted solution was deposited at the centre of the Petri dish.
  • the fungus was used at 10 5 CFU/ml.
  • Figure 14 presents the distance effect of chemical compounds IV, V, VI, VII, VII, and X on the growth of B. Cinerea.
  • Fig.14, part B compound V and B. Cinerea (BC)
  • Fig.14, part C compound VII and B. Cinerea (BC)
  • Fig.14, part D compound VIII and B. Cinerea (BC)
  • Fig.14, part E compound VI and B. Cinerea (BC)
  • Fig.14, part F compound X and B. Cinerea (BC).
  • a mother solution was prepared (45.76 mM in MeOH), then 1 ml of this solution was added in 99 ml sterile distilled water to obtain a diluted solution and l ⁇ l of said diluted solution was deposited on the sides of the Petri dish.
  • the fungus was used at 10 5 CFU/ml.
  • Figure 15 presents the effect of compounds IV and X on the plant protection (grapevine) against B. cinerea.
  • Fig.15 part B : B. cinerea
  • Fig.15 part C : B. cinerea and compound IV,
  • Fig. 15, part D B. cinerea and compound X.
  • Figures 16 and 17 present the expression in grapevine of a few genes coding for PR proteins induced by compounds produced by SS37.
  • each histogram (control and each product) from left to right corresponds to the induction factor: stilbene synthase (STS), chitinase (Chit4c), proteinase inhibitor (PR6 ) and lipoxygenase (Lox) respectively.
  • STS stilbene synthase
  • Chit4c chitinase
  • PR6 proteinase inhibitor
  • Lix lipoxygenase
  • the histograms correspond to the induction factor of glucanase expression (Glue), all the compound being active, in particular compound IV.
  • Example 1 Isolation of actinomycetes strains fromixie soil a) Sampling
  • Samples were collected using Pochon & Tardieux method (1962, Technique d'analyse en microbiologie du sol, Edition de Ia Tourtourelle, Saint-Mande) from fourixie Rhizosphere soils of healthy vineyard fields (Tinghir). The samples from each of the rhizosphere soils were taken with an auger (up to 10 cm depth) after removing approximately 3cm of the soil surface. Samples were placed in sterile polyethylene bags, closed tightly and stored in the refrigerator at 4°C until use.
  • Actinomycetes Isolation Agar (Olson's medium; 5 % glycerol, 0.2 % sodium caseinate, 0.01 % L-asparagin, 0.4 % sodium propionate, 0.05 % K2HPO4, 0.0001 % FeSO4 and 1.5 % agar Difco), (Olson 1968; Actinomycetes Isolation Agar, In: Difco: Supplementary Literature. Difco Lab., Detroit, (Michi.).
  • Actinomycetes were recognized on the basis of morphological features following directions given by International Streptomyces Project (ISP) (Shirling & Gottlieb 1966; Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16:313-340).
  • ISP International Streptomyces Project
  • S37 colonies were isolated, purified and conserved in 20 % Glycerol at -20 oC.
  • Example 2 Characterisation of the S37 isolate (or other above obtained isolates) a) Morphological, physiological and chemotaxonomic characterization of selected bacterial isolates
  • the exemplary strain of the invention shows the following properties when incubated at
  • ISP4 Inorganic Salt Starch Agar
  • ISP6 Pepton Yeast Extract Iron Agar
  • ISP7 Tyrosine Agar
  • SS37 were able to use citrate, sucrose, fructose, glucose, maltose, mannitol, mannose, melibiose, lactose arabinose and glycerol as sole carbon sources whereas inositol, D-raffinose, sorbitol, rhamnose, galactose, and xylose were not used (Table 1).
  • SS37 was sensitive to Novobiocin (30 ⁇ g), Gentamycin (10 ⁇ g), Bacitracin (10 U) and
  • Polymyxin B (300 U) but resistant to Sulfamides and Cefalotin (30 ⁇ g).
  • the purified SS37 actinomycete isolate was grown for 2 days at 28°C with agitation in 500 ml flasks containing 100 ml of Hickey-Tresner medium containing 1 g/1 yeast extract, 1 g/1 beef extract, 2 g/1 N-Z-amine A (Sigma-Aldrich), 10 g/1 Dextrin, 20 mg/1 CoC12.6H2O (Hopwood et al. 1985; Genetic manipulation of Streptomyces: a laboratory manual. John Innes Foundation, Norwich, United Kingdom). Biomass was harvested by centrifugation (8000 g for 10 min) and washed twice with double-distilled water. 200 mg of mycelia was used for DNA extraction as described in Liu et al. (2000; Rapid mini-preparation of fungal DNA for PCR. J. Clin. Microbiol. 38: 471-1471).
  • the 16S rDNA was amplified using the PCR method with T ⁇ q DNA polymerase (Qiagen,
  • Amplification was carried out in 50 ⁇ l reaction mixture containing 1.5U of AmpliTaq Gold T ⁇ q polymerase (Applied Biosystems), 10 ⁇ l of 5x AmpliTaq Gold reaction buffer (Applied Biosystems), 2.5 mM of each dNTP, 1 ⁇ M of each primer and 100 ng of genomic DNA.
  • Reaction conditions were: 97°C for 4 min, (97°C for 45s, 52°C for 45s and 72°C for 45s) x 35 cycles followed by an incubation at 72°C for 10 min.
  • the amplified products were visualized on a 0.8% (w/v) agarose gel stained with ethidium bromide. Sequencing reactions were performed by Macrogen (Seoul, Korea).
  • the obtained sequence (SEQ ID NO: 1) was compared for similarity with sequences present in the genomic database banks, using the 'NCBI Blast' program available at the ncbi- nlm-nih.gov Web site and exhibited 99% sequence identity to Streptomyces ⁇ nul ⁇ tus.
  • Example 3 Determination of the antifungal activity of S37 strain (or other isolates above obtained) a) Antifungal assays
  • the antifungal activity of the S37 isolate was determined by the plate diffusion method
  • Botrytis cinerea B. cinerea
  • Fusarium oxyysporum f. sp. Albedinis Foa
  • Sclerotium rolfsii SR
  • Verticillium dahliae VD
  • Pythium ultimum PU
  • Isolates were grown on Bennett medium (beef extract [Merck, Germany] lg/1; glucose [Merck, Germany] lOg/1; peptone [Merck, Germany] 2g/l; yeast extract [Merck, Germany] lg/1 and agar [Difco, USA] 15g/l) for 14 days and six mm diameter agar disks containing actinomycetes colonies mass was prepared by using sterile cork borers. Disks were then aseptically transferred to PDA plates having fungal mycelial-disk (diameter of 6mm) in the centre. Plates were first kept in a refrigerator (4°C) for at least 4h to allow the diffusion of any antibiotics produced, then incubated at 30oC. Inhibition zones were determined after 4 days of incubation. Only the isolates showed an inhibition zone superior to 8 mm were considered as active isolates.
  • Plantlets of Vitis vinifera L. cv. 'Chardonnay' clone 7535 were micropropagated by nodal explants grown on 15 ml semi-solid medium (Martin et al. 1987, The vine and techniques of in vitro cultivation. Bull Org Int Vigne 675-676:447-458), in 25-mm culture tubes under 200 ⁇ mol/m2/s white fluorescent light, 16 h photoperiod at 26°C (Ait Barka et al. 2000, Enhancement of in vitro growth and resistance to gray mould of Vitis vinifera L. co-cultured with plant growth promoting rhizobacteria. FEMS Microbiol Lett 186:91-95).
  • B. cinerea 105 spores/ml suspended in sterile distilled water
  • Example 4 Test for antagonistic activity, for chitinase and indole acetic acid (IAA) production and for detection of siderophore of SS 37 actinomycete isolate, a) Test for antagonistic activity The plate diffusion method was used to assess the global anti-microbial activity of the strains (Bauer et al. 1966, Antibiotic susceptibility testing by standard single disk method. Am J Clin Pathol.
  • SS37 actinomycete isolate was grown on grapevine tissue extract agar medium for 14 days then three disks (diameter 10 mm) were cut out and placed on 48 h grown lawns of the different microorganisms tested grown on nutrient agar (Difco, USA) for bacteria and yeasts and Sabouraud agar medium (Biorad, France) for fungi. Plates were first stored at 4 oC for at least 2 h to allow the diffusion of any substances produced, then incubated at 28 oC. Sizes of the inhibition zones were determined after 24 h of incubation for bacteria and yeasts and 48 h for fungi. Controls involved the use of sterile agar plugs. Three replicates were performed per isolate by each microorganism test.
  • the MICs of "Ymycine” for clinic yeast and phytopathogenic mould Botrytis cinerea were ranged from 0.060 to 0.070 ⁇ g/m; whereas the MICs of amphotericin B were ranged between 3 and 4 ⁇ g/ml.
  • a colloidal chitin suspension was, prepared from crab shell chitin as follows: 40 g of chitin were dissolved, under stirring for 30 to 50 min, in 400 ml of HCl 6M (Hsu and Lockwood, 1975, Powdered Chitin Agar as a Selective Medium for Enumeration of Actinomycetes in Water and Soil. Appl Environ Microbiol. 29(3): 422-426). The chitin was precipitated as a colloidal suspension by diluting it slowly with 2 1 of deionized water at 5-10 oC. The colloidal suspension was filtered and washed until the pH of the suspension was about 3.5 and then was dried.
  • Dry chitin (4 g) was added as sole carbon source, in replacement of glucose to 1 1 of synthetic minimum medium (SMM) containing 0.5 g 1-1 Rock Phosphate (RP). After autoclaving, the melted agar medium was adjusted to pH 7.8. The positive growth control experiment was made with SMM containing glucose as sole carbon source. Only chitinase excreting isolates could grow on the chitin SMM medium.
  • SMM synthetic minimum medium
  • RP Rock Phosphate
  • Indoleacetic acid (IAA) production was analysed using a modification of the qualitative method developed by Brie et al. (1991, Rapid in situ assay for indole acetic acid production by bacteria immobilized on a nitrocellulose membrane. Appl Environ Microbiol 57:535-538).
  • the growth medium used contained 1O g tryptone, 5 g yeast exctract, 5 g NaCl, 1,02 g L-tryptophane and 20 g agar for 1 litter of deionized water. This medium was sterilized at 121°C for 20 min.
  • Actinomycetes strains were plated on the surface of the medium overlaid with a nitrocellulose membrane (Amersham Pharmacia) and incubated at 28°C for 7 days. After Actinomycetes growth had occurred, the membrane was removed from the plate and treated with Salkowski reagent (2% (w/v) 0.5 M FeCB in 35% perchloric acid) for 15 min at room temperature. Actinomycetes strains producing IAA are identified by a red halo surrounding the colony.
  • SS37 actinomycete isolate they were grown for 5 days under the conditions described above. The supernatants were centrifuged at 10, 000 xgmin-1, filtered and concentrated filtrates were deposited on sterile cellulose disks (5 mm diameter) placed aseptically on blue CAS-agar plates as described by Schwyn and Neilands (1987, Universal chemical assay for the detection and determination of siderophores. Analytical Biochem. 160, 47-56.) and incubated at 30 oC for 3 days.
  • the disks impregnated with solutions containing siderophore were surrounded by a zone of colour change (blue to yellow-orange) of the CAS that was due to iron chelation (Schwyn and Neilands, 1987, cited above).
  • the size of the zones and the intensity of the colour change were estimated and compared to the controls.
  • the table III shows the results obtained with SS37 for the antagonistic activity, the production of chitinase and IAA, and the detection of siderophore (activity against fungi, Gram+ bacteria, Gram- bacteria and Yeast: +: low; ++: medium and +++: high; other activities: 1 means production or detection, 0 means no production or detection).
  • Example 5 purification of chemical compounds from SS37 actinomycetes isolate, a) Actinomycete sp. cell culture
  • the resulting dry extracts were dissolved in methanol and subjected to biological assay (disk of 6 mm in diameter) against Bacillus subtilis (40 ⁇ l per disk) and Botrytis cinerea (60 ⁇ l per disk).
  • the n-butanol extract gave the highest inhibition diameter were then retained for the detection of antibiotics by bioautography [Petersen, M.; Simmonds, M. Rosmarinic acid. Phytochemistry 2003, 62, 121-125].
  • 40 ⁇ l (Bacillus subtilis) or 60 ⁇ l (Botrytis cinerea) of n-butanol extract were spotted onto 20 x 20-cm silica gel plates (Merck Art.
  • TLC plates were air-dried overnight at 37 oC to remove all traces of solvent. They were then placed in plastic bioassay dish (23 x 23 x 2.2 cm3, Fisher Scientific Labosi) and overlaid with 150 ml of nutrient agar or PDA agar media (containing 7 g/1 of agar) seeded respectively with Bacillus subtilis or Botrytis cinerea. After the agar had set, the plate was inoculated at 30 oC.
  • the inhibition zones of the active fraction were determined after 24 h of incubation for the Botrytis cinerea and after 48 h for Bacillus subtilis. Clear areas due to inhibition of microorganism growth indicated the location of antibiotic compounds on the TLC plates. The retention factor (Rf) of each spot was recorded.
  • n-butanol extract was evaporated to dryness, the dry residue (8,8 g) was dissolved in a small volume of methanol and was submitted to liquid-liquid fractionation using for each extraction, 60OmL of the ternary biphasic solvent mixture n- heptane/ methano I/water (5: 1.5: 3.5, v/v). After 4 time extractions, the collected upper organic phases were regrouped and evaporated to dryness. About 1.4 g of the later residue was obtained. It was then stored for 24 hours at - 10 oC prior to use in CPC.
  • Rotation speed can be adjusted from 200 to 2000 rpm, producing a centrifugal force field in the partition cell of about 120 g at 1000 rpm and 470 g at 2000 rpm.
  • the solvents were pumped by a Dionex P580HPG 4-ways binary high-pressure gradient pump (Sunnyvale, CA, USA).
  • the samples were introduced into the CPC column via a low pressure injection valve (Upchurch, CIL Cluzeau, Sainte-Foy-La-Grande, France) equipped with a 21 mL sample loop.
  • the effluent was monitored with a Dionex UVD 170S detector equipped with a preparative flow cell (6 ⁇ L internal volume, path length of 2 mm). Fractions were collected by a Pharmacia Superfrac collector (Uppsala, Sweden). The experiments were conducted at room temperature (22 ⁇ 1°C).
  • the Arizona quaternary solvent system (R) composed of n-Heptane/ Ethyl acetate/ methanol/ water (2: 1: 2: 1, v/v) (experiment A) and the second biphasic sytem n-Heptane / acetonitrile/ water (5: 5: 4, v/v) (experiment B) were prepared by mixing the appropriate solvent in a separatory funnel, shaking them vigorously and allowing them to settle until the phases became limpid.
  • experiment A the CPC column was filled with the aqueous stationary phase of the biphasic system.
  • the sample solution was injected in the sample loop, the rotation speed was adjusted to 1300 rpm and the organic mobile phase was pumped at 6 mL/min.
  • experiment B separation was performed with the organic as the mobile phase at 1200 rpm and 3mL/min.
  • the outgoing phase was monitored at 270 nm by the online UV detector for experiments A and B.
  • the stationary phase retention was 75 and 70%
  • the back pressure was about 45 and 40 bars.
  • CPC fraction analysis was performed using normal phase TLC (Merck Si60) with CHC13/ methanol (99: 1, v/v) as eluent. Plate visualization were realised thanks to sulphuric vanilline reagent (vanillin / H2SO4 concentrated/ Ethanol, 3/ 3/ 100, w/ v/v). The fractions containing pure compounds from the TLC analysis were regrouped. These fractions were submitted to 1H and 13C NMR experiments. The spectra were recorded in CDCl 3 (1H at 500 MHz and 13C at 125 MHz) on a Bruker Avance DRX 500. 2D (COSY, HSQC, HMBC .) experiments were performed using BRUKER microprograms. ESI-MS and HRESI-MS were obtained in a Micromass Q-TOF micro spectrometer apparatus.
  • the bioassay guided liquid-liquid pre-fractionation had to simplify the initial n-butanol extract while enriching it of the active constituent.
  • the primary extract could be thus fractioned in groups of compounds of distinct polarities.
  • a first system of solvents has been selected from the systems of the Arizona range in order to be used in CPC.
  • the R system (n-Hept / AcOEt / CH 3 OH / Water, 2 : 1 :2 :1, v/v) Arizona range has been kept in first intention. Partition coefficients about 1 have been obtained with this system for the majority of the active compounds of the prefractionated extract.
  • Example 6 effect of SS37 actinomycetes strain on the growth of pathogen B. cinerea. a) Botrytis cinerea culture
  • the B. cinerea strain 630 used in this experiment was provided by Dr. Brygoo (INRA, Paris, France). Filamentous fungi were maintained on potato dextrose agar medium (PDA). Fungal inoculum was prepared by growing the fungus for one week on fresh PDA medium. After this period, the B. cinerea isolate had developed abundant hyphal swellings. The mycelium was removed aseptically from the PDA plates and was briefly homogenized in a disinfected blender in the presence of sterile distilled water. The density of the fungal inoculum was determined and the concentration of the fungus was adjusted to 105 spores/ml.
  • the actinomycetes were inoculated on the sides of the Petri dishes containing the grapevine tissue extract agar (1 kg sheath chopped into small pieces was submersed in 1000 ml boiling tap water for 1 h and 15 g agar was added). After inoculation at 28 oC for 4 days, Botrytis cinerea was inoculated on the centre of the Petri plates. After 4 days under the same culture conditions, the distance between the edge of actinomycete colony and the edge of fungal colony was evaluated for evidence of inhibition.
  • Calcofluor white (Sigma F-3397) and Nile red (Sigma N-3013).
  • Stock solutions of the stains were made at 1 mg ml-1 for Nile red in DMSO while Calcofluor white (Sigma Fluorescent Brightener 28) was made at 1 mg ml-1 in 50 mM phosphate buffer.
  • the working concentration in staining solutions ( ⁇ l stock solution ml-1 MiIIiQ water) and staining times were 15 min for Calcofluor white at 3 ⁇ l ml— 1, and 20 min for Nile red at 2 ⁇ l ml— 1.
  • the effect of actinomycete on vitality of B. cinerea was tested by adding the fluorescent stains to fungus mycelium in eppendorf.
  • V. vinifera L. 'Chardonnay' were obtained by growing nodal explants on Murashige and Skoog medium (1962, A revised medium of rapid growth and biomass assays with tobacco tissue cultures. Physiol. Plant. 15, 473-497). Vitroplantlets were grown under 200 ⁇ mol m 2 s -1 white fluorescent light for a 16 h photoperiod at 25 oC day and night temperatures.
  • Multiplication was done in 25-mm-diameter test tubes containing a 15 mL medium. In each of the following experiments 24 nodal explants were used per treatment.
  • Plant growth was measured and the effect of bacterial inoculation on the plant was assessed (Fig. 10).
  • the strains that promoted greater plant growth were SS37.
  • the length of the principal root was boosted when the plantlets were inoculated with SS37.
  • NN node number
  • SFW shoot fresh weight
  • RFW root fresh weight
  • TB total biomass.
  • Values per plantlets are means of 24 replicates based on 12-week-old-plantlets.
  • Inoculated plantlets (first generation) were subcultured in vitro obtain the second generation. Inoculation was carried out only on the initial explants, not on the second and the third generation explants.
  • SS37 isolate is capable of establishing endophytic and epiphytic populations, allowing clonal multiplication of plantlets by nodal explants in perpetuum without the need for reinoculation (Fig. 1OA and B).
  • the response of plant toward bacterization was maintained and amplified after the second and the third generation.
  • the actinomycete has the advantages of escaping microbial competition and influencing the host's response to pathogens attack.
  • Enzyme activities were determined by the method of the Reinhold-Hurek et al (1993, Cloning, expression in Escherichia coli, and characterization of cellulo lytic enzymes of Azoarcus sp., a root invading diazotroph. J. Bacteriol. 175, 7056-7065) with some modifications. Briefly, plates containing Bennett solid medium, with either 0,2% carboxymethyl cellulose (CMC) or 0,5% polygalacturonic acid, were spot inoculated with actinomycetes and incubated at 30oC for 4 days.
  • CMC carboxymethyl cellulose
  • Endopolygalacturonase activity was determined by the appearance of intense purple-red halos on a colourless background at the point where the bacterium was inoculated (McKay, 1988, above cited).
  • the experiment unit in all experiment was an individual plant, contained in test tubes. Plants (24) were evaluated per treatment. An antagonistic test was done using six plates for each treatment. All experiments were repeated three times.

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Abstract

The present invention relates to new actinomycetes strain compositions and their use for the prevention and/or the control of micro organism inducing plant diseases.

Description

NEW ACTINOMYCETES STRAIN COMPOSITIONS AND THEIR USE FOR THE PREVENTION AND/OR THE CONTROL OF MICRO ORGANISM INDUCING PLANT
DISEASES
The present invention relates to new actinomycetes strain compositions and their use for the prevention and/or the control of micro organism inducing plant diseases.
The causal agent of grey mould, Botrytis cinerea, is a ubiquitous necrotrophic pathogen and can infect almost every plant part on a wide range of plants worldwide (Rosslenbroich and Stuebler, 2000, Botrytis cinerea - history of chemical control and novel fungicides for its management. Crop Prot. 19, 557-561). The control of this and other fungal diseases of grapevine is mainly achieved by use of chemical fungicides. Widespread use of chemical fungicides has certainly decreased the incidence of fungal diseases, but at the same time has contributed to the appearance of fungicide resistant strains of the pathogens. Due to consumer resistance to chemical residues in food and public concern for environmental safety, there is an increasing demand to develop alternative methods for disease control.
In recent years, microbe-based biological control of plant-pathogenic fungi has increased in agricultural importance as replacements or supplements for agrochemicals, which may pollute the environment. Public concern about chemical pesticides has fostered an interest in application of bacteria for biological control to protect agricultural crops against pathogenic fungi. Microbe- based biological control of plant diseases has increased in agricultural importance as replacements or supplements for agrochemicals, which may pollute the environment and affect non-target organisms (Gerhardson et al., 2002, Biological substitutes for pesticides. Trends in Biotechnology. 20(8) 338-343; Welbaum et al., 2004, Fertilizing soil microorganisms to improve productivity of agroecosystems. Crit. Rev. Plant Sci. 23, 175-193.).
The search for alternatives to chemical control of plant pathogens, such as biological control, has also gained momentum due to the emergence of fungicide-resistant pathogens and health concerns for the producer and the consumer (Compant et al. 2005; Biocontrol of plant diseases using plant growth-promoting bacteria (PGPB): principles, mechanisms of action and future prospects. Appl. Environ. Microbiol. 71 :4951-4959). The control of pathogens by biological agents is not widely practiced on a commercial scale, but there have been a number of experimental approaches which show potential for further development. In targeting fungal diseases, endophytic bacteria have shown significant control for different types of pathogens. To date, many promising microbes have been reported as candidate biocontrol agents against soilborne and airborne plant pathogenic fungi (Hallman et al., 1997, Bacterial endophytes in agricultural crops. Can. J. Microbiol. 43, 895-914; Sivasithamparam, 1998, Root cortex-the final frontier for the biocontrol of root-rot with fungal antagonists: a case study on a sterile red fungus. Ann. Rev. Phytopathol. 36, 439-452; Sturz et al., 2000, Endophytic communities of rhizobacteria and the strategies required to create yield enhancing associations with crops. Appl. Soil Ecol. 15,183-190; Mucciarelli et al., 2003, In vitro and in vivo peppermint {Mentha piperita) growth promotion by nonmycorrhizal fungal colonization. New Phytologist 158, 579-591; Narisawa et al., 2004, Control of Verticillium yellows in Chinese cabbage by the dark septate endophytic fungus LtVB3, Phytopathology 94, 412-418; Jain and Jain, 2007, Isolation, characterization and antifungal activity of Streptomyces sampsonii GS 1322. Indian J. Exp. Biol. 45, 203-206).
Beneficial bacteria, known as plant growth-promoting rhizobacteria (PGBR) (Kloepper et al., 1991, Plant promotion mediated by rhizosphere colonizers. In: Keister, D.L., Cregan, P.B. (Eds.), The Rhizosphere and Plant Growth. Kluwer Academic Publishers, Dordrecht, pp. 315- 326), colonize roots, enhance shoot emergence, and stimulate plant growth either directly, by producing plant hormones and improving nutrient uptake, or indirectly, by changing the microbial balance in the rhizosphere in the favor of the beneficial microorganisms (Nowak et al., 1988, Benefits of in vitro "biotization" of tissue cultures with microbial inoculants. In vitro Cell. Dev. Biol. Plant 34, 122-130; Lazarovits and Nowk, 1997, Rhizobacteria for improvement of plant growth and establishment. HortScience 32, 188-192; Ait Barka et al., 2000, Enhancement of in vitro growth and resistance to gray mould of Vitis vinifera L. co-cultured with plant growth- promoting rhizobacteria. FEMS Microbiol. Lett. 186, 91-95; Ait Barka et al, 2002, Inhibitory effect of endophyte bacteria on Botrytis cinerea and its influence to promote the grapevine growth. Biol. Control 24,135-142; Compant et al., 2005a, Use of plant growthpromoting bacteria for biocontrol of plant diseases: principles, mechanisms of action, and future prospects. Appl. Environ. Microbiol. 71, 4951-4959 and 2005b), Endophytic colonization of Vitis vinifera L. by plant growth-promoting bacterium Burkholderia sp. strain PsJN. Appl. Environ. Microbiol. 71: 1685-1693).
Actinomycetes, especially Streptomyces spp., are among the most fascinating microorganisms. Actinomycetes are well-known isolated from the rhizosphere and have been the focus for excellent biocontrol agents to soilborne plant diseases (Fabre et al., 1988, A simple screening method for insecticidal substances from actinomycetes. J. Antibiot. (Tokyo) 41, 12- 219; Tokala et al., 2002, Novel plant microbe rhizosphere interaction involving Streptomyces Lydicus WYEC108 and the pea plant (Pisum sativum). Appl. Environ. Microbiol. 68, 2161-2171; Ikeda, 2003, Pharmacological effects of ivermectin, an antiparasitic agent for intestinal strongyloidiasis: its mode of action and clinical efficacy. Nippon Yakurigaku Zasshi 122, 527- 538; El-Tarabily et al., 2000, Biological control of Sclerotinia minor using a chitinolytic bacterium and actinomycetes. Plant Pathology 49, 573-583; Errakhi et al., 2007, Evidences of biological control capacities of Streptomyces spp. against Sclerotium rolfsii responsible for damping-off disease in sugar beet {Beta vulgaris L.). World J. Microbiol. Biotechnol. 23, 1503- 1509; Jain and Jain, 2007, Isolation, characterization and antifungal activity of Streptomyces sampsonii GS 1322. Indian J. Exp. Biol. 45, 203-206).
The actinomycetes are important producers of bioactive compounds (Lange et al. 1993; Microbial Fungicides - the Natural choice. Pestic. Sci. 39:155-160) and constitute a potential as biocontrol agents (Kim et al. 2000, Structure elucidation and fungal activity of an anthracycline antibiotic, daunomycin, isolated from Actinomadura roseola. J. Agr. Food Chem. 48:1875-1881; Gomes et al. 2000, Chitinolytic activity of actinomycetes from a cerrado soil and their potential in biocontrol. Letters Appl.. Microbiol. 30:146-150; Ouhdouch et al. 2001, Actinomycetes from Maroccan Habitats: Screening for antifungal activites. Eur. J. soil Biol. 37:1-6). Actinomycetes represent a high proportion of soil microbial biomass, and appear to be of importance among the microbial flora of the rhizosphere (Sardi et al. 1992, Isolation of endophytic Streptomyces strains from surface-sterillized roots. Appl. Environ. Microbiol. 58:2691-2698). Associations between actinomycetes and plant organs could be deleterious or beneficial for the host. Some actinobacteria are also known to inhabit the tissues of healthy plants. By cultivation independent techniques based on 16S rDNA genes, the actinomyctes were found to colonize inside the roots of barley (Thirup et al, 2001, Succession of Indigenous Pseudomonas spp. and Actinomycetes on Barley Roots Affected by the Antagonistic Strain Pseudomonas fiuorescens DR54 and the Fungicide Imazalil. Appl. Environ. Microbiol. 67, 1147-1153 and stems of potato (Sessitsch et al, 2002, Cultivation- independent population analysis of bacterial endophytes in three potato varieties based on eubacterial and Actinomyces-specific PCR of 16S rRNA genes. FEMS Microbiol. Ecol. 39, 23-32).
The actinomycetes residing in healthy plant tissues without causing symptoms of disease were defined as endophytic actinomycetes. The first endophytic actinomycetes were studied from nonleguminous plants (Benson et al, 1993, Biology of Frankia strains actinomycete symbionts of actinorhizal plant. Microbiological Reviews 57, 293-319). Other endophytic actinomycetes such as Streptomyces , Streptoverticillium, Nocardia, Micromonospora, Microbispora, and Streptosporangium strains were isolated surface-sterilized roots of different plant species in Italy (Sardi et al., 1992, Isolation of endophytic Streptomyces strains from surface-sterilized roots. Appl. Environ. Microbiol. 58, 2691-2693) and of maize in Brazil (Araujo et al, 2000, Diversity of endophytic bacterial populations and their interaction with Xylella fastidiosa in citrus plants. Appl. Environ. Microbiol. 68,4906-4914). The use of endophytic actinomycetes as biocontrol agents of soil-borne roots disease is of interest through their ability to colonize healthy plant tissue and produce antibiotics in situ (Kunoh et al, 2002, Endophytic actinomycetes: attractive biocontrol agents. Journal of General Plant Pathology 68, 249-252; Cao et al, 2004, Isolation and characterization of endophytic Streptomyces strains from surface-sterilized tomato (Lycopersicon esculentum) roots. Lett. Appl. Microbial. 39, 425-430; Conn et al, 2008, Endophytic actinobacteria induce defence pathways in Arabidopsis thaliana. MoI. Plant Microbe Interact. 21, 208-218).
Therefore, there is a need to develop new fungicides for which on one hand pathogens are not resistant and on the other hand which do not show the drawbacks of chemical fungicides.
One of the aims of the invention is to provide new compositions comprising actinomycetes strains or compounds thereof and their use for their stimulation and/or fungicide properties against micro organisms responsible of plant diseases without showing chemical fungicide drawbacks.
Another aim of the invention is to provide an extraction and purification process of compounds contained in said actinomycetes strains. Another aim of the invention is to provide prevention and/or control process of micro organism inducing plant diseases.
The present invention relates to the use of a composition comprising one or more actinomycetes strains, said composition having stimulation properties of plant natural defences and/or fungistatic properties, for the prevention and/or the control of micro organism inducing plant diseases, provided that when said plant is soybean, and when only one strain of actinomycetes is used, said actinomycetes strain is different from Streptomyces melanosporofaciens (EF-76).
Actinobacteria or actinomycetes are a group of Gram-positive bacteria. They include some of the most common soil life, playing an important role in decomposition of organic materials, such as cellulose and chitin and thereby playing a vital part in organic matter turnover and carbon cycle.
Most actinobacteria of medical or economic significance are in subclass Actinobacteridae, order Actinomycetales, and several genus are found, the most important of them being the following: Actinomyces, Arthrobacter, Corynebacterium, Frankia, Micrococcus, Micromonospora, Mycobacterium, Nocardia, Propionibacterium, Streptomyces .
Actinomycetes can be isolated from soil of various origin and in particular from the Moroccan soil according to procedures well-known from a man skilled in the art, and for instance, according to example 1. In the following of the specification, the term "strain" also refers to one of its variant or mutant.
A mutant of a strain contains at least one modification of the genotype of said strain provided that said mutant belongs to the same species of the strain.
A variant of a strain contains one or more modification of the genotype of said strain without altering the phenotype of said strain. According to the Cambridge Advanced Learner's Dictionary, Third edition Cambridge University press, the term "stimulation" means: "something that causes someone or something to become more active or to develop or function".
The term "plant" means living organisms belonging to the kingdom Plantae. They include familiar organisms such as trees, herbs, bushes, grasses, vines, ferns, mosses, and green algae.
By the expression "plant natural defences" is meant the implementation of one or more mechanism(s) of defence by the plant itself during an infection of said plant by a pathogen agent, such as a fungi, i.e. the implementation of mechanisms of control of the pathogen by the plant.
Said involved mechanisms of defence are chosen among the following: - the hypersensitive reaction (HR), i.e. a localised and induced cellular death of infected tissues, the "Local Acquired Resistance" (LAR), i.e. an induction phenomenon. LAR is induced by the cells in a state of cellular death via the transmission of signal molecules such as jasmonic acid (JA), salicylic acid (SA) or ethylene (ET), - the "systemic acquired resistance" (SAR) or the "induced systemic resistance" (ISR).
Therefore, the expression "stimulation properties of plant natural defences" means that said actinomycetes strains are able to trigger said mechanisms of defence in order to control pathogens.
Said stimulation properties of plant natural defence can be evaluated according to Walters et al., 2005, (Induced Resistance for Plant Disease Control: Maximizing the Efficacy of
Resistance Elicitors. Phytopathology, 95 : 1368-1383 Fagard et al, 2007, Arabidopsis thaliana Expresses Multiple Lines of Defense to Counterattack Erwinia chrysanthemi MPMI, Vol. 20, No. 7, pp. 794-805.)
The term "fungistatic" refers to any agent that inhibits fungal growth without killing the fungus.
Therefore, the expression "fungistatic properties" means that said actinomycetes strains are able to inhibit fungal growth without killing the fungus.
Said "fungistatic properties" can be evaluated according to Ouhdouch et al. 2001, (Actinomycetes from Maroccan Habitats: Screening for antifungal activites. Eur. J. soil Biol. 37:1-6) The term "control" means to limit, to protect or regulate a plant against a pathogen. By "micro organism induced disease" is meant a disease caused by a micro organism or a microbe, i.e. by an organism that is microscopic (usually too small to be seen by the naked human eye). Microorganisms are very diverse. They include bacteria, fungi, archaea, and protists; microscopic plants (called green algae); and animals such as plankton, the planarian and the amoeba.
Such diseases are various and can be for example, but without being limited to: the mildew (or blew spot, or downy mildew or blue mold) caused by fungi selected from the list consisting of phytophthora, plasmopara or peronospora, such as phytophthora infestans on potato or tomato, or Peronospora tabacina on tobacco, or Plasmora viticola on vine, the powdery mildew caused by a fungus of the family Erysiphaceae such as Erysiphe necator on vine, or a fungus of the genus Oidium distinguished by an abundant powdery conidia produced on the host, eutypiose disease of the vine caused by the fungus Eutypa lata after pruning, black dead arm (BDA) caused by Botrytosphaeria sp., grey mould of the vine caused by Botrytis cinerea, and other wood diseases or ESCA (Galet , 1977, Les Maladies et Les Parasites de La Vigne. Tome I, Imprimerie du Paysan du Midi, France, pp. 313-360) caused by various fungi such as
Phaemonacremonium sp., Phaemoniella sp., Phellinus sp...),
It must be noted that in the case of soybean plant, that is a species of legume native to East Asia and from the family of Fabaceae, the following actynomycetes strain: Streptomyces melanosporofaciens (EF-76), is excluded from the compositions of the invention and cannot therefore be used for the prevention and/or the control of micro organism induced plant diseases.
Thus, one of the advantages of the compositions of the present invention is their capacity to develop or promote the natural defences of the plant and to inhibit or stop the growth of the fungi.
Interestingly, the compositions of the invention have prevention properties or fungistatic properties but can also possess both properties. As a result, the composition is, in one hand, able to prevent or to protect plants from a the aggression of a pathogen agent, i.e. to avoid a pathogen agent such as a micro organism from attacking said plant and further causing a disease but it is also able, on the other hand to control, i.e. to fight against said micro organism, i.e. to protect the plant against the micro organism leading to the development control of said micro organism without having harmful impact neither on the growth of said plant and thus on the biomass yield, nor on the environment.
In another embodiment, the present invention relates to the use of a composition comprising one or more actinomycetes strains, said composition having stimulation properties of plant natural defences and/or fungistatic properties, for the prevention and/or the control of micro organism induced plant diseases, provided that when said plant is soybean, said actinomycetes strain is different from Streptomyces melanosporofaciens (EF-76).
In an advantageous embodiment, said actinomycetes strains above defined are of Streptomyces or Micromonospora genus.
The Streptomyces genus comprised more than 500 species, for example, but without being limited to the following species:
S. ambofaciens, S. achromogenes, S. anulatus, S. avermitilis, S. coelicolor, S. clavuligerus, S. felleus, S.ferralitis, S.filamentosus, S. griseus, S. hygroscopicus, S. iysosuperficus, S. lividans, S. noursei, S. scabies, S. somaliensis, S. thermoviolaceus, S. venezuelae and S. violaceoruber.
The Micromonospora genus comprised several species, for example, but without being limited to the following species:
Micromonospora aurantiaca, Micromonospora carbonacea, Micromonospora chalcea, Micromonospora chersina, Micromonospora citrea, Micromonospora coerulea, Micromonospora echinaurantiaca, Micromonospora echinofusca, Micromonospora echinospora, Micromonospora fulviviridis, Micromonospora gallica, Micromonospora halophytica, Micromonospora inositola, Micromonospora inyonensis, Micromonospora nigra, Micromonospora olivasterospora, Micromonospora pallida, Micromonospora peucetia, Micromonospora purpureochromogenes, Micromonospora rosaria, Micromonospora sagamiensis, Micromonospora viridifaciens .
In an advantageous embodiment, said Streptomyces genus strains defined above are chosen among Streptomyces spp., i.e. among the 500 species above defined, provided that when only one strain is used in the composition, the Streptomyces melanosporofaciens (EF-76) strain is excluded from the compositions of the present invention.
In an advantageous embodiment, the micro organisms above defined are chosen among Botrytis sp., Fusarium sp., Pythium sp. or Candida tropicalis R2, in particular Botrytis cinerea. Botrytis sp. is a genus of Ascomycete fungi causing several plant diseases. The genus contains 22 species and one hybrid.
Plant diseases caused by Botrytis species appear primarily as blossom blights and fruit rots but also as leaf spots and bulb rots in the field and in stored products. The fungi induce host cell death resulting in progressive decay of infected plant tissue, when they take nutrients. Fusarium sp. is a large genus of filamentous fungi widely distributed in soil and in association with plants. Most species are harmless saprobes and are relatively abundant members of the soil microbial community.
The genus includes a number of economically important plant pathogenic species.
Fusarium graminearum commonly infects barley under unstable weather conditions such as late rain. Fusarium contamination in barley can result in head blight and in extreme contaminations the barley can appear pink
Fusarium graminearum can also cause root rot and seedling blight.
Pythium sp. is a genus of micro organism classified among oomycetes, previously classified as fungi. Pythium root rot is a common crop disease caused by a genus of organisms called "Pythium". These are commonly called water moulds.
Pythium damping off is a very common problem in fields and greenhouses, where the organism kills newly emerged seedlings.
Candida tropicalis R2 is an amphotericin B and nystatin resistant fungal strain.
Botrytis cinerea is a necrotrophic fungus that affects many plant species, such as tomato or sunflower and in particular wine grapes.
This fungi exists under four forms in function of its biological cycle: sclerotia compact mass of hardened mycelium stored with reserve food material), mycelia (vegetative part of a fungus, consisting of a mass of branching, thread-like hyphae), conidia, sometimes termed conidiospores that are asexual non-motile spores of a fungus and apothecia that are particular bowl-shape sexed formation. The stimulation properties of plant natural defences and/or the fungistatic properties of the compositions of the invention allows to prevent and/or control the micro organism induced plant diseases by interacting with one, two, three or four of the four forms of Botrytis cinerea fungi. In an advantageous embodiment said micro organisms are in particular fungi responsible of wood diseases or ESCA and include Phaemonacremonium sp., Phaemoniella sp., Phellinus sp, or Eutypa lata ...
In an advantageous embodiment, the actinomycetes strain above defined is used at a concentration from about 10 cfu/ml to about 10 cfu/ml, preferably from about 107 cfu/ml to about 10 cfu/ml, more preferably from about 107 cfu/ml to about 10 cfu/ml, in particular 107 cfu/ml.
Below 106 cfu/ml, the concentration is too low to observe an activity.
The maximal observed effect does not change above 10 cfu/ml.
At this range dose, the compositions of the invention comprising the actinomycetes strains defined above are liable to prevent and/or control the micro organism induced plant diseases.
For instance, the emergence of grey mold caused by Botrytis cinerea in leaves of V. vinifera (common grape vine) is prevented by pre treating leaves or plantlets with the SS37 actinomycetes strain of example 1 (see example 3 and figures 2 and 3) and the figure 6 demonstrates that the hyphal growth of Botrytis cinerea is inhibited in the presence of SS37 on a grapevine tissue extract showing thus the fungistatic effect of this strain.
In an advantageous embodiment, the composition above defined comprises or is constituted of the culture supernatant isolated from a culture medium of said actinomycetes strain. By "culture medium" or "growth medium" is meant a liquid or gel designed to support the growth of microorganisms or cells.
The "culture medium" may be commercially available. The most common growth media for microorganisms are nutrient broths (liquid nutrient medium) or Luria Bertani medium (LB medium or Lysogeny Broth). Liquid media are often mixed with agar and poured into petri dishes to solidify. They may be also specifically designed for a particular micro organism.
By "culture supernatant isolated from a culture medium" is meant the liquid fraction surmounting the micro organism after growing in the culture medium and that has been removed from the culture medium. Thus, in the present invention, the compositions comprise or consist in either the actinomycetes strains or in the supernatant of the culture medium of said actinomycetes strains and are both liable to prevent and/or control the micro organism induced plant diseases.
In an advantageous embodiment, the culture supernatant isolated from a culture medium of said actinomycetes strain above defined is at a concentration from about 10- M to about 10- M, preferably from about 10- M to about 10- M, more preferably from about 10- M to about 10-1 M, in particular 10-2 M.
Above 10-1 M, the concentration of the supernatant is too high and has harmful impact on the plant.
Below 10- M, the concentration of the supernatant is too low to be still active. In an advantageous embodiment, the composition above defined comprises or is constituted of one or more chemical compounds extracted and purified from said culture supernatant, or synthesized by a chemical way.
For instance, following the procedure of example 5, the man skilled in the art is able to extract and purify more than six fungicidal compounds, in particular against B. cinerea, belonging to several different chemical families, in particular from two to more than three different chemical families, such as compounds IV, V, VI, VII, VIII, IX and X (see example 5).
Therefore, in the present invention, the composition can comprise a mixture of said above seven defined compounds, or a mixture of six, five, four, three or two compounds selected from the chemical compound list above defined, or a single compound from said list. Therefore, another advantage of the invention is to provide a composition comprising several compounds from different chemical families allowing the prevention and/or the control of plant diseases induced by one or more micro organisms.
In an advantageous embodiment, the composition above defined comprises or is constituted of one or more chemical compounds belonging to one of the following chemical families: tryptophan derivatives, 2-terpenyl pyridines, ionophoretic polyethers, and chlorinated lipid compounds, extracted and purified from said culture supernatant.
Tryptophan (abbreviated as Trp or W) is one of the 20 standard amino acids, as well as an essential amino acid in the human diet and has the following formula (I):
Figure imgf000013_0001
The term "tryptophan derivatives" represents every chemical compound of formula I-a bearing an indole moiety and an oxazole moiety that can be substituted by one to eight substituents:
Figure imgf000013_0002
in which Rl to R7 are selected independently from each other from the group consisting in: hydrogen, halogen group such as chlorine, bromine, fluorine or iodine, alkyl or heteroalkyl group in Cl to ClO, linear or branched, saturated or not saturated, substituted or not with an hydroxyl, alkoxy or acyl group, hydroxyl, alkoxy or acyl group, or primary, secondary or tertiary amine,
R8 being selected from the group consisting in: hydrogen, alkyl group in Cl to ClO, linear or branched, saturated or not saturated, substituted or not with an hydroxyl, alkoxy or acyl group,
The expression "2-terpenyl pyridines" refers to a molecule that comprises a pyridine moiety substituted with Rl to R4 and a terpene moiety being into brackets with m and having the following general formula II,
Figure imgf000014_0001
in which Rl to R4 are selected independently from each other from the group consisting in:
- hydrogen,
- halogen group such as chlorine, bromine, fluorine or iodine, - alkyle, alkoxy or acyl group, or
- heteroalkyle group in Cl to ClO, linear or branched, saturated or not saturated, substituted or not with an hydroxyl, alkoxy or acyl group,
- hydroxyl, alkoxy or acyl group, or
- primary, secondary or tertiary amine, m being equal to 2 to 8, and R5 to R9 being selected independently from each other from the group consisting in:
- hydrogen,
- halogen group such as chlorine, bromine, fluorine or iodine,
- alkyle or heteroalkyle group in Cl to ClO, linear or branched, saturated or not saturated, substituted or not with an hydroxyl, alkoxy, acyl, alkyl or heteroalkyl group. - hydroxyl, alkoxy or acyl group,or
- primary, secondary or tertiary amine.
The dashed line means that a double bond is present or not.
The expression "ionophoric polyethers" refers to chemical compounds comprising at least two cyclic polyethers with 5 or 6 atoms linked between them by a bond or by a common carbon atom having the property to form a complex with an ion, and transport said ion across the membrane, with the general formula III:.
Figure imgf000015_0001
in which Rl to R7 are any chemically acceptable substituent.
The chemical compounds above defined of this embodiment can be extracted and purified by methods known from a man skilled in the art, or according to example 5, from the supernatant of the micro organism culture medium, or synthesized by a chemical or other way.
Therefore, in this embodiment, the composition comprises a mixture of two or several compounds belonging to one or two or three chemical families.
In an advantageous embodiment, said chemical compounds, are at a concentration from about 10- M to about 10- M, preferably from about 10- M to about 10- M, more preferably from about 10-5 M to about 10-9 M, more preferably from about 10-6 M to about 10-9 M, more preferably from about 10-7 M to about 10-9 M, more preferably from about 10-8 M to about 10-9 M, more preferably from about 10- M to about 10- M, and more preferably from about 10- M to about 10-7 M.
At a concentration below 10-9 M, the chemical compound is no more active. At a concentration above 10-3 M, the chemical compound concentration is too high and has harmful impact on the plant, such as decrease of the growth or death of the plant. In an advantageous embodiment, said actinomycetes strain, comprised or constituted in the above defined composition, is the actinomycetes strain S37, as deposited at the BCCM on the 03rd Mai 2007 under the number LMG P-24130.
In the following description, the actinomycetes strain can be named S37 or SS37 and refers to the same strain. It can be also named S37 or SS37 actinomycete isolate.
The S37 actinomycetes strain has been tested for taxonomical diversity using morphological, cultural, physiological and biochemical criteria as a well as others features (example 2a) as shown in the table I below of example 2.
The analysis of cellular constituents of the SS37 isolate revealed the presence of the L- diaminopimelic acid (DAP) isomer (table I) and the isolate was assigned to the genus of
Streptomyces. That has been confirmed by the sequencing of the 16S RNA of these strains confirmed this classification (SEQ ID NO:1 corresponding to the 16S rDNA and SEQ ID NO:2 corresponding to the 16S rRNA).
SS37 isolate exhibited 99% sequence identity with Streptomyces anulatus. In an advantageous embodiment, said chemical compounds are chosen among 4-chloro-5-
(3'-indolyle) oxazole, piericidine or abierixine.
4-chloro-5-(3'-indolyle) oxazole has the following structure (IV):
θ-"^ N
(IV)
Cl
H
Piericidin has the following structure (V):
Figure imgf000016_0001
Abierixin has the following structure (VI):
Figure imgf000017_0001
These three compounds have been isolated according to example 5.
In this embodiment, the composition can comprise one single compound selected from compounds list IV, V and VI or two compounds selected from said compound list, i.e. compounds IV and V, or compounds IV and VI, or compounds V and VI, or the three compounds IV, V and VI.
For instance, compounds of the invention, in particular compounds IV, V and VI are able to control B. cinerea in grapevine in vitro (Figure 14) as well as in vivo, see for example compound IV, figure 15C). In an advantageous embodiment, said stimulation properties of plant natural defences with the composition above defined, are obtained by the signalling pathway activation, in particular leading to a local and systemic induction of at least one of the following genes: VvGIuC, VvChi4C, VvPRό and VvStSy with an induction factor higher than about 3, except for the activation of the fiavonoid synthesis pathway, the induction factor of which being less than 3, in particular the gene inducing the phenylalanine ammonia lyase (PAL).
By the expression "stimulation properties of plant natural defences" is meant that the plant synthesizes one or more defence compounds such as PR (pathogenesis related) protein (van
Loon et al, 2006; Ethylene as a modulator of disease resistance in plants. Trends in Plant Science
11: 184-191; Significance of inducible defence related proteins in infected plants. Annual Review of Phytopathology 44: 135-162; see figure 9), by means of signal molecules. The « signalling pathway activation» means that genes implicated in the plant natural defences and causing the resistance of the plant near and around the pathogen infection site, have an increased expression, i.e. they are stimulated or inducted by means of signal molecules such as jasmonic acid (JA), salicylic acid (SA), ethylene, systemin or abscissic acid. The expression "local and systemic induction" means that the induction, i.e. the activation of the gene implicated in the plant natural defences is carried out near the pathogen infection site and activated by interaction of the plant with the pathogen agent.
VvGIuC is a gene coding for a basic PR-2 (β-1,3-glucanase).
VvChi4C is a gene coding for an acidic PR-3 (chitinase). VvPR6 is a gene coding for a PR-6 (proteinase inhibitor).
VvStSy is a gene coding for a stilbene synthase.
VvChi4C et VvGIuC are induced or regulated by both SA and JA.
VvPR6 is induced by JA.
The activation of said gene can be measured by quantitative RT-PCR (Bezier et al. 2002, Study of defence-related gene expression in grapevine leaves and berries infected with Botrytis cinerea. European Journal of Plant Pathology 108: 111-120)
The expression "induction factor" measures the activation of the gene induced by means of signal molecules to produce a defence compounds such as PR.
Above 3, said induction factor is efficient for the activation of said gene coding for said protein.
Below 3, said induction factor is inefficient.
Thus in the invention, the composition comprising either one or more actinomycetes strain, or the culture supernatant isolated from a culture medium of said actinomycetes strain or one or more different chemical molecules is thus able to induce one of the genes selected from the list above defined or two genes such as VvGIuC and VvCi4C, or VvGIuC and VvPR6, or VvGIuC and VvStSy , or VvCM4C and VvPR6, or VvCM4C and VvStSy, or VvPR6 and VvStSy, or three genes such as VvGIuC, VvChi4C and VvPR6, or VvGIuC, VvPR6 and VvStSy, or VvChi4C, VvPRό and VvStSy, or the four genes of said list, leading thus to an improved resistance of the plants toward one or more micro organism induced plant diseases and thus to the prevention of one or more micro organism induced plant diseases and/or an improved pathogen control.
For instance, compound of the invention, in particular compounds IV, V and VI are able to induce different genes, in particular STS, Chit4c, PR6 or Glue with different induction factors (figure 16 and 17).
In an advantageous embodiment, the ratio corresponding to the activation of the flavonoid synthesis pathway:
[isoflavones after stimulation] - [isoflavones before stimulation]
[isoflavone before stimulation] is lower than 0.45 and in particular the isoflavone quantity being present after stimulation of the natural defences is lower than 680μg/g of dry matter.
Said ratio and said isoflavone quantity correspond to the activation of the expression of phenylalanine ammonia lyase (PAL). A ratio lower than 0.45 means that said PAL has not been induced by the actinomycetes strains of the invention and that the protection against micro-organism induced diseases is not due to the induction of PAL expression.
In an advantageous embodiment, said composition used has also fungicidal properties and/or growth promoting properties. Fungicidal properties can be evaluated according to Bauer et al. 1966, (Antibiotic susceptibility testing by standardized single disk method.Am J Clin Pathol 45 :493-497)
Growth promoting properties can be evaluated according to Ait Barka et al., (2000,
Enhancement of in vitro growth and resistance to gray mold of Vitis vinifera L. co-cultured with plant growth-promoting rhizobacteria. FEMS Microbiology Letters 186: 91-95) The term "fungicidal" refers to any agent that inhibits fungal growth and kills the fungus.
For instance, the actinomycete strain of example 1 (SS37) is able to inhibit the development of Botrytis cinerea when it is introduced on potato dextrose agar medium (PDA, figure 1) previously inoculated with Botrytis cinerea or the compounds of example 5 are able to inhibit the development of Botrytis cinerea when they are introduced on PDA previously inoculated with Botrytis cinerea (see example 5 and figure 8). Table II (example 4b) shows the fungicidal activity against B. cinerea.
In another example, the compound X alone has fungicidal properties on B. Cinerea as shown by a local effect on B. Cinerea (figure 13) and compounds IV, V, VI, and to a lesser extent, compounds VII and VIII have a distance effect on the growth of B. cinerea, while compound X has no distance effect.
Another advantage of said composition comprising a mixture of chemical compounds above defined is to exhibit a synergistic effect against one or more fungi compared to the effect of each compound taken alone against the same fungus or fungi (figure 13).
The expression "growth promoting properties" refers to an agent able to increase the development of a plant.
For instance, plant growth was measured and the effect of bacterial inoculation on the plant was assessed (Fig. 10). The shoot and root fresh weights were increased by the treatment with the selected SS37 isolate (Table IV).
The length of the principal root was boosted when the plantlets were inoculated with SS37.
Thus, in this embodiment the composition have plant natural defences and/or fungistatic properties as well as fungicidal properties and/or growth promoting properties and thus have two, three or four of the properties list above defined.
In an advantageous embodiment, said composition above defined allows the implementation of a systemic acquired resistance (SAR, Durrant W.E et al. 2004, Systemic acquired resistance. Annual Review of Phytopathology 42: 185-209) and/or the implementation of an induced systemic resistance (ISR, Van Loon et al., 1998, Systemic resistance induced by rhizosphere bacteria. Annual Review of Phytopathology 36: 453-483, Pieterse, CMJ. et al., 2001,
Rhizobacteria-mediated induced systemic resistance: triggering, signalling, and expression. European Journal of Plant Pathology 107: 51-61) in said plants.
The "systemic acquired resistance (SAR)" is a resistance mechanism activated by a first interaction with a pathogen leading to long time resistance of the plant (several weeks) against the pathogen infections caused by various pathogen micro organisms.
It consists in the activation of some genes allowing the induction and the maintaining of a resistance state of the whole plant against a large spectrum of pathogen agents. The ISR is induced following the plant interaction with beneficial bacteria.
For instance, the presence of actinomycete in the root tips (Fig. 1 IA), after inoculation, supports the possibility of entry of actinomycetes strain of the invention, in particular SS37, via root tips and the continuous presence of actinomycetes in the plant even in the second or third generation, allowing thus the plant to resist to other micro-organism attacks.
In another example, vitroplants of Chardonnay (grapevine), that are susceptible to fungal attack, inoculated with B. cinerea produced characteristic gray mold symptoms within 7 days (Fig. 12A and B). In contrast, under the same inoculation and growth conditions, endophyte actinomycete bacterized vitroplants appeared healthy (Fig. 12C and D) showing thus not only the stimulation properties of plant natural defences with SS37 but also the implementation of an induced systemic resistance (ISR) of said plant toward the disease and thus the prevention of said disease.
In an advantageous embodiment, said plants are chosen among grapevine, or cereals such as wheat or rape. In another aspect, the present invention relates to a composition comprising the actinomycetes strain S37 as deposited at the BCCM on the 03rd Mai 2007 under the number LMG P-24130, as defined above, or a culture supernatant of a culture medium of said actinomycetes strain.
In an advantageous embodiment, said strain comprised in the composition above defined is at a concentration from about 106 cfu/ml to about 109 cfu/ml, preferably from about 107 cfu/ml to about 10 cfu/ml, from about 10 cfu/ml to about 10 cfu/ml, in particular 10 cfu/ml.
In an advantageous embodiment, said supernatant of the composition above defined is at a concentration from about 10-4 M to about 10-1 M, preferably from about 10-3 M to about 10-1 M, more preferably from about 10-2 M to about 10-1 M, in particular 10-2M. In another aspect, the present invention relates to a composition comprising or being constituted of one or more chemical compounds belonging to one of the following chemical families: tryptophan derivatives, 2-terpenyl pyridines, ionophoretic polyethers, and chlorinated lipid compounds such as defined above, extracted and purified from the culture supernatant isolated from an actinomycetes strain culture medium, and when only one chemical compound is present in the composition, the following compounds 4-chloro-5-(3'-indolyle) oxazole, piericidine and abierixine are excluded.
Thus in this embodiment, the composition is composed of chemical compounds from an extracted and purified origin, or synthesized by a chemical way.
In another embodiment, said composition comprising or being constituted of one or more of said chemical compounds does not comprise 4-chloro-5-(3'-indolyle) oxazole, piericidine and abierixine.
In an advantageous embodiment, said chemical compounds of the compositions above defined, are at a concentration from about 10- M to about 10- M, preferably from about 10- M to about 10- M, more preferably from about 10- M to about 10- M, more preferably from about 10- 6 M to about 10-9 M, more preferably from about 10-7 M to about 10-9 M, more preferably from about 10- M to about 10- M, more preferably from about 10-7 M to about 10- M, and more preferably from about 10- M to about 10-7 M. In another aspect, the present invention relates to an actinomycetes stain S37 as deposited at the BCCM on the 03rd Mai 2007 under the number LMG P-24130 such as defined in claim 10.
In another aspect, the present invention relates to culture medium allowing to cultivate the strain defined above, comprising or constituted from glucose, peptone (Difco) 2 g/1, yeast extract (Difco) 1 g/1, and meat extract (Difco) 1 g/1.
Glucose is commercially available and can be obtained for example from Merck (Merck Chemicals Limited, Padge Road, Nottingham, NG9 2JR) and is at a concentration comprised from about 1 g/1 to about 100 g/1, preferably from about 1 g/1 to about 50 g/1, more preferably from about 1 g/1 to about 20 g/1, in particular about 10 g/1. Peptone, yeast extract and meat extract are commercially available and can be obtained for example from Difco (USA).
Peptone is used at a concentration from about 0.1 g/1 to about 10 g/1, preferably from about 0.1 g/1 to about 5 g/1, more preferably from about 0.5 g/1 to about 1 g/1, in particular about 2 g/1. Yeast extract is used at a concentration from about 0.1 g/1 to about 10 g/1, preferably from about 0.1 g/1 to about 5 g/1, more preferably from about 0.5 g/1 to about 2 g/1, in particular about 1 g/1.
Meat extract is used at a concentration from about 0.1 g/1 to about 10 g/1, preferably from about 0.1 g/1 to about 5 g/1, more preferably from about 0.5 g/1 to about 2 g/1, in particular about
1 g/1.
The pH is adjusted from about 7.0 to about 7.5, in particular 7.2 before sterilization at about 100ºC to 120ºC, in particular 110ºC for about 10 to about 60 minutes, preferably about 10 to about 50 minutes, more preferably about 20 to about 40 minutes, in particular about 30 minutes.
In another aspect, the present invention relates to an extraction and purification process of chemical compounds, such as defined above, from a culture supernatant isolated from a culture medium of one or more actinomycetes strains such as defined above, comprising a chromatography step, in particular a centrifugal partition chromatography of one or several fractions of chemical compounds obtained by liquid liquid pre- fractionating of a culture supernatant solvent extract of said culture medium of one or more actinomycetes strain, said fractions presenting fungicidal properties, to obtain one or more said pure chemical compounds and, optionally, a mixture of other said chemical compounds.
The chromatography is a well known technique for a man skilled in the art. The expression "centrifugal partition chromatography" is liquid-liquid chromatography without "solid-support", requiring two immiscible solvent phases.
The expression" liquid liquid pre-fractionating" refers to the use of a biphasic or triphasic system composed of organic solvents or water, the solvent system comprising at least 2 immiscible solvents. Organic solvents are chosen among liquid alkanes (hexane, cyclohexane, heptane, petroleum ethers,...) aromatic hydrocarbons (toluene) ketones (acetone, methyl-isobutyl ketone, methyl-ethyl ketone,...) alcohols (methanol, ethanol, butanols) - esters (ethyl acetate, butyl acetate,...) halogenated solvents (chloroform, dichloro methane,...) ethers (methyl terbutyl ether, ethyl terbutyl ether, ...) acetonitrile
The expression "pure chemical compounds" refers to a compound with more than about
95% of purity.
The fungicidal properties of said fractions can be determined by techniques well known from the man skilled in the art.
The extraction and purification process of the compounds of the invention is given in example 5.
In an advantageous embodiment, the fungicidal properties of said fraction(s) of chemical compounds of the extraction and purification process defined above are determined by bioautography.
The bioautography technique is detailed in Petersen, M.; Simmonds, M. Rosmarinic acid. Phyto chemistry 2003, 62, 121-125) and allows to determine the activities of the products adsorbed on plate by chromatography on thin layer.
In an advantageous embodiment, the extraction and purification process defined above comprises a second chromatography, in particular a centrifugal partition chromatography of said mixture of other chemical compounds, different from the previous centrifugal partition chromatography.
In certain cases, the first chromatography does not allow to separate all the compounds produced by the actinomycetes strains.
Therefore, a second chromatography, in particular a centrifugal partition chromatography, with a solvent system different from the one used in the first chromatography, of the collected fractions containing a mixture of chemical compounds not separated must be carried out to obtain all the compounds in a pure state.
The expression "pure state" refers to a compound with more than about 95% of purity.
In an advantageous embodiment, said actinomycetes extract being subjected to the extraction and purification process above defined, has been obtained by the culture supernatant extraction of culture medium of one or more actinomycetes strain by means of a solvent, in particular 1-butanol, then evaporating said solvent containing said strain extract.
In an advantageous embodiment, the extraction and purification process defined above, comprises the following steps: a. culture supernatant extraction of a culture medium of one or more actinomycetes strain, previously cultivated, by means of a solvent, in particular 1-butanol, to obtain a solution containing a culture supernatant extract of a culture medium of one or more actinomycetes strain, b. liquid liquid pre- fractionation of said culture supernatant extract by means of a triphasic or biphasic extraction system, after evaporation of said solvent containing extract, to obtain one or more fraction(s) containing chemical compounds from said culture supernatant extract, c. determination of the fungicidal properties of said fraction(s) containing said chemical compounds by bioautography, d. centrifugal partition chromatography of said fraction(s) above obtained and having fungicidal properties, by means of a solvent biphasic mixture, to obtain one or more said pure chemical compounds and optionally a mixture of other said chemical compounds.
The first step a. allows the extraction of a mixture of compounds contained in the culture medium of the strain. This step can be performed with different organic solvents such as butanol, ethyl acetate, hexane or derivatives, dichloromethane, methanol, ethanol, propanol, chloroform...., and in particular with 1-butanol that leads to the best results considering the detection of the compounds having a biological activity, i.e. the fungicidal activity, by bioautography.
The second step b.) allows to obtain one more fractions containing one or more chemical compound in order to evaluate the biological activity of each fraction. This step corresponds to the partial purification of the butanolic extract obtained in step a.)
The step c.) corresponds to the fungicidal evaluation of each fraction obtained in step b. The step d.) allows to obtain pure chemical compounds from each biological active fraction determined in step c.
The step d.) of the process does not lead always to the purification of all the biological obtained fractions of step c. and some of the chemical compounds contained in the biological active fractions are therefore not separated.
In an advantageous embodiment, the process defined above comprises a further step of centrifugal partition chromatography of said mixture of other said chemical compounds above obtained, by means of a biphasic mixture different from the one used for the first centrifugal partition chromatography carried out in step d). The chemical compounds contained in the biological active fractions that have not been separated in step d. of the previous defined process are collected, then evaporated and subjected to a further centrifugal partition chromatography using a biphasic mixture different from the previous one in order to separate the different chemical compounds of said biological active fractions that have not been separated in step d. In an advantageous embodiment, said strain being subjected to the process above defined is the actinomycetes strain S37 as deposited at the BCCM on the 03rd Mai 2007 under the number LMG P-24130.
In another aspect, the present invention relates to products obtained by process as defined above, except for the compounds 4-chloro-5-(3'-indolyle) oxazole, piericidine and abierixine. In another aspect, the present invention relates to a prevention and/or control process of plant diseases induced by microorganisms such as Botrytis sp., Fusarium sp., Pythium sp. or Candida tropicalis R2, in particular Botrytis cinerea induced diseases, comprising a spraying step of the aerial parts and/or an inoculation step of the underground parts of said plants with a composition comprising one or more actinomycetes strain such as defined above, preferably at a concentration from about 10 cfu/ml to about 10 cfu/ml, preferably from about 10 cfu/ml to about 109 cfu/ml, from about 107 cfu/ml to about 108 cfu/ml, in particular 107 cfu/ml.
In this aspect, the strains are the same as defined above provided that the Streptomyces melanosporofaciens is excluded from the species that can be used in the compositions of the present invention. The spraying step of the aerial part allows to stop the development of the fungi, either by fungistatic activity of the composition (see figure 6), either by killing the fungi (see figure 8). Said spraying allows also (figure 3-5) to prevent the disease and develop a resistance by pre- treatment with the compositions of the invention The inoculation of the underground parts allows to prevent the disease and develop a resistance by pre-treatment with the compositions of the invention (see figure 11, 12).
In an advantageous embodiment, said spraying step of the aerial parts and/or an inoculation step of the underground parts of said plants in the process above defined, is carried out with a composition comprising or constituted of the culture supernatant isolated from a culture medium of one or more actinomycetes strains, preferably at a concentration from about 10~4 M to about 10-1 M, preferably from about 10~3 M to about 10-1 M, more preferably from about 10~2 M to about 10-1 M, in particular 10~2 M.
In an advantageous embodiment, said spraying step of the aerial parts and/or an inoculation step of the underground parts of said plants of the process above defined is carried out with one or more chemical compounds belonging to one of the following chemical families: tryptophan derivatives, 2-terpenyl pyridines, ionophoretic polyethers, and chlorinated lipid compounds such as defined above, and preferably at a concentration from about 10- M to about 10- M, preferably from about 10- M to about 10- M, more preferably from about 10- M to about 10-9 M, more preferably from about 10-6 M to about 10-9 M, more preferably from about 10-7 M to about 10-9 M, more preferably from about 10-8 M to about 10-9 M, more preferably from about 10- M to about 10- M, and more preferably from about 10- M to about 10- M.
Figure 13 and 14 shows the in vitro fungicidal effect of the compounds of the invention.
Figures 11, 12, 15 shows that the inoculation with the compounds of the invention allows to protect the plant and to prevent the disease and develop a resistance against said disease.
In another aspect, the present invention relates to a promoting growth process of plant, said plants being chosen among grapevine, or cereals such as wheat or rape, comprising a spraying step of the aerial parts and/or an inoculation step of the underground parts of said plants with a composition comprising or constituted of strain above defined, or of culture supernatant isolated from a culture medium of one or more actinomycetes strains above defined, or of one or more chemical compounds belonging to one of the following chemical families: tryptophan derivatives, 2-terpenyl pyridines, ionophoretic polyethers, and chlorinated lipid compounds above defined.
In an advantageous embodiment, said chemical compounds of the process above defined are chosen among 4-chloro-5-(3'-indolyle) oxazole, piericidine or abierixine.
In an advantageous embodiment, said strain of the process above defined is the actinomycetes strain S37 as deposited at the BCCM on the 03rd Mai 2007 under the number LMG P-24130.
DESCRIPTION OF THE FIGURES
Figure 1 presents the interaction between selected SS37 actinomycete isolate and B. cinerea when inoculated together on PDA (distance confrontation).
The fungus has been placed in the centre of the Petri dish. This figure shows the significant zone of inhibition around the fungus inoculums due to the effect of SS37 strain against B. cinerea.
The SS37 strain was used at 107CFU/ml. The fungus was used at 105 CFU/ml.
Figure 2 presents the hyphal mycelium from the zone of interaction between B. cinerea and SS37 actinomycete isolate.
Part a: normal hypha of B. cinerea.
Parts b, c, d: mycelium taken in the zone of interaction between B. cinerea and isolate
SS37. Part b: partially emptied mycelium, part c: big vesicles in the mycelium, part d: emptied mycelium ( black arrow). Black bar = 40 μm (a), 100 μ (b-d),
Actinomycete isolates were effective in restricting radial growth of B. cinerea.
Microscopic observations showed a marked morphological changes and severe structural alterations of the fungal mycelium (Fig.2, parts b-d). Furthermore, the coagulation into the fungus cytoplasm was observed and characterized by the appearance of small vesicles or in extreme cases larger vesicles or even empty cells devoid of cytoplasm (Fig. 2, parts b-d). SS37 strain was used at 107CFU/ml. The fungus was used at 105 CFU/ml.
Figure 3 presents the inoculation experiments with SS37 actinomycete (spraying of the leaves) on detached leaves of V. vinifera with B. cinerea (White bar = 2 cm): (Figure 3, part a): leaves from control plantlets; (Figure 3, part b): leaves inoculated with actinomycetes (isolate SS37); (Figure 3, part c): leaves inoculated with B. cinerea and exhibiting the symptoms of gray mold disease;
(Figure 3, part d): leaves pre-treated with isolate SS37 before their inoculation with B. cinerea showing enhanced resistance toward the pathogen.
The control leaves were healthy (Fig. 3, part a), whereas leaves treated with B. cinerea exhibit the classic symptoms of gray mold (Fig. 3, part c). However, symptoms development on detached leaves did not occur when plantlets were inoculated with actinomycetes (Fig. 3, part b). When in vitro detached leaves (from control plantlets and plantlets inoculated with SS37 actinomycetes isolate) were challenged with B. cinerea, the fungus growth was suppressed on leaves treated subsequently with isolates (Fig. 3, part d), indicating that the presence of the actinomycetes restrict the spread of the pathogen. The SS37 strain was used at 107CFU/ml. The fungus was used at 10 CFU/ml.
Figure 4 presents the effect of the pre- treatment (spraying of the leaves) with SS37 actinomycete isolate of grapevine plantlets inoculated with B. cinerea (White bar = 1 cm):
(Figure 4, part a): control plantlets;
(Figure 4, part b): control plantlets+ B. cinerea showing the visual symptoms of gray mould;
(Figure 4, part c) plantlets inoculated with isolate SS37; (Figure 4, part d) plantlets pre -treated with actinomycete isolate before their inoculation with B. cinerea showing enhanced resistance toward the pathogen.
The same protection as in figure 3 toward B. cinerea was observed when the whole plantlets were subsequently inoculated with actinomycetes (Fig. 4). The SS37 strain was used at 107CFU/ml.
The fungus was used at 105 CFU/ml.
Figure 5 presents the light micrographs of samples from grapevine leaves.
(Fig 5, part a) sample from the untreated (control) grapevine leaves; (Fig 5, part b) leaves inoculated with actinomycete (spraying of the leaves) (isolate
SS37);
(Fig 5, part c) leaves inoculated with B. cinerea and exhibiting the symptoms of gray mold disease; The hyphae of the pathogen abundantly colonize the epidermis and the parenchyma. Fungal growth occurs both intracellularly and intercellularly. Pathogen ingress inside the leaf coincides with local cell wall alterations (black arrows = B. cinerea).
(Fig 5, part d) leaves pre -treated with isolate SS37 (spraying of the leaves) before their inoculation with (black arrow = B. cinerea) showing enhanced resistance toward the pathogen as fungal growth is mainly restricted to the epidermis. Control leaves show unaltered tissues (Fig. 5, part a). Treatment of leaves with isolates has no incidence on leaf structure (Fig. 5, part b), while the parenchyma of pathogen-inoculated leaves showed an intensive colonization by B. cinerea (Fig. 5, part c). Pathogen ingress toward the inner tissues coincided with extensive plant cell disorganization such as host cell wall alteration (Fig. 5, part c). In massively invaded areas cell walls are no longer discernible in extreme cases (Fig. 5, part c). Nevertheless, leaves pre -treated with actinomycetes isolates before their inoculation with B. cinerea showed that pathogen growth was stopped at the leaf surface (Fig. 5, part d). The SS37 strain was used at 107CFU/ml.
The fungus was used at 105 CFU/ml. Figure 6 presents the effect of SS37 Actinomycete isolate on hyphal mycelium growth of B. cinerea when inoculated together on grapevine tissue extract agar medium.
Diamond: B. cinerea inoculated alone
Triangle: B. cinerea and SS37 inoculated together It shows the fungistatic effect of SS37 on the development of B. cinerea.
The SS37 strain was used at 107CFU/ml. The fungus was used at 105 CFU/ml.
Figure 7 presents the chromatograms obtained from CPC experiments A (Fig.7, partA) and B (Fig.7, part B).
Fig. 7, part A shows the compounds IV, VIII and IX obtained by experiment A. Compound X can be obtained by dual mode (see example 5)
Fig.7, part B shows the compounds V, VI and VII obtained by experiment B after collecting the fraction containing these compounds in experiment A.
Figure 8 presents the fungicidal activity against B. cinerea of compounds control, IV, V, VI, VII, VIII and X.
Fig.8, part A shows the fungicidal activity against B. cinerea of compounds control, VI, VII, and X. Fig.8, part B shows the fungicidal activity against B. cinerea of compounds IV, V, and
VII.
For each compound tested, a mother solution was prepared (45.76 mM in MeOH), then 1 ml of this solution was added in 99 ml sterile distilled water to obtain a dilute solution and lμl of said diluted solution was deposited on the sides of the Petri dish. The fungus was used at 105 CFU/ml.
Figure 9 presents the different types of PR protein according to Van Loon et al. (2006). Figure 10 presents the in vitro responses of grapevine plantlets co-cultured with SS37 strain; the bacterisation process of plantlets was accomplished according to Ait Barka et al, 2000, Enhancement of in vitro growth and resistance to gray mould of Vitis vinifera L. co- cultured with plant growth-promoting rhizobacteria. FEMS Microbiol. Lett. 186, 91-95) Figure 10, part A: control
Figure 10, part B: grapevine plantlets co-cultured with SS37 strain
This figure shows that SS37 promotes the growth of treated grapevine plantlets with SS37.
The SS37 strain was used at 107CFU/ml.
Figure 11 presents the microscopic observation showing emergence of SS37 mycelia from disinfected root cuttings (figure 11, part A) and a test of the presence of actinomycete colonies in leaf tissues sampled from bacterized plantlets after four days of incubation on solid Bennett medium (figure 11, part B): (1) First generation, (2) Second generation, (3) Third generation, (T) control plantlets.
This figure shows that the presence of actinomycete in the root tips (Fig. 11, part A) after inoculation supports the possibility of entry of SS37 via root tips and that the actinomycetes is still present in the plant even in the second or third generation allowing thus the plant to resist to an micro-organism attack. The SS37 strain was used at 107CFU/ml.
Figure 12 presents the effect of inoculation with B. cinerea on grapevine plantlets bacterized or nonbacterized with SS37.
(fig. 12, part a) nonbacterized plantlets; (fig. 12, part b) nonbacterized plantlets + B. cinerea; (fig. 12, part c) bacterized plantlets; (fig. 12, part d) bacterized plantlets + B. cinerea.
This figure shows the stimulation properties of plant natural defences with SS37 but also the implementation of a resistance of said plant toward the disease and thus the prevention of said disease.
The SS37 strain was used at 107CFU/ml. The fungus was used at 105 CFU/ml. Figure 13 presents the effect of chemical compounds IV, V, VI, VII, VII, and X on the growth of B. Cinerea (chemical compounds were deposited simultaneously with the fungus at the centre of the Petri dish). Fig.13, part A: compounds IV, V, VI, VII, VIII and B. Cinerea.
Fig.13, part B: compounds X and B. Cinerea. Fig.13, part C: control B. Cinerea.
The figure 13, part A shows that a synergistic effect is observed between compounds IV, V, VI, VII, VIII. and the figure 13, part B shows that the compound X alone has fungicidal (or fungistatic) properties on B. Cinerea
For each compound tested, a mother solution was prepared (45.76 mM in MeOH), then 1 ml of this solution was added in 99 ml sterile distilled water to obtain a diluted solution and lμl of said diluted solution was deposited at the centre of the Petri dish. The fungus was used at 105 CFU/ml.
Figure 14 presents the distance effect of chemical compounds IV, V, VI, VII, VII, and X on the growth of B. Cinerea.
(The fungus was inoculated in the center of Petri dish and chemical compounds were deposited on the sides of the Petri dish) Fig.14, part A: compound IV and B. Cinerea (BC),
Fig.14, part B: compound V and B. Cinerea (BC), Fig.14, part C: compound VII and B. Cinerea (BC), Fig.14, part D: compound VIII and B. Cinerea (BC), Fig.14, part E: compound VI and B. Cinerea (BC), Fig.14, part F: compound X and B. Cinerea (BC).
This figure shows that compounds IV, V, VI, and to a lesser extent, compounds VII and VIII have a distance effect on the growth of B. cinerea, while compound X has no distant effect.
For each compound tested, a mother solution was prepared (45.76 mM in MeOH), then 1 ml of this solution was added in 99 ml sterile distilled water to obtain a diluted solution and lμl of said diluted solution was deposited on the sides of the Petri dish. The fungus was used at 105 CFU/ml.
Figure 15 presents the effect of compounds IV and X on the plant protection (grapevine) against B. cinerea.
Fig 15, part A : Control,
Fig.15, part B : B. cinerea,
Fig.15, part C : B. cinerea and compound IV,
Fig. 15, part D : B. cinerea and compound X. Fig.15, part C and to a lesser extent fig 15, part D shows that compounds IV and X protect the grapevine inoculated with B. cinerea.
For each compound tested, a mother solution was prepared (45.76 mM in MeOH), then 1 ml of this solution was added in 99 ml sterile distilled water and 4 ml per container containing 4 plants (1 ml/ plant) was sprayed.
Figures 16 and 17 present the expression in grapevine of a few genes coding for PR proteins induced by compounds produced by SS37.
In figure 16, each histogram (control and each product) from left to right corresponds to the induction factor: stilbene synthase (STS), chitinase (Chit4c), proteinase inhibitor (PR6 ) and lipoxygenase (Lox) respectively. This figure shows that all the compounds induced one or more gene, compounds IV and VI being the more active.
In figure 17, the histograms correspond to the induction factor of glucanase expression (Glue), all the compound being active, in particular compound IV.
EXAMPLES
Example 1: Isolation of actinomycetes strains from Moroccan soil a) Sampling
Samples were collected using Pochon & Tardieux method (1962, Technique d'analyse en microbiologie du sol, Edition de Ia Tourtourelle, Saint-Mande) from four Moroccan Rhizosphere soils of healthy vineyard fields (Tinghir). The samples from each of the rhizosphere soils were taken with an auger (up to 10 cm depth) after removing approximately 3cm of the soil surface. Samples were placed in sterile polyethylene bags, closed tightly and stored in the refrigerator at 4°C until use.
b) Isolation of actinomycetes
Samples of each soil were first mixed, suspended in sterile distilled water (Ig in 100 ml) homogenized by vortexing and finally treated 10 to 15 min by sonication according to Ouhdouch et al. (Ouhdouch Y, Barakate M, Finace C (2001) Actinomycetes from Maroccan Habitats: Screening for antifungal activites. Eur J soil Biol 37: 1-6). All treated samples were serially diluted up to 10- and spread (0.1ml) over the surface of nutrient agar (Difco, USA), soil extract agar (Barakate et al. 2002; Characterization of rhizospheric soil streptomycetes from Maroccan habitats and their antimicrobial activities. World J. Microbiol. Biotechnol. 17: 49-54) and Actinomycetes Isolation Agar (Olson's medium; 5 % glycerol, 0.2 % sodium caseinate, 0.01 % L-asparagin, 0.4 % sodium propionate, 0.05 % K2HPO4, 0.0001 % FeSO4 and 1.5 % agar Difco), (Olson 1968; Actinomycetes Isolation Agar, In: Difco: Supplementary Literature. Difco Lab., Detroit, (Michi.).
The two media were supplemented with 40 μg/ml actidione to inhibit the development of fungi (Olson 1968, above referenced), and 10 μg/ml nalidixic acid to inhibit the bacteria capable of swarming without affecting the growth of actinomycetes (Nonomura & Hayakawa 1988; New methods for selective isolation of soil actinomycetes. In: Okami Y, Beppu T, Ogawara H,(eds) Biology of Actinomycetes. pp. 288-293. Japan Scientific Societies Press, Tokyo, ISBN 4-7622- 1552-X; Bulina et al. 1997; A novel approach to isolation of actinomycetes involving irradiation of soil samples with microwaves. Microbiology 66: 231-234).
The plates were incubated at 28 ºC and the number of colonies was determined for total bacteria after 7 days and 21 days for actinomycetes. Actinomycetes were recognized on the basis of morphological features following directions given by International Streptomyces Project (ISP) (Shirling & Gottlieb 1966; Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16:313-340).
S37 colonies were isolated, purified and conserved in 20 % Glycerol at -20 ºC. Example 2: Characterisation of the S37 isolate (or other above obtained isolates) a) Morphological, physiological and chemotaxonomic characterization of selected bacterial isolates
The morphological, cultural, physiological and biochemical characteristics of the SS37 selected isolate was evaluated as described in the International Streptomyces Project (ISP)
(Shirling and Gottlieb, 1966, Methods for characterization of Streptomyces species. Int. J. Syst.
Bacteriol. 16:313-340).
Cultural characteristics were observed on yeast extract-malt extract agar (ISP2), oatmeal agar (ISP3) and inorganic salts-starch agar (ISP4) media at 28°C for 7-21 days and the colour series were determined according to the system proposed by Nonomura (1974). The assimilation of carbohydrates was studied by using the medium ISP9, containing 16 different carbohydrates at a concentration of 1% (w/v) as sole carbon source.
The exemplary strain of the invention shows the following properties when incubated at
28°C in the following various solid media: On Yeast Extract-Malt Extract Agar (ISP2), abundant white-grey growth; yellow- brownish substrate mycelium, no soluble pigment.
On Oatmeal Agar (ISP3), moderate gray growth for both aerial and substrate mycelia, no soluble pigment.
On Inorganic Salt Starch Agar (ISP4), moderate white aerial growth, brown substrate mycelium, no soluble pigment.
On Glycerol-Asparagine Agar (ISP5), abundant white growth, yellow-brownish substrate mycelium, no soluble pigment.
On Pepton Yeast Extract Iron Agar (ISP6), thin white growth, creamy vegetative mycelium, no soluble pigment. On Tyrosine Agar (ISP7), abundant white-gray growth, creamy substrate mycelium, no soluble pigment.
On Sucrose Nitrate Agar, abundant white growth, white-yellowish substrate mycelium, no soluble pigment. On Sucrose Czapeck Agar (Shirling and Gottlieb, 1966, Methods for characterization of
Streptomyces species. Int. J. Syst. Bacteriol. 16:313-340), thin white growth for both aerial and substrate mycelia, no soluble pigment.
On Glucose Asparagine Agar, thin creamy growth for both aerial and substrate mycelia, no soluble pigment.
On Olson's medium (Olson 1968, actinomycetes Isolation Agar: in Difco ; supplementary literature), abundant white growth, yellow-brownish substrate mycelium, no soluble pigment.
On Bennett's Agar (composed as mentioned above, example 5), abundant white-gray aerial mycelium, yellow vegetative mycelium, no soluble pigment. On Nutrient Agar, moderate yellow growth for both aerial and substrate mycelia, no soluble pigment.
The chemical analyses of the diaminopimelic acid isomer were performed as described by
Becker et al. (1964; Rapid differentiation between Nocardia and Streptomyces by paper chromatography of whole-cell hydrolisates. Applied Microbiology 12: 12421-12423). Spore chains morphology and spore shapes were observed on the same media using light microscopy.
SS37 were able to use citrate, sucrose, fructose, glucose, maltose, mannitol, mannose, melibiose, lactose arabinose and glycerol as sole carbon sources whereas inositol, D-raffinose, sorbitol, rhamnose, galactose, and xylose were not used (Table 1). SS37 was sensitive to Novobiocin (30 μg), Gentamycin (10 μg), Bacitracin (10 U) and
Polymyxin B (300 U) but resistant to Sulfamides and Cefalotin (30 μg).
The analysis of cellular constituents of SS37 revealed the presence of the L- diaminopimelic acid (DAP) isomer (Table I).
In function of the results of the tests above carried out, the strain SS37 was assigned to the genus of Streptomyces. 10
15
20
25
Figure imgf000038_0001
TABLE I
30 b) Amplification and sequencing of the 16S rDNA of the selected strains
The purified SS37 actinomycete isolate was grown for 2 days at 28°C with agitation in 500 ml flasks containing 100 ml of Hickey-Tresner medium containing 1 g/1 yeast extract, 1 g/1 beef extract, 2 g/1 N-Z-amine A (Sigma-Aldrich), 10 g/1 Dextrin, 20 mg/1 CoC12.6H2O (Hopwood et al. 1985; Genetic manipulation of Streptomyces: a laboratory manual. John Innes Foundation, Norwich, United Kingdom). Biomass was harvested by centrifugation (8000 g for 10 min) and washed twice with double-distilled water. 200 mg of mycelia was used for DNA extraction as described in Liu et al. (2000; Rapid mini-preparation of fungal DNA for PCR. J. Clin. Microbiol. 38: 471-1471). The 16S rDNA was amplified using the PCR method with Tαq DNA polymerase (Qiagen,
USA) and universal primers PA (5'-AGAGTTTGATCCTGGCTCAG-S') and PH (5'- AAGGAGGTGATCCAGCCGCA- 3').
Amplification was carried out in 50 μl reaction mixture containing 1.5U of AmpliTaq Gold Tαq polymerase (Applied Biosystems), 10 μl of 5x AmpliTaq Gold reaction buffer (Applied Biosystems), 2.5 mM of each dNTP, 1 μM of each primer and 100 ng of genomic DNA.
Reaction conditions were: 97°C for 4 min, (97°C for 45s, 52°C for 45s and 72°C for 45s) x 35 cycles followed by an incubation at 72°C for 10 min. The amplified products were visualized on a 0.8% (w/v) agarose gel stained with ethidium bromide. Sequencing reactions were performed by Macrogen (Seoul, Korea).
The obtained sequence (SEQ ID NO: 1) was compared for similarity with sequences present in the genomic database banks, using the 'NCBI Blast' program available at the ncbi- nlm-nih.gov Web site and exhibited 99% sequence identity to Streptomyces αnulαtus.
Example 3: Determination of the antifungal activity of S37 strain (or other isolates above obtained) a) Antifungal assays
The antifungal activity of the S37 isolate was determined by the plate diffusion method
(Barakate et al. 2002; Characterization of rhizospheric soil streptomycetes from Maroccan habitats and their antimicrobial activities. World J. Microbiol. Biotechnol. 17:49-54) against Botrytis cinerea (B. cinerea), Fusarium oxyysporum f. sp. Albedinis (Foa), Sclerotium rolfsii (SR), Verticillium dahliae (VD) and Pythium ultimum (PU) (strains collection of the Phytopathology Laboratory, Marrakech).
Isolates were grown on Bennett medium (beef extract [Merck, Germany] lg/1; glucose [Merck, Germany] lOg/1; peptone [Merck, Germany] 2g/l; yeast extract [Merck, Germany] lg/1 and agar [Difco, USA] 15g/l) for 14 days and six mm diameter agar disks containing actinomycetes colonies mass was prepared by using sterile cork borers. Disks were then aseptically transferred to PDA plates having fungal mycelial-disk (diameter of 6mm) in the centre. Plates were first kept in a refrigerator (4°C) for at least 4h to allow the diffusion of any antibiotics produced, then incubated at 30ºC. Inhibition zones were determined after 4 days of incubation. Only the isolates showed an inhibition zone superior to 8 mm were considered as active isolates.
In order to test the effect of actinomycete on B. cinerea structure, a thin layer of PDA was aseptically removed from the zone of contact between B. cinerea and the isolate and placed in a drop of sterile water on a microscope glass slide. A cover slip was placed on the film, and observations were made under the microscope (Model BH2, Olympus, Japan). Actinomycetes isolates showing the highest antagonist activity have been selected to further study their biological effect against gray mold of grapevines.
b) Inoculation plant assays
Plantlets of Vitis vinifera L. cv. 'Chardonnay' clone 7535 were micropropagated by nodal explants grown on 15 ml semi-solid medium (Martin et al. 1987, The vine and techniques of in vitro cultivation. Bull Org Int Vigne 675-676:447-458), in 25-mm culture tubes under 200 μmol/m2/s white fluorescent light, 16 h photoperiod at 26°C (Ait Barka et al. 2000, Enhancement of in vitro growth and resistance to gray mould of Vitis vinifera L. co-cultured with plant growth promoting rhizobacteria. FEMS Microbiol Lett 186:91-95). Uniform plantlets (n=24) were selected for each treatment in each experiment. Fungal pathogen or actinomycetes isolates were applied to detached leaves maintained in Petri dishes or to the whole grapevine plantlet (Vitis vinifera L. cv. Chardonnay). Using a spectrophotometer (600 nm), the actinomycete concentration was adjusted to 106 c.f.u./ml with PBS as described by Ait Barka et al. (2000, above cited). The concentration was confirmed by plate counting. Leaves were inoculated by depositing 20 μl of B. cinerea (105 spores/ml suspended in sterile distilled water) on their surface. Two days after treatment, 30 μl of the actinomycete suspension (106 c.f.u./ml) was deposited on the top of each leaf. Control plants were treated similarly but only with sterile distilled water. Gray mold symptoms were evaluated 5 days after inoculation with B. cinerea and samples were processed for light microscope investigations.
c) Microscopy analysis
Fresh plant leaves inoculated with actinomycetes isolates, B. cinerea, both actinomycetes isolates and B. cinerea or control (water) were collected and prepared for microscopy analysis according to Gognies et al. (2001, Saccharomyces cerevisiae, a potential pathogen towards grapevine, Vitis vinifera. FEMS Microbiol Ecol 1271: 143-150). Briefly, plant organs were fixed for 24 h at room temperature in 2% (w/v) glutaraldehyde in 0.1 M phosphate buffer (pH 7.24) with 2% (w/v) sucrose and 0.1% (v/v) Tween 20. After 3 rinses (5 min each) with the phosphate buffer and 2% (w/v) sucrose, samples were fixed for 4h in 1% (w/v) osmium tetroxyde in the phosphate buffer with 2% (w/v) sucrose. The samples were then dehydrated in an alcohol series, transferred to acetone and embedded in araldite. Semi- thin sections (lμm) of different treatments were collected on glass slides and stained with 0.1% toluidine blue and examined under microscope (model BH2; Olympus, Japan).
(I) Statistical analyses
Unless stated otherwise, replicates of 24 plants were used per treatment. Collected data were analysed statistically using ANOVA. Means for each treatment were separated with a least significant difference (LSD, P<0.05) multiple comparison test according to Student's test. All experiments were repeated at least 3 times.
Example 4: Test for antagonistic activity, for chitinase and indole acetic acid (IAA) production and for detection of siderophore of SS 37 actinomycete isolate, a) Test for antagonistic activity The plate diffusion method was used to assess the global anti-microbial activity of the strains (Bauer et al. 1966, Antibiotic susceptibility testing by standard single disk method. Am J Clin Pathol. 45, 493-496.) against the Gram negative bacteria Escherichia coli K12 (Wl 130) and Pseudomonas aureus CCMM/B11, the Gram positive bacteria Micrococcus luteus ATCC 381, Bacillus subtilis ATCC 9524 and Staphylococcus aureus DSM 20231 (ATCC 12600), the yeast Candidas albicans ATCC 2091, Rhodotorula rubra Tu 8093, and the phytopathogenic fungi Mucor ramannianus, Fusarium oxysporum f. sp. albedinis and Pythium ultimum (strains collection of Microbiology Laboratory, Marrakech). SS37 actinomycete isolate was grown on grapevine tissue extract agar medium for 14 days then three disks (diameter 10 mm) were cut out and placed on 48 h grown lawns of the different microorganisms tested grown on nutrient agar (Difco, USA) for bacteria and yeasts and Sabouraud agar medium (Biorad, France) for fungi. Plates were first stored at 4 ºC for at least 2 h to allow the diffusion of any substances produced, then incubated at 28 ºC. Sizes of the inhibition zones were determined after 24 h of incubation for bacteria and yeasts and 48 h for fungi. Controls involved the use of sterile agar plugs. Three replicates were performed per isolate by each microorganism test.
b) Comparative antifungal activities of compounds produced by a strain of Streptomyces sp.S37 and amphotericin B
TABLE II
MIC (μg/ml) Bacterial strain ∞∞∞∞∞∞∞∞∞∞^^
S37 compounds Amphotericin B
Yeast* 0.060 4
Botrytis c 0.070 3
*Clinic yeast {Candida albicans ) was isolated from various sample of hospitalized sick patients in Marrakech's Military Hospital and their resistance confirmed. MIC: Minimum inhibitory concentration. SS37 showed antibacterial activity against gram positive tested bacteria and its MICs are lower than that of amphoericin B.
The MICs of "Ymycine" for clinic yeast and phytopathogenic mould Botrytis cinerea, were ranged from 0.060 to 0.070μg/m; whereas the MICs of amphotericin B were ranged between 3 and 4 μg/ml.
c) Assay for chitinase
In order to assay chitinase production by the selected strains, a colloidal chitin suspension was, prepared from crab shell chitin as follows: 40 g of chitin were dissolved, under stirring for 30 to 50 min, in 400 ml of HCl 6M (Hsu and Lockwood, 1975, Powdered Chitin Agar as a Selective Medium for Enumeration of Actinomycetes in Water and Soil. Appl Environ Microbiol. 29(3): 422-426). The chitin was precipitated as a colloidal suspension by diluting it slowly with 2 1 of deionized water at 5-10 ºC. The colloidal suspension was filtered and washed until the pH of the suspension was about 3.5 and then was dried. Dry chitin (4 g) was added as sole carbon source, in replacement of glucose to 1 1 of synthetic minimum medium (SMM) containing 0.5 g 1-1 Rock Phosphate (RP). After autoclaving, the melted agar medium was adjusted to pH 7.8. The positive growth control experiment was made with SMM containing glucose as sole carbon source. Only chitinase excreting isolates could grow on the chitin SMM medium.
d) Determination of Indol Acetic Acid (IAA) produced
Indoleacetic acid (IAA) production was analysed using a modification of the qualitative method developed by Brie et al. (1991, Rapid in situ assay for indole acetic acid production by bacteria immobilized on a nitrocellulose membrane. Appl Environ Microbiol 57:535-538). The growth medium used contained 1O g tryptone, 5 g yeast exctract, 5 g NaCl, 1,02 g L-tryptophane and 20 g agar for 1 litter of deionized water. This medium was sterilized at 121°C for 20 min. Actinomycetes strains were plated on the surface of the medium overlaid with a nitrocellulose membrane (Amersham Pharmacia) and incubated at 28°C for 7 days. After Actinomycetes growth had occurred, the membrane was removed from the plate and treated with Salkowski reagent (2% (w/v) 0.5 M FeCB in 35% perchloric acid) for 15 min at room temperature. Actinomycetes strains producing IAA are identified by a red halo surrounding the colony.
e) Detection of siderophore In order to determine whether siderophore were present in the culture supernatants of the
SS37 actinomycete isolate, they were grown for 5 days under the conditions described above. The supernatants were centrifuged at 10, 000 xgmin-1, filtered and concentrated filtrates were deposited on sterile cellulose disks (5 mm diameter) placed aseptically on blue CAS-agar plates as described by Schwyn and Neilands (1987, Universal chemical assay for the detection and determination of siderophores. Analytical Biochem. 160, 47-56.) and incubated at 30 ºC for 3 days. Disks impregnated with 2, 4, 6, 8 or 10 μg ml-1 Desferrioxamine B (Sigma- Aldrich, Germany), a well known siderophore (Tunca et al, 2007, Transcriptional regulation of the desferrioxamine gene cluster of Streptomyces coelicolor is mediated by binding of DmdRl to an iron box in the promoter of the desA gene. FEBS J. 274, 1110-1122.), placed aseptically on blue CAS-agar plates and incubated under the same conditions, were used as positive controls. The disks impregnated with solutions containing siderophore were surrounded by a zone of colour change (blue to yellow-orange) of the CAS that was due to iron chelation (Schwyn and Neilands, 1987, cited above). The size of the zones and the intensity of the colour change were estimated and compared to the controls. The table III shows the results obtained with SS37 for the antagonistic activity, the production of chitinase and IAA, and the detection of siderophore (activity against fungi, Gram+ bacteria, Gram- bacteria and Yeast: +: low; ++: medium and +++: high; other activities: 1 means production or detection, 0 means no production or detection).
Figure imgf000045_0001
TABLE III
Example 5: purification of chemical compounds from SS37 actinomycetes isolate, a) Actinomycete sp. cell culture
SS37 Streptomyces sp. used in this study was previously isolated from rhizospherical soil of Maroccan Vitis vinifera soil. Spores of this strain, stored in 20% sterile glycerol at - 20ºC, were used to inoculate (at 106 spores/ml) cultures of liquid Bennett medium consisting of beef extract (Merk) 1 g/1; glucose (Merk) 10 g/1; peptone (Merk) 2 g/1; yeast extract (Merk) lg/L, pH
7.3.
3 liter cultures were grown for 6 days at 28°C under constant agitation on a rotary shaker (180 rpm / minute). b) Chemical compounds extraction
As maximum antibiotic production was observed on the 6th day of the incubation, cultures was terminated on the 6th day and the broth was centrifuged at 10,000 x g for 20 min to separate the mycelial bio mass. Different solvents were tested for the extraction of the chemical compounds from the culture supernatant. The solvents used were n-butanol, ethyl acetate, nhexane, dichloromethane, methanol, chloroform. The organic extracts were evaporated to dryness using a Rotavapor (Laborota 4000). The resulting dry extracts were dissolved in methanol and subjected to biological assay (disk of 6 mm in diameter) against Bacillus subtilis (40 μl per disk) and Botrytis cinerea (60 μl per disk). The n-butanol extract gave the highest inhibition diameter were then retained for the detection of antibiotics by bioautography [Petersen, M.; Simmonds, M. Rosmarinic acid. Phytochemistry 2003, 62, 121-125]. For this, 40 μl (Bacillus subtilis) or 60 μl (Botrytis cinerea) of n-butanol extract were spotted onto 20 x 20-cm silica gel plates (Merck Art. 5735, Kiesselgel 60F254), and then developed with CH2Cl2/CH3OH (1 :9 v/v). The developed TLC plates were air-dried overnight at 37 ºC to remove all traces of solvent. They were then placed in plastic bioassay dish (23 x 23 x 2.2 cm3, Fisher Scientific Labosi) and overlaid with 150 ml of nutrient agar or PDA agar media (containing 7 g/1 of agar) seeded respectively with Bacillus subtilis or Botrytis cinerea. After the agar had set, the plate was inoculated at 30 ºC. The inhibition zones of the active fraction were determined after 24 h of incubation for the Botrytis cinerea and after 48 h for Bacillus subtilis. Clear areas due to inhibition of microorganism growth indicated the location of antibiotic compounds on the TLC plates. The retention factor (Rf) of each spot was recorded.
c) Bioassay guided liquid-liquid pre-fractionation
Two different systems of solvents have been studied in view of the pre-fractionation: the triphasic system n-Hept / MtBE / CH3CN / Water (1 :1 :2 : 1, v/v) and of the biphasic system n-Hept / CH3OH / Water (5 : 1.5 :3.5, v/v).
The n-butanol extract was evaporated to dryness, the dry residue (8,8 g) was dissolved in a small volume of methanol and was submitted to liquid-liquid fractionation using for each extraction, 60OmL of the ternary biphasic solvent mixture n- heptane/ methano I/water (5: 1.5: 3.5, v/v). After 4 time extractions, the collected upper organic phases were regrouped and evaporated to dryness. About 1.4 g of the later residue was obtained. It was then stored for 24 hours at - 10 ºC prior to use in CPC.
d) CPC Apparatus The separations were performed on a FCPC® Kromaton Technologies apparatus (Angers,
France), using a rotor of 20 circular partition disks (1320 partition cells, column capacity: 200 mL). Rotation speed can be adjusted from 200 to 2000 rpm, producing a centrifugal force field in the partition cell of about 120 g at 1000 rpm and 470 g at 2000 rpm.
The solvents were pumped by a Dionex P580HPG 4-ways binary high-pressure gradient pump (Sunnyvale, CA, USA). The samples were introduced into the CPC column via a low pressure injection valve (Upchurch, CIL Cluzeau, Sainte-Foy-La-Grande, France) equipped with a 21 mL sample loop. The effluent was monitored with a Dionex UVD 170S detector equipped with a preparative flow cell (6 μL internal volume, path length of 2 mm). Fractions were collected by a Pharmacia Superfrac collector (Uppsala, Sweden). The experiments were conducted at room temperature (22±1°C).
e) Preparation of Solvent Systems
The Arizona quaternary solvent system (R) composed of n-Heptane/ Ethyl acetate/ methanol/ water (2: 1: 2: 1, v/v) (experiment A) and the second biphasic sytem n-Heptane / acetonitrile/ water (5: 5: 4, v/v) (experiment B) were prepared by mixing the appropriate solvent in a separatory funnel, shaking them vigorously and allowing them to settle until the phases became limpid.
f) Preparation of Sample Solutions 1.4 g of the fractionated butanolic extract was dissolved in 1OmL of organic phase and
1OmL of aqueous phase.
g) CPC Experimental Conditions
First, in experiment A, the CPC column was filled with the aqueous stationary phase of the biphasic system. The sample solution was injected in the sample loop, the rotation speed was adjusted to 1300 rpm and the organic mobile phase was pumped at 6 mL/min. In the same way, in experiment B, separation was performed with the organic as the mobile phase at 1200 rpm and 3mL/min. The outgoing phase was monitored at 270 nm by the online UV detector for experiments A and B. For experiments A and B, respectively, the stationary phase retention was 75 and 70%, the back pressure was about 45 and 40 bars.
h) Fraction Analysis
CPC fraction analysis was performed using normal phase TLC (Merck Si60) with CHC13/ methanol (99: 1, v/v) as eluent. Plate visualization were realised thanks to sulphuric vanilline reagent (vanillin / H2SO4 concentrated/ Ethanol, 3/ 3/ 100, w/ v/v). The fractions containing pure compounds from the TLC analysis were regrouped. These fractions were submitted to 1H and 13C NMR experiments. The spectra were recorded in CDCl3 (1H at 500 MHz and 13C at 125 MHz) on a Bruker Avance DRX 500. 2D (COSY, HSQC, HMBC .) experiments were performed using BRUKER microprograms. ESI-MS and HRESI-MS were obtained in a Micromass Q-TOF micro spectrometer apparatus.
RESULTS:
Bioassay guided liquid-liquid pre-fractionation
The bioassay guided liquid-liquid pre-fractionation had to simplify the initial n-butanol extract while enriching it of the active constituent. For it, a system of triphasic solvent of n- Heptane / Methyl tert-buty\ ether (MtBE) / acetonitrile (CH3CN) / Water (1 : 1 : 2: 1, v/v) classically employed in the industry for the dividing of surrounding culture (communication of Dr R. Margraff) was first used. The primary extract could be thus fractioned in groups of compounds of distinct polarities. The examination of each of the liquid phases collected, done by bioautography, meaning chromatography on thin layer assessment of the activities of the products adsorbed on plate (Botz L, NagyS, Kocsis B (2001) Detection of microbio logically active compounds In Sz Nyiredy ed., Proc Int Symp Planar Chromatography, Planar Chromatography 2001 Lillafured, Hungary, 489- 516) revealed that the least polar compounds contained in the superior heptane phase and the intermediate phase rich in MtBE and CH3CN were the most active. Indeed, these inhibit the growth of Botrytis cinerea in the middle of culture.
In order to simplify this pre- fractionation, a second biphasic solvent system has been tested with success to separate the active of those inactive substances. It is about the system constituted with the mixture of: n-Hept / methanol (CH30H) / Water, 5 : 1 ,5 :3,5, v/v. This last system presents the advantage to be more selective of the active compounds of the extract and is less expensive in solvent that the triphasic system previously used. It has been preferred therefore at last for the liquid pre-fractionation.
Isolation of the active molecules (IV, V, VI) by CPC
Following the pre-fractionation of the butanolic extract of Streptomyces sp., a first system of solvents has been selected from the systems of the Arizona range in order to be used in CPC.
The R system (n-Hept / AcOEt / CH3OH / Water, 2 : 1 :2 :1, v/v) Arizona range has been kept in first intention. Partition coefficients about 1 have been obtained with this system for the majority of the active compounds of the prefractionated extract.
On the one hand, isolation of pure compounds of formula IV, VIII and IX was performed using conditions A in elution mode CPC with the biphasic solvent system composed of n- Heptane/ Ethyl acetate/ methanol/ water as describe above (Fig 7A).
The subsequent use of the aqueous mobile phase when applying the strategy of the dual mode succeeded in the purification of the compound X that was until there retained by the stationary aqueous phase. Its chromatographic behavior is the opposite in CCM and with respect to that in CPC ("polar" in CPC but "apolar" in CCM).
Compounds V, VI and VII could not be separated within these conditions.
However, the collected fractions containing these molecules were gathered and then reinjected in the CPC column so as to submit elution mode using conditions B.
In this manner, compounds of formula V, VI and VI were each successfully purified (Fig. 7, partB).
The collected fractions containing the same compounds have been gathered.
In vitro activity of compounds IV, V and VI The in vitro antimicrobial activities, against Botrytis cinerea of these pure compounds were evaluated. Four of the six tested compounds show high activities against Botrytis cinerea (Fig. 8).
Example 6: effect of SS37 actinomycetes strain on the growth of pathogen B. cinerea. a) Botrytis cinerea culture
The B. cinerea strain 630 used in this experiment was provided by Dr. Brygoo (INRA, Paris, France). Filamentous fungi were maintained on potato dextrose agar medium (PDA). Fungal inoculum was prepared by growing the fungus for one week on fresh PDA medium. After this period, the B. cinerea isolate had developed abundant hyphal swellings. The mycelium was removed aseptically from the PDA plates and was briefly homogenized in a disinfected blender in the presence of sterile distilled water. The density of the fungal inoculum was determined and the concentration of the fungus was adjusted to 105 spores/ml.
b) Confrontation tests
The actinomycetes were inoculated on the sides of the Petri dishes containing the grapevine tissue extract agar (1 kg sheath chopped into small pieces was submersed in 1000 ml boiling tap water for 1 h and 15 g agar was added). After inoculation at 28 ºC for 4 days, Botrytis cinerea was inoculated on the centre of the Petri plates. After 4 days under the same culture conditions, the distance between the edge of actinomycete colony and the edge of fungal colony was evaluated for evidence of inhibition.
In order to test the effect of actinomycete on B. cinerea structure, a thin layer of fungal mycelium was aseptically removed from the zone of contact between the isolate and B. cinerea and placed in a drop of sterile water on a microscope glass slide. A cover slip was placed on the film, and observations were made under the microscope (Model BH2, Olympus, Japan).
c) Fluorescent staining of Botrytis cinerea mycelium
For vital observations, mycelium of Botrytis cinerea was stained with two vital fluorescent stains,
Calcofluor white (Sigma F-3397) and Nile red (Sigma N-3013). Stock solutions of the stains were made at 1 mg ml-1 for Nile red in DMSO while Calcofluor white (Sigma Fluorescent Brightener 28) was made at 1 mg ml-1 in 50 mM phosphate buffer. The working concentration in staining solutions (μl stock solution ml-1 MiIIiQ water) and staining times were 15 min for Calcofluor white at 3 μl ml— 1, and 20 min for Nile red at 2 μl ml— 1. The effect of actinomycete on vitality of B. cinerea was tested by adding the fluorescent stains to fungus mycelium in eppendorf.
Three replicates per treatment were performed.
d) Plant material and in vitro growth conditions
Disease- free plantlets of V. vinifera L. 'Chardonnay' were obtained by growing nodal explants on Murashige and Skoog medium (1962, A revised medium of rapid growth and biomass assays with tobacco tissue cultures. Physiol. Plant. 15, 473-497). Vitroplantlets were grown under 200 μmol m2 s-1 white fluorescent light for a 16 h photoperiod at 25 ºC day and night temperatures.
Multiplication was done in 25-mm-diameter test tubes containing a 15 mL medium. In each of the following experiments 24 nodal explants were used per treatment.
Plant growth was measured and the effect of bacterial inoculation on the plant was assessed (Fig. 10). The strains that promoted greater plant growth were SS37.
The shoot and root fresh weights were increased by the treatment with the selected SS37 isolate (Table P/).
The length of the principal root was boosted when the plantlets were inoculated with SS37.
TABLE IV: influence of SS37 Actinomycete isolate on in vitro growth parameters of V Vinefera L.
Figure imgf000051_0001
NN, node number; SFW, shoot fresh weight; RFW, root fresh weight; TB, total biomass. Values per plantlets are means of 24 replicates based on 12-week-old-plantlets.
e) In vitro bacterization with Actinomycetes The bacterial inoculum was produced by transferring tow loops of actinomycetes to 60 ml Bennett liquid medium in a 250 ml Erlenmeyer flask and incubated at 28°C at 150 rpm for 4 days as previously described (Barakate et al., 2002). Bacteria were collected by centrifugation (3000xg. 15 min) and washed twice with phosphate saline buffer (PBS) (pH 6,5). The pellet was resuspended in the same PBS buffer, then used as inoculum. About 1-cmlong nodal explants, taken from 6-week-old plantlets with removed leaves, were dipped in the inoculum for 1 min, blotted with sterile filter paper, and propagated on Murashige and Skoog medium as above. Nonbacterized controls were dipped only in PBS. The plants were grown in the growth chamber as above.
f) In vitro growth responses of the first and second and third plantlet generations after inoculation
Inoculated plantlets (first generation) were subcultured in vitro obtain the second generation. Inoculation was carried out only on the initial explants, not on the second and the third generation explants.
g) Endophytic colonization
To determine endophytic colonization, plantlets were removed from the agar and the roots were gently rinsed in sterile distilled water. The samples were surface sterilized with 70% ethanol for 5 min, followed by 1% commercial bleach and a 0,01% Tween 20 solution for 1 min, and then washed three times in distilled water (1 min each time).
SS37 isolate is capable of establishing endophytic and epiphytic populations, allowing clonal multiplication of plantlets by nodal explants in perpetuum without the need for reinoculation (Fig. 1OA and B). The response of plant toward bacterization was maintained and amplified after the second and the third generation. Furthermore, as endophyte, the actinomycete has the advantages of escaping microbial competition and influencing the host's response to pathogens attack.
To prove the presence of endophyte actinomycete inside the leaf tissues, disk of fifth leaves from bacterized plantlets were deposited on Bennett medium. After 4 days of incubation, colonies of bacteria were observed around bacterized leaf and tissues (Fig. 11). h) Analysis of the cell wall-degrading enzymes activities
Enzyme activities were determined by the method of the Reinhold-Hurek et al (1993, Cloning, expression in Escherichia coli, and characterization of cellulo lytic enzymes of Azoarcus sp., a root invading diazotroph. J. Bacteriol. 175, 7056-7065) with some modifications. Briefly, plates containing Bennett solid medium, with either 0,2% carboxymethyl cellulose (CMC) or 0,5% polygalacturonic acid, were spot inoculated with actinomycetes and incubated at 30ºC for 4 days. Then the cells were removed from the plates, and the CMC- containing plates were stained Congo red (0,1%) for 30 min; this was followed by several washes with IM NaCl to improve the contrast (Reinhold-Hurek et al., 1993, above cited). Similarly, the polygalacturonic acid-containing plates were stained with ruthenium red
(0,1%) and washed with IM NaCl (McKay, 1988, A plate assay method for the detection of fungal polygalacturonase secretions. FEMS Microbiol. Lett. 56, 355-358). Endoglucanase (CMC-degrading cellulase) activity was determined by the appearance on a red background of clear yellowish halos around the points where the actinomycete was inoculated (Reinhold-Hurek et al, 1993, above cited).
Endopolygalacturonase activity was determined by the appearance of intense purple-red halos on a colourless background at the point where the bacterium was inoculated (McKay, 1988, above cited).
i) Statistical analyses
The experiment unit in all experiment was an individual plant, contained in test tubes. Plants (24) were evaluated per treatment. An antagonistic test was done using six plates for each treatment. All experiments were repeated three times.

Claims

1. Use of a composition comprising one or more actinomycetes strains, said composition having stimulation properties of plant natural defences and/or fungistatic properties, for the prevention and/or the control of micro organism inducing plant diseases, provided that when said plant is soybean, and when only one strain of actinomycetes is used, said actinomycetes strain is different from Streptomyces melanosporofaciens (EF-76).
2. Use according to claim 1, wherein said actinomycetes strains are of Streptomyces or
Micromonospora genus.
3. Use according to claim 1 or 2, wherein said micro organisms are chosen among Botrytis sp., Fusarium sp., Pythium sp. or Candida tropicalis R2, in particular Botrytis cinerea.
4. Use according to anyone of claim 1 to 3, wherein said actinomycetes strain is at a concentration from about 106 cfu/ml to about 109 cfu/ml, preferably from about 107 cfu/ml to about 109 cfu/ml, more preferably from about 107 cfu/ml to about 108 cfu/ml, in particular 107 cfu/ml.
5. Use according to anyone of claims 1 to 4, wherein said composition comprises or is constituted of the culture supernatant isolated from a culture medium of said actinomycetes strain.
6. Use according to claim 5, wherein said composition comprises or is constituted of one or more chemical compounds belonging to one of the following chemical families: tryptophan derivatives, 2-terpenyl pyridines, ionophoretic polyethers, and chlorinated lipid compounds.
7. Use according to anyone of claims 1 to 6, wherein said actinomycetes strain is the actinomycetes strain S37, as deposited at the BCCM on the 03rd Mai 2007 under the number LMG P-24130.
8. Use according to anyone of claims 1 to 7, wherein said chemical compounds are chosen among 4-chloro-5-(3'-indolyle) oxazole, piericidine or abierixine.
9. Use according to anyone of claims 1 to 8, wherein said stimulation properties of plant natural defences are obtained by the signalling pathway activation, in particular leading to a local and systemic induction of at least one of the following genes: VvGIuC, VvChi4C,
VvPR6 and VvStSy with an induction factor higher than about 3, except for the activation of the flavonoid synthesis pathway, the induction factor of which being less than 3, in particular the one inducing the phenylalanine ammonia lyase (PAL).
10. Use according to anyone of claims 1 to 9, wherein said composition has also fungicidal properties and/or growth promoting properties
Composition comprising the actinomycetes strain S37 as deposited at the BCCM on the 03rd Mai 2007 under the number LMG P-24130, as defined in claim 7, or a culture supernatant of a culture medium of said actinomycetes strain.
Composition according to claim 11, wherein said strain is at a concentration from about 106 cfu/ml to about 109 cfu/ml, preferably from about 107 cfu/ml to about 109 cfu/ml, more preferably from about 107 cfu/ml to about 108 cfu/ml, in particular 107 cfu/ml.
Composition comprising or being constituted of one or more chemical compounds belonging to one of the following chemical families: tryptophan derivatives, 2-terpenyl pyridines, ionophoretic polyethers, and chlorinated lipid compounds such as defined in claim 6, and when only one chemical compound is present in the composition, the following compounds 4-chloro-5-(3'-indolyle) oxazole, piericidine and abierixine are excluded.
Composition according to claim 13, wherein said chemical compounds are at a concentration from about 10~3 M to about 10~9 M, preferably from about 10~4 M to about 10- M, more preferably from about 10-5 M to about 10- M, more preferably from about 10- M to about 10- M, more preferably from about 10-7 M to about 10- M, more preferably from about 10-8 M to about 10-9 M, more preferably from about 10-7 M to about 10-8 M, and more preferably from about 10-6 M to about 10-7 M.
Actinomycetes stain S37 as deposited at the BCCM on the 03rd Mai 2007 under the number LMG P-24130 such as defined in claim 7.
Extraction and purification process of chemical compounds, such as defined in claim 6, from a culture supernatant isolated from a culture medium of one or more actinomycetes strains such as defined in claim 1, comprising a chromatography step, in particular a centrifugal partition chromatography of one or several fractions of chemical compounds obtained by liquid liquid pre-fractionating of a culture supernatant solvent extract of said culture medium of one or more actinomycetes strain, said fractions presenting fungicidal properties, to obtain one or more said pure chemical compounds and, optionally, a mixture of other said chemical compounds.
Extraction and purification process according to claim 16, comprising a second chromatography, in particular a centrifugal partition chromatography of said mixture of other chemical compounds, different from the previous centrifugal partition chro matography.
Extraction and purification process according to claim 16 or 17, comprising the following steps: a. culture supernatant extraction of a culture medium of one or more actinomycetes strain, previously cultivated, by means of a solvent, in particular 1-butanol, to obtain a solution containing a culture supernatant extract of a culture medium of one or more actinomycetes strain, b. liquid liquid pre- fractionation of said culture supernatant extract by means of a triphasic or biphasic extraction system, after evaporation of said solvent containing extract, to obtain one or more fraction(s) containing chemical compounds from said culture supernatant extract, c. determination of the fungicidal properties of said fraction(s) containing said chemical compounds by bioautography, d. centrifugal partition chromatography of said fraction(s) above obtained and having fungicidal properties, by means of a solvent biphasic mixture, to obtain one or more said pure chemical compounds and optionally a mixture of other said chemical compounds.
19 Process according to claim 18, comprising a further step of centrifugal partition chromatography of said mixture of other said chemical compounds obtained in claim 18, by means of a biphasic mixture different from the one used for the first centrifugal partition chromatography carried out in step d) of claim 18.
20 Prevention and/or control process of plant diseases induced by microorganisms such as
Botrytis sp., Fusarium sp., Pythium sp. or Candida tropicalis R2, in particular Botrytis cinerea induced diseases, comprising a spraying step of the aerial parts and/or an inoculation step of the underground parts of said plants with a composition comprising one or more actinomycetes strain such as defined in claim 1, preferably at a concentration from about 10 cfu/ml to about 10 cfu/ml, preferably from about 10 cfu/ml to about 10 cfu/ml, from about 107 cfu/ml to about 108 cfu/ml, in particular 107 cfu/ml.
21 Process according to claim 20, wherein said spraying step of the aerial parts and/or an inoculation step of the underground parts of said plants is carried out with one or more chemical compounds belonging to one of the following chemical families: tryptophan derivatives, 2-terpenyl pyridines, ionophoretic polyethers, and chlorinated lipid compounds such as defined in claim 6, and preferably at a concentration from about 10- M to about 10- M, preferably from about 10- M to about 10- M, more preferably from about 10-5 M to about 10-9 M, more preferably from about 10-6 M to about 10-9 M, more preferably from about 10-7 M to about 10-9 M, more preferably from about 10-8 M to about 10- M, more preferably from about 10-7 M to about 10- M, and more preferably from about 10-6 M to about 10-7 M.
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