WO2017125775A1 - Prevention or treatment in algae of diseases induced by protistan pathogens - Google Patents

Prevention or treatment in algae of diseases induced by protistan pathogens Download PDF

Info

Publication number
WO2017125775A1
WO2017125775A1 PCT/IB2016/000151 IB2016000151W WO2017125775A1 WO 2017125775 A1 WO2017125775 A1 WO 2017125775A1 IB 2016000151 W IB2016000151 W IB 2016000151W WO 2017125775 A1 WO2017125775 A1 WO 2017125775A1
Authority
WO
WIPO (PCT)
Prior art keywords
pyrenocine
pathogen
compound
protistan
pyrenochaetic
Prior art date
Application number
PCT/IB2016/000151
Other languages
French (fr)
Inventor
Soizic Prado
Marine VALLET
Claire GACHON
Martina STRITTMATTER
Gwang Hoon Kim
Original Assignee
Museum National D'histoire Naturelle
The Scottish Association For Marine Science
Kongju National University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Museum National D'histoire Naturelle, The Scottish Association For Marine Science, Kongju National University filed Critical Museum National D'histoire Naturelle
Priority to PCT/IB2016/000151 priority Critical patent/WO2017125775A1/en
Publication of WO2017125775A1 publication Critical patent/WO2017125775A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
    • C07D309/34—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D309/36—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with oxygen atoms directly attached to ring carbon atoms
    • C07D309/38—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with oxygen atoms directly attached to ring carbon atoms one oxygen atom in position 2 or 4, e.g. pyrones
    • 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
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/10—Aromatic or araliphatic carboxylic acids, or thio analogues thereof; Derivatives thereof
    • 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
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
    • A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
    • A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
    • A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • the present invention relates to the use of a pyrenocine compound or a pyrenochaetic acid compound for the prevention or the treatment in algae of diseases induced by a broad spectrum of protistan pathogens.
  • the present invention also relates to a new pyrenocine compound.
  • Algae in particular marine algae belonging to the class Phaeophyceae or the division Rhodophyta play a fundamental role in coastal ecosystems and form the first link in the food chain.
  • These two classes of algae are complex multicellular photosynthetic eukaryotes.
  • Agars, carrageenan and alginates obtained from Phaeophyceae or Rhodophyta are hydrocolloids, which are present in a variety of consumer products. 55 000 tones thereof are produced annually. The market value is estimated at US $ 585 million. Alginate and other cell wall polymers are used in pharmaceutical, cosmetic and food products. The market value is estimated at US $ 213 million.
  • the alginates are used in the manufacture of biomaterials and implants. They are also components of toothpaste, ointment for treatment of wounds and burns, compresses and dental implants.
  • Mannitol obtained from Phaeophyceae is a polyol used in solution for treating renal failure, increased intracranial pressure and for determining the hematocrit of a patient or for diagnosing a vasodilatation.
  • Phloroghicinol extracted from brown algae has antispasmodic and analgesic effects and is used in the treatment of digestive, urinary and gynecological spasmodic pain.
  • Laminarin obtained by extraction of the alga Laminaria digitata, belonging to the genus Laminaria belonging to the class of Phaeophyceae is used in priming plant defenses against diseases, for example the treatment of septoria in wheat and in the treatment of mildew in barley.
  • Red algae belonging mainly to the genus Pyropia and Porphyra (laver, also known as Nori in Japan) of the division of Rhodophyta is an important marine crop, extensively cultivated in Japan, Korea, and other oriental countries, with a market currently worth over US $ 1 billion per year and which accounts for 60% of the entire seaweed industry.
  • Pyropia yezoensis (formerly known as Porphyra yezoensis) is the most economically valuable species of Pyropia due to its high quality.
  • Protists are a polyphyletic group encompassing all eukaryotes, to the exclusion of animals, plants and fungi. Protists are eukaryotic organisms with typically unicellular, filamentous or parenchymatous organization. They have little or no vegetative tissue differentiation, except for reproduction (Adl et al, 2007).
  • Protists include the clade SAR.
  • SAR derives from the acronym of the three groups composing this clade: Stramenopiles, Alveolata and Rhizaria.
  • the Stramenopiles comprise oomycete and hyphochytrid micro-organisms and Rhizaria comprises phytomyxean micro-organisms (e.g. Plasmodiophora micro-organisms) (Adl et al, 2012).
  • Oomycetes are among the pathogens responsible of the algal diseases previously cited. Oomycetes are fungus-like organisms widely present in the phycosphere (i.e., the physicochemical environment in which an alga lives) and constitute a major cause of disease in phycoculture and against which no treatment is truly effective. For example, serious damage is caused by the oomycete pathogens, Pythium porphyrae and Olpidiopsis spp.fsubspecies), which decrease the productivity of Pyropia and Porphyra sea farms. In Seocheon sea farms, an outbreak of Olpidiopsis spp.
  • Olpidiopsis disease resulted in approximately US $ 1.6 million in loss, representing approximately 24.5% of total sales during the 2012-2013 season (Kim et al, 2014). Olpidiopsis disease also concerns brown algae, for example Undaria pinnatifida (Akiyama et al, 1977).
  • E. dicksonii In laboratory cultures, the oomycete Eurychasma dicksonii infects virtually all species of brown algae tested. Phylogenetically, E. dicksonii represents the most basal member of the oomycete lineage. It is frequently found infecting brown algae in the field, throughout the world. In particular, E. dicksonii infects the genome model seaweed Ectocarpus siliculosus (Gachon et al., 2009).
  • Oomycetes propagate and infect their host through flagellated zoospores, that are typically biflagellated. Marine genera such as Eurychasma and Olpidiopsis are obligate biotrophs, that penetrate their host and develop intracellularly. The penetration mechanism has been well described in the oomycete Eurychasma dicksonii, which cannot live without its host.
  • the infection mechanism comprises: adhesion of a flagellate zoospore to the host cell surface, encystment on the surface, formation of an appressorial pad, germination of the cyst and penetration into the host cell.
  • the thallus grows intracellularly, undergoes synchronised nuclear divisions to form a syncytium, and typical of holocarpic pathogens, ultimately converts itself into a sporangium that releases new propagules.
  • Olpidiopsis has an intracellular, development cycle, well understood on the pathogens of Pyropia and Porphyra, Olpidiopsis pyropiae and Olpidiopsis porphyrae (Sekimoto et al., 2008b; Klochkova et al., 2015).
  • Olpidiopsis pyropiae infects Pyropia yezoensis, and is responsible for the red rot disease.
  • Pythium is an oomycete genus containing over a hundred species with contrasting lifestyle ranging from saprophytes to parasites of plants, mammals, and marine algae (Levesque et al, 2004).
  • Pythium porphyrae which belongs to the same species complex as Pythium chondricola, form a germ tubes and appressoria, and colonise the thallus of their host with hyphae (Kazama et al, 1977).
  • P. marinum also grows on Pyropia and closely related red algae.
  • Hyphochytriales share many similar morphological features with obligate biotrophic oomycetes such as Olpidiopsis oomycetes, but are characterised by a single anterior flagellum.
  • the brown algae belonging to the class of Phaeophyceae are also infected by protistan pathogens that are only distantly or unrelated to oomycetes.
  • protistan pathogens that are only distantly or unrelated to oomycetes.
  • large kelps such as Durvillea antartica (Goecke et al., 2012) were infected by phytomyxea (Phytomyxea, Rhizaria (Adl et al, 2012)) pathogens, closely related to another pathogen of Ectocarpus, namely Maullinia ectocarpi (Maier et al, 2000).
  • Maullinia ectocarpii is also able to infect multiple brown algal species belonging to the class Phaeophyceae, including the kelp Macrocystis pyrifera.
  • Phytomyxea are also biotrophic parasites that alternate the production of two types of intracellular Plasmodia (sporogenic and sporangia!). These plasmodia result from the development of biflagellated primary and secondary zoospores. Hie Plasmodium derived from a secondary zoospore forms resting spores.
  • Pyrenocines and pyrenochaetic acids are mycotoxins produced by fungi isolated from plants or algae.
  • Pyrenocine compounds are pyran-2-one derivatives, substituted at position 4 by a methoxy group, at position 5 by a saturated or unsaturated, substituted or unsubstituted butyryl group and said pyran-2-one derivatives being further substituted at position 6 by a substituted or unsubstituted alkyl chain (such as a methyl group).
  • Pyrenochaetic acid compounds are benzoic acid derivatives, substituted at position 3 by a methoxy group, at position 4 by a saturated or unsaturated, substituted or unsubstituted butyryl group and said benzoic acid derivatives being further substituted at position 5 by a methyl group. Some of these compounds are reported to have antibacterial, antifungal or algicidal properties. Pyrenocines A and B and pyrenochaetic acids A, B and C have been isolated from culture filtrates of Pyrenochaeta terrestris, the causal fungus of onion pink root disease (Sato et al., 1981).
  • Pyrenocines A, B, D and £ are produced by a strain of Penicillium waksmanii Zaleski OUPS-N133 separated from the brown alga Sargassum ringgoldianum (Amagata et al, 1998). More recently, pyrenocines J, K, L and M have been isolated from the fungus Pkomopsis sp., from the plant Cistus salvifolius internal strain 7852 (Hidayat et al, 2012). Methods for obtaining these pyrenocine and pyrenochaetic acid are described in Sato et al, 1981, Amagata et al., 1998 and Hidayat et al., 2012.
  • Pyrenocines A, B and pyrenochaetic acid A have also been obtained by chemical synthesis (Ichihara et al., 1981 and Ichihara et al., 1983).
  • the biological activity of these pyrenocine compounds has already been described against fungal and bacterial pathogens.
  • pyrenocine A inhibits the mycelium growth of Fusarium oxysporum, the spores germination of F. oxysporum forma specialis Cepae (F. oxysporum f. sp. Cepae), F, solani f. sp. pisi, Mucor hiemalis and Rhizopus stolonifer.
  • Pyrenocine A also inhibits the growth of positive- gram bacteria such as Bacillus subtilis, Staphylococcus aureus and Escherichia coli (Sparace et al., 1987).
  • the cytotoxicity of pyrenocines A, B and E has also been tested in a lymphocytic leukemia test system (Amagata et al, 1998).
  • the biological activities of pyrenocines J, K, L and M have been assessed against bacteria, plant fungi, plant fungi-like and algae (Hidayat et al, 2012).
  • the anti-oomycete, anti-hypho chytrid and anti -phytomyxean activities of pyrenocine compounds and pyrenochaetic acids has never been reported in algae.
  • pyrenocine compounds and pyrenochaetic acid C compounds have a broad-spectrum anti-protistan activity, in particular an anti- oomycete, anti-hyphochytrid and/or anti-phytomyxean activity (i.e., has properties useful for preventing or treating diseases induced by oomycete pathogens, hyphochytrid pathogens or phytomyxean pathogens), in algae, in particular, brown algae belonging to the class Phaeophyceae and red algae belonging to the division Rhodophyta.
  • the inventors have also isolated a new compound belonging to the pyrenocine family, called pyrenocine N having an anti-oomycete activity.
  • the present invention provides the use of a pyrenocine compound or a pyrenochaetic acid compound for the prevention or the treatment in an alga, preferably in a brown alga belonging to the class Phaeophyceae or a red alga belonging to the division Rhodophyta, of a disease induced by a protistan pathogen (microorganism).
  • the present invention also provides a method for preventing or treating a disease induced by a protistan pathogen (microorganism) in an alga, preferably in a brown alga belonging to the class Phaeophyceae or a red alga belonging to the division Rhodophyta, comprising contacting an effective amount of a pyrenocine compound or a pyrenochaetic acid compound with said alga or portion thereof.
  • a protistan pathogen microorganism
  • the alga is in a culture pool.
  • An effective amount refers to an amount of a pyrenocine compound or a pyrenochaetic acid compound which is effective in preventing or treating in an alga a disease induced by a protistan pathogen.
  • One skilled in the art can determine what an effective amount is appropriate for preventing or treating a disease induced by a protistan pathogen.
  • treating refers to the inhibition of the infection by a protistan pathogen in an alga, by slowing down or stopping the growth of the pathogen, and/or inducing the death of the pathogen.
  • preventing refers to the induction of the resistance to an infection by a protistan pathogen in an alga (i.e. inhibition of the appearance of an infection).
  • a pyrenocine compound is a pyran-2-one derivative, substituted at position 4 by a methoxy group, at position 5 by a saturated or unsaturated, substituted or unsubstituted butyryl group and said pyran-2-one derivative being further substituted at position 6 by substituted or unsubstituted alkyl chain.
  • Said alkyl chain is preferably a methyl group. It includes pyrenocine A, B, D, E, F, G, H, J, K, L, M and N.
  • a pyrenochaetic acid compound is a benzoic acid derivative, substituted at position 3 by a methoxy group, at position 4 by a saturated or unsaturated, substituted or unsubstituted butyryl group and said benzoic acid derivative being further substituted at position 5 by a methyl group. It includes pyrenochaetic acid A, B and C.
  • the pyrenocine compound is selected from the group consisting of pyrenocine A, pyrenocine B, pyrenocine E and pyrenocine N.
  • the pyrenochaetic acid compound is pyrenochaetic acid C.
  • Preferred pyrenocine and pyrenochaetic acid compounds of the present invention have the following formula:
  • a pyrenocine can be used alone or in combination with one or more other pyrenocine and/or pyrenochaetic acid.
  • pyrenochaetic acid in particular pyrenochaetic acid C, can be used alone or in combination with one or more pyrenocine.
  • these compounds are isolated from a Phaeosphaeria sp. strain in particular the strain Phaeosphaeria sp. AN596H, which is an endophyte fungus, isolated from Ascophyllum nodosum, a brown alga belonging to the class Phaeophyceae.
  • the strain Phaeosphaeria sp. AN596H was deposited according to the Budapest Treaty at CNCM (Collection Nationale de Culture de Microorganismes, 25 rue du Dondel Roux, 75724 Paris Cedex 15) on January 14, 2016, under the number CNCM 1-5041.
  • Red rot disease Diseases induced by a protistan pathogen include red rot disease, "chytrid blight” disease, preferably red rot disease.
  • Protist responsible of a disease in a red alga and/or a brown alga include:
  • the brown alga belonging to the class Phaeophyceae includes the alga of the following orders: Laminariales, Fucales, Ectocarpales, Ascoseirales, Desmarestiales, Dictyotales, Nemodermatales, Ralfsiales, Scytothamnales, Sphacelariales, Sporochnales, Syringodermatales, Tilopteri dales and Disco sporangiales.
  • the order Laminariales comprises the following families: Laminariaceae, Lessoniaceae and Alariaceae.
  • the order Fucales comprises the family Fucaceae, Hormosiraceae and Durvilleaceae.
  • the order Ecto carpal es comprises the family Ectocarpaceae.
  • the order Ectocaipales and more precisely the species Ectocarpus siliculosus is phylogenetically close to the species belonging to the order Laminariales.
  • Laminariaceae comprises the following genera: Laminaria, Saccharina, Macrocystis and Nereocystis.
  • the family Lessoniaceae comprises the following genera: Ecklonia and Eisenia.
  • the family Alariaceae comprises the following genera: Undaria and Alaria.
  • the family Fucaceae comprises the following genera: Ascophyllum and
  • the family Hormosiraceae comprises the genus Hormosira.
  • the family Durvilleaceae comprises the genus Durvillea.
  • the family Ectocarpaceae comprises the genus Ectocarpus.
  • Laminaria comprises the following species: Laminaria digitata,
  • Saccharina The genus Saccharina comprises the following species: Saccharina japonica and Saccharina latissima.
  • the genus Macrocystis comprises the following species: Macrocystis pyrifera and Macrocystis pomifera.
  • Macrocystis pyrifera is the strain deposited at CCAP (The Culture Collection of Algae and Protozoa, Scottish Marine institute, OBAN, Argyll PA37 1QA Scotland), under the number CCAP 1323/1.
  • Nereocystis comprises the species Nereocystis luetkeana.
  • the genus EcMonia comprises the following species: Ecklonia kurome and
  • the genus Eisenia comprises the species Eisenia bicyclis ⁇ Ecklonia bicyclis).
  • the genus Undaria comprises the following species: Undaria pinnatifida, Undaria crenata, Undaria undarioides and Undaria peterseniana.
  • the genus Alaria comprises the species Alaria esculenta.
  • the genus Ascophyllum comprises the species Ascophyllum nodosum.
  • the genus Pelvetia comprises the species Pelvetia canaliculata.
  • the genus Durvillea comprises the following species: Durvillea antartica and Durvillea poha.
  • Ectocarpus comprises the following species: Ectocarpus siliculosus,
  • Ectocarpus fasciculatus Ectocarpus crouaniorum, Ectocarpus subulatus and Ectocarpus mitchellae.
  • An example of Ectocarpus siliculosus is the strain deposited at the CCAP, under the number CCAP 1310/4.
  • the brown alga is preferably selected from the genus Laminaria, Saccharina, Ascophyllum, Pelvetia and Ectocarpus, more preferably selected from the species Laminaria digitata, Saccharina latissima, Ascophyllum nodosum, Pelvetia canaliculata and Ectocarpus siliculosus.
  • the red alga belonging to the division Rhodophyta includes the alga of the following orders: Bangiales, Palmariales, Gelidiales, Gracilariales, Nemaliades and Gigartinales.
  • the order Bangiales comprises the family Bangiaceae.
  • the order Palmariales comprises the family Palmariaceae.
  • the order Gelidiales comprises the family Gelidiaceae.
  • the order Gracilariales comprises the family Gracilariaceae.
  • the order Nemaliades comprises the family Bonnemaisoniaceae.
  • the order Gigartinales comprises the family Solieriaceae.
  • the family Bangiaceae comprises the following genera: Pyropia and Porphyra.
  • the family Palmariaceae comprises the genus Palmaria.
  • the family Gelidiaceae comprises the genus Gelidium.
  • the family Gracilariaceae comprises the following genera: Gracilaria and Gracilariopsis.
  • the family Bonnemaisoniaceae comprises the genus Asparagopsis.
  • the family Solieriaceae comprises the following genera: Kappaphycus and Eucheuma.
  • the genus Pyropia comprises the following species: Pyropia abbottiae, Pyropia acanthopohora, Pyropia aeodis, Pyropia bajacaliforniensis, Pyropia brumalis, Pyropia cinnamomea, Pyropia columbiensis, Pyropia columbina, Pyropia conwayae, Pyropia crassa, Pyropia dentate, Pyropia denticulate, Pyropia elongate, Pyropia endiviifolia, Pyropia fallax, Pyropia francissii, Pyropia fiicicola, Pyropia gardneri, Pyropia haitanensis, Pyropia hiberna, Pyropia hollenbergii, Pyropia ishigecola, Pyropia kanakaensis, Pyropia katadae, Pyropia kinositae, Pyropia koreana, Pyropia kuniedae, Pyropia
  • Porphyra comprises the following species: Porphyra akasakae, Porphyra angusta, Porphyra argentinensis, Porphyra atropurpurea, Porphyra augustinae, Porphyra autumnalis, Porphyra bangiaeformis, Porphyra bulbopes, Porphyra capensis, Porphyra cornea, Porphyra ceylanica, Porphyra chauhanii, Porphyra conwayae, Porphyra corallicola, Porphyra cordata, Porphyra cucullata, Porphyra cuneiformis, Porphyra americanula, Porphyra dentimarginata, Porphyra diocia, Porphyra drachii, Porphyra fiijianensis, Porphyra gardneri, Porphyra grateloupicola, Porphyra grayana, Porphyra guangdongen
  • Palmaria callophylloides The genus Palmaria comprises the following species: Palmaria callophylloides,
  • Palmaria centrocarpa Palmaria decipiens, Palmaria georgica, Palmaria hecatensis, Palmaria integrifolia, Palmaria marginicrassa, Palmaria mollis, Palmaria moniliformis, Palmaria palmata and Palmaria stenogona.
  • the genus Gelidium comprises the following species: Gelidium amansii, Gelidium robustum, Gelidium chilense, Gelidium sesquipedale, Gelidium pristoides and Gelidium canariense.
  • the genus Gracilaria comprises the following species: Gracilaria chilensis, Gracilaria gracilis, Gracilaria gigas, Gracilaria tenuisti, Gracilaria edulis, Gracilaria multipartita and Gracilaria bursa-pastoris,
  • the genus Gracilariopsis comprises the following species: Gracilariopsis lemaneiformis, Gracilariopsis longissimi, Gracilariopis andersonii and Gracilaiopsis sjoestedtii.
  • the genus Asparagopsis comprises the following species: Asparagopsis armata, Asparagopsis svedelii and Asparagopsis taxiformis.
  • the genus Kappaphycus comprises the following species: Kappaphycus alvarezii, Kappaphycus cottonii, Kappaphycus inermis, Kappaphycus malesianus, Kappaphycus procrusteanus and Kappaphycus striatus.
  • the genus Eucheuma comprises the following species: Eucheuma adhaerens, Eucheuma amakusaense, Eucheuma arnoldii, Eucheuma cartilagineum, Eucheuma cervicorne, Eucheuma chondriforme, Eucheuma crassum, Eucheuma crustiforma, Eucheuma deformans, Eucheuma denticulatum, Eucheuma dichotonum, Eucheuma edula, Eucheuma horizontals Eucheuma horridum, Eucheuma isiforme, Eucheuma johnstonii, Eucheuma jugata, Eucheuma kraftianum, Eucheuma leeuwenii, Eucheuma nodulosum, Eucheuma nudum, Eucheuma odontophorum, Eucheuma perplexum, Eucheuma platycladum, Eucheuma serra, Eucheuma simplex, Euche
  • the red alga is Pyropia yezoensis or Porphyra purpurea.
  • the red and the brown alga of the present invention can be a natural or artificial hybrid between 2 species.
  • a hybrid can be a cultivar obtained from the crossing between Laminaria longissima x Saccharina japonica (Zhang et al., 2007) or obtained after a protoplast fusion between Pyropia yezoensis and Pyropia haitanensis (Dai etal, 1993).
  • the oomycete pathogen comprises:
  • Sirolpidium that are suspected to be closely related or indeed overlap with the aforementioned orders.
  • the order Eurychasmales comprises the genus Eurychasma.
  • the order Olpidiopsidiales comprises the genus Olpidiopsis.
  • the order Pythiales comprises the genus Pythium.
  • the order Peronosporales comprises the following species: Pythium porphyrae, Pythium chondricola and Pythium marinum.
  • the order Myzocytiopsidales comprises the following genera: Pontisma, Petersenia and Sirolpidium.
  • the genus Eurychasmidium comprises the following species: Eurychasmidium joycei, Eurychasmidium sacculus and Eurychasmidium tumefaciens.
  • the genus Petersenia comprises the following species: Petersenia lobota, Petersenia pollagaster and Petersenia palmariae.
  • the genus Pontisma comprises the following species: Pontisma antithamnionis, Pontisma dangeardii, Pontisma feldmanii, Pontisma inhabilis, Pontisma lagenidioles and Pontisma magnusii.
  • the genus Sirolpidium comprises the species Sirolpidium ectocarpii.
  • the genus Eurychasma comprises the species Eurychasma dicksonii.
  • Eurychasma dicksonii are the strains CCAP 4018/1 and the CCAP 4018/3.
  • Olpidiopsis comprises the following species: Olpidiopsis bostrychiae, Olpidiopsis feldmannii, Olpidiopsis porphyrae, Olpidiopsis pyropiae, Olpidiopsis antithamnionis, Olpidiopsis dangeardii, Olpidiopsis magnusii, and Olpidiopsis tumefaciens.
  • the genus Pythium comprises the species Pythium porphyrae.
  • the oomycete pathogen is preferably selected from the species
  • Eurychasma dicksonii and Olpidiopsis pyropiae Eurychasma dicksonii and Olpidiopsis pyropiae.
  • Eurychasma dicksonii infects a brown alga belonging to the class Phaeophyceae, more particularly, belonging to the genus Laminaria, Saccharina, Macrocystis, and Ectocarpus.
  • Eurychasma dicksonii infects the species such as Macrocystis pyrifera, Laminaria digitata, Saccharina latissima, and Ectocarpus siliculosus, preferably, Laminaria digitata, Saccharina latissima, and Ectocarpus siliculosus, more preferably Ectocarpus siliculosus.
  • Olpidiopsis pyropiae infects a red alga belonging to the division Rhodophyta, preferably the family Bangiaceae, more preferably the species such as Pyropia yezoensis and Pyropia haitanensis, and even more preferably Pyropia yezoensis.
  • the hyphochytriale comprises the family Anisolpidiaceae (Adl et al, 2012).
  • the family Anisolpidiaceae comprises the genus Anisolpidium.
  • Anisolpidium comprises the following species: Anisolpidium ectocarpii, Anisolpidium joklianum, Anisolpidium rosenvingii, Anisolpidium minutum, Anisolpidium olpidium and Anisolpidium sphacellarum.
  • An example of Anisolpidium ectocarpii is the strain Anisolpidium ectocarpii CCAP 4001/1.
  • Anisolpidium ectocarpii infects a brown alga belonging to the class Phaeophyceae, more particularly, belonging to the genus Laminaria, Saccharina, and Ectocarpus.
  • Anisolpidium ectocarpii infects the species such as Laminaria digitata, Saccharina latissima, Ectocarpus mitchellae and Ectocarpus siliculosus, preferably, Laminaria digitata, Saccharina latissima, Ascophyllum nodosum, Pelvetia canaliculate and Ectocarpus siliculosus, more preferably Ectocarpus siliculosus.
  • the hyphochytrid pathogen is preferably the species Anisolpidium ectocarpii.
  • the phytomyxea includes the following genera: Maullinia, Phagomyxa and
  • the genus Maullinia comprises the species Maullinia ectocarpii.
  • Maullinia ectocarpii is the strain Maullinia ectocarpii CCAP 1538/1.
  • the genus Phagomyxa comprises the species Phagomyxa algarum
  • Labyrinthula comprises the following species: Labyrinthula pohlia and Labyrinthula roscoffiensis.
  • Maullinia ectocarpii infects a brown alga belonging to the class Phaeophyceae, more particularly, belonging to the genus Laminaria, Saccharina, and Ectocarpus.
  • Maullinia ectocarpii infects the species such as Laminaria digitata, Saccharina latissima, and Ectocarpus siliculosus, preferably, Laminaria digitata, Saccharina latissima, or Ectocarpus siliculosus, more preferably Ectocarpus siliculosus.
  • the phytomyxean pathogen is preferably the species Maullina ectocarpii.
  • the pyrenocine compound is selected from the group consisting of pyrenocine A and pyrenocine B and the protistan pathogen is selected from the group consisting of the genus Eurychasma and Anisolpidium, preferably selected from the species Eurychasma dicksonii and Anisolpidium ectocarpii.
  • the pyrerocine compound is the pyrenocine A and the protistan pathogen is selected from the species Eurychasma dicksonii and Anisolpidium ectocarpii.
  • the pyrenocine compound is selected from the group consisting of pyrenocine A, pyrenocine B and pyrenocine E and the protistan pathogen is of the genus Maullinia, preferably the species Maullinia ectocarpii.
  • the pyrerocine compound is the pyrenocine A and the protistan pathogen is the species Maullinia ectocarpii.
  • the pyrenocine or pyrenochaetic acid compound is selected from the group of pyrenocine A, pyrenocine N and pyrenochaetic acid C and the protistan pathogen is of the genus Olpidiopsiale, preferably the species Olpidiopsis pyropiae.
  • the pyrenocine compound is selected from pyrenocine A and pyrenochaetic acid C and the protistan pathogen is the species Olpidiopsis pyropiae. More preferably, the pyrenocine compound is the pyrenocine A and the protistan pathogen is the species Olpidiopsis pyropiae.
  • the pyrenochaetic acid compound is pyrenochaetic acid C and the protistan pathogen is of the genus Pythium, preferably the species Pythium porphyrae.
  • pyrenocine A has an anti-protistan activity (i.e., has properties useful for preventing or treating, preferably treating, a disease induced by a protistan pathogen) against the following species of a protistan pathogen: Eurychasma dicksonii, Anisolpidium ectocarpii and Maullinia ectocarpii, in a brown alga belonging to the class Phaeophyceae, preferably in Ectocarpus siliculosus.
  • pyrenocine A has an anti-oomycete activity against Olpidiopsis pyropiae, in a red alga belonging to the division Rhodophyta, preferably in Pyropia yezoensis.
  • pyrenocine B has an anti-protistan activity against the following species of a protistan pathogen: Eurychasma dicksonii, Anisolpidium ectocarpii, Maullinia ectocarpii, in a brown alga belonging to the class Phaeophyceae, preferably in Ectocarpus siliculosus.
  • pyrenocine E has an anti- phytomyxean activity against Maullinia ectocarpii, in a brown alga belonging to the class Phaeophyceae, preferably in Ectocarpus siliculosus.
  • pyrenochaetic acid C has an anti- oomycete activity against a following species of oomycete pathogen: Olpidiopsis pyropiae and Pythium porphyrae, in a red alga belonging to the division Rhodophyta, preferably Pyropia yezoensis.
  • the pyrenocine compound or pyrenochaetic acid compound used for the prevention or the treatment, in a brown alga belonging to the class Phaeophyceae or in a red alga belonging to the division Rhodophyta, of a disease induced by a protistan pathogen can be formulated in a liquid, emulsion, foam, paste, powder or gel form.
  • the pyrenocine or pyrenochaetic acid compound as defined above can be included into a cyclodextrine.
  • Methods of encapsulation of a compound into cyclodextrines are well-known in the art Munin et al, 2011 describes a method for encapsulating polyphenolic compounds into cyclodextins. Such method can apply for pyrenocine and pyrenochaetic acid compounds.
  • the pyrenocine or pyrenochaetic acid compound as defined above can be formulated as stable water-dispersible nanoassemblies of homogeneous size, as described in the art for derivatives of curcumin (Cheikh-Ali et al, 2015).
  • the pyrenocine or pyrenochaetic acid compound as defined above can be in solution with a polar aprotic solvent or a polar protic solvent.
  • the polar aprotic solvent is preferably selected from the group consisting of acetone, N,N- dimethylforrnamide (DMF), acetonitrile and dimethylsulfoxide (DMSO), more preferably dimethylsulfoxide (DMSO).
  • the polar protic solvent is preferably selected from the group consisting of methanol, ethanol and butanol, more preferably methanol.
  • the concentration of a pyrenocine compound or a pyrenochaetic acid compound, used for the prevention or the treatment, in a brown alga belonging to the class Phaeophyceae, of a disease induced by a protistan pathogen (microorganism) is advantageously preferably between and 100 ⁇ , more preferably between 1 and 10 ug/ml.
  • the concentration of a pyrenocine compound or a pyrenochaetic acid compound, used for the prevention or the treatment, in a red alga belonging to the division Rhodophyta, of a disease induced by a protistan pathogen is advantageously between and 200 ⁇ g/ml, preferably between 5 and more
  • the pyrenocine compound or the pyrenochaetic compound can be applied once a day, for one week, every day, to repeat every month for 6 months.
  • the present invention also provides the isolated compound pyrenocine N, of formula:
  • the present invention also provides a composition, such as an anti -protistan composition, comprising the compound pyrenocine N.
  • Said composition can comprise a polar protic or aprotic solvent as described above.
  • Pyrenocine N can be formulated as stable water-dispersible nanoassemblies of homogeneous size as described above.
  • Pyrenocine N can also be included into a cyclodextrine as described above.
  • Pyrenocine N is obtainable by the method described below.
  • the present invention also provides a method for obtaining the compound pyrenocine N, comprising the steps of:
  • the elution occurred with buffer A (95:5 Water+0.5% TFA / ACN) and buffer B (5:95 Water+0.5% TFA (Trifluoroacetic acid) / (ACN)) according the following gradient method: starting at 95/5 of A/B, 75/25 of A/B at 10 min, 50/50 of A/B at 20 min, 10/90 of A/B at 24 min and 95/5 of A/B at 27 min.
  • buffer A 95:5 Water+0.5% TFA / ACN
  • buffer B 5:95 Water+0.5% TFA (Trifluoroacetic acid) / (ACN)
  • step a) the strain of Phaeosphaeria can be cultured in a solid medium, such as the culture medium TUA+ASW+AN (described in Example 2 below).
  • the culture can be extracted with a solvent optionally under mechanical agitation.
  • the solvent can be an organic polar aprotic solvent, such as, ethyl acetate and acetonitrile, preferably ethyl acetate or an organic polar prone solvent, preferably, methanol.
  • the chromatography can include an exclusion chromatography and/or
  • HPLC preferably an exclusion chromatography followed by a HPLC.
  • the exclusion chromatography is performed on a column made of a gel filtration resin and elution with methanol.
  • the column can be a silica gel column or a reverse-phase CI 8 silica gel column.
  • the column is made of Sephadex* G, Sephadex* LH, Bio-Gel* P, Bio-Gel* A, Sephacryl*, Sepharose e or Fractogel* TSK and more preferably the column is made of Sephadex* LH20.
  • the HPLC is performed on a stationary phase CI 8 column and elution with buffer A (95:5 Water+0.5% Trifluoroacetic acid (TFA) / Acetonitrile (ACN)) and buffer B (5:95 Water+0.5% TFA / ACN).
  • the stationary phase is a bonded silica, zirconium oxide, titan oxide or aluminum oxide CI 8 phases with polar, non polar endcapping, di-isobutyl side chains or reverse phases with phenyl, phenyl-hexyl or pentafluorophenyl and more preferably the stationary phase is a bonded silica Zorbax XBD CI 8 column.
  • the method for obtaining the compound pyrenocine N is described in details in Example 2 below.
  • Pyrenocine N could also be obtained from pyrenocine B by hydrogenation.
  • the present invention also provides an isolated extract of a Phaeosphaeria strain comprising the compound pyrenocine N.
  • the Phaeosphaeria strain is isolated from Ascophyllum nodosum, in particular the strain Phaeosphaeria sp. AN596H (CNCM 1-5041).
  • the present invention also provides a Phaeosphaeria strain wherein it is the strain Phaeosphaeria sp. AN596H CNCM 1-5041, deposited according to the Budapest Treaty at CNCM on January 14, 2016.
  • the invention further comprises other features which will emerge from the following description, which refers to an example illustrating the present invention, as well as to the appended figures.
  • Figure 1 shows the experimental setup of test of a brown alga resistance against a protist pathogen.
  • Figure 2 shows the infection score assigned with the light microscopy observations.
  • Figure 3 shows the average scores of infection of Ectocarpus siliculosus obtained during tests of biological assessments of endophytic extract AN596H at 10 against E. dicksonii CCAP4018/1 (point), E. dicksonii CCAP4018/3 (grey), Maullinia ectocarpii (hatching), Anisolpidium ectocarpii (black) after counting by optical microscopy. Extracts from endophytic strains isolated and identified and the positive and negative controls are represented on the ordinate. The score scale is represented on the abscissa.
  • Figure 4 shows the DNA quantification using qPCR.
  • Figure 5 shows the linear correlation between the values of ACt (ordinate) obtained by qPCR and microscopic scores (abscissa) obtained by optical microscopy observations of quantification of infection by E. dicksonii CCAP4018/1 and CCAP4018/3.
  • Figure 6 shows the steps of the protocol of isolation of the active compounds against a protist pathogen.
  • Figure 7 shows the chromatographic profile of fraction C5 comprising pyrenocine compounds and acid pyrenochaetic C, obtained by HPLC.
  • Figure 8 shows the average of scores obtained by microscopy scores of infection of Ectocarpus siliculosus by the different pathogens: E. dicksonii CCAP4018/1 (point), E. dicksonii CCAP4018/3 (grey), Maullinia ectocarpii (hatching), Anisolpidium ectocarpii (black) in the presence of purified pyrenocine compounds at 1 ⁇ g/ml.
  • Figure 9 shows the average of ACt values obtained by quantification of E. dicksonii infection in E. siliculosus in the presence of pyrenocine A at a final concentration of 1 ⁇ g/ml.
  • Tests were performed in 6 -well plates, in which 7 ml of culture medium and 7 ⁇ of the endophyte extract ANS96H (final concentration: 10 ⁇ g/ml ) were added to each well ( Figure 1). Subsequently, Ectocarpus siliculosus CCAP 1310/4 was added to each well. The inoculum Macrocystis pyrifera CCAP 1323/1 infected by the pathogen Eurychasma dicksonii CCAP 4018/1 was placed in a cell strainer with a pore size of 70 ⁇ and put into the well. Positive controls (omitting the addition of endophyte extract) and negative controls (omitting the pathogen-infected inoculum) were also added. Each sample was run in triplicates and the experiments were performed three times independently. The plates were sealed with parafilm and incubated at 15°C with a photoperiod of 12 hours (light intensity: for
  • E. siliculosus was transferred to a microscope slide and analysed via microscopy (Zeiss Axiovert 2, DIC III apochromatic).
  • the degree of infection was determined by applying an arbitrary score scale based on the number of infected algal cells. Thus, a score of 0 was assigned if the algal culture was not infected (no infected algal cell), a score of 1 for low infection (1- 10 infected cells), a score of 2 for moderate infection (11-100 algal cells infected) and a score of 3 for a dense infection (> 101 algal cells infected) ( Figure 2).
  • qPCR quantitative PCR
  • Ectocarpus material that was previously for microscopy scoring was then harvested, frozen in RNAlater® (Thermo Fisher) at - 80 °C buffer for analysis by qPCR.
  • the Ct value (threshold cycle) which is the intersection between the amplification curve and a given fluorescence threshold line, allows measuring the relative concentration of amplicons of the target DNA.
  • the qPCR reactions were performed in triplicates on cyclers Quantica (technetium Barloworld, UK) and LightCycler® (Roche 96SW1.1, UK).
  • the qPCR reaction consisted of 10 ⁇ qPCR buffer (qPCR MasterMix Mesagreen Plus for SYBR Assay, Eurogentec), 1 ⁇ of each sense and anti-sense primer at a final concentration of 300 nM and 8 ⁇ of DNA in a final volume of 20 ⁇ .
  • the amplification program was as follows: denaturation for 10 min at 95°C and then 45 amplification cycles of 15 s at 95°C followed by incubation at 60°C for 1 min, with the reading of fluorescence to monitor the PCR reaction in real time.
  • the melting curve characteristics were obtained with a final cycle defined by an increase of temperature of 70 to 95°C ( ⁇ 1°C every 20 seconds).
  • the infection was quantified for each sample by calculating a ACt value (Eurychasma Ct - Ectocarpus Ct) obtained from the amplification of algal and pathogen DNA ( Figure 4).
  • Endophyte extract AN596H completely inhibits the infection of E. siliculosus CCAP 1310/4 by E. dicksonii CCAP 4018/1, at in all experiments preformed as assessed by microscopy. Subsequent quantification of infection via qPCR confirmed the visual scoring via microscopy ( Figure 3). Thus, the ACt value obtained by the molecular quantification allows obtaining very similar results to those obtained by the determination of the infection score as shown in Figure 5.
  • the value of the ACt is weak ( ⁇ 20) when infection of the alga is high and this value is high (> 20) when there is a weak infection or no infection
  • endophyte extract AN596H had inhibitory activity in a wide range since it prevents infection of E. siliculosus by four different pathogen strains belonging to the oomycete, hyphochytriale and phytomyxea, i.e., the two E. dicksonii, A. ectocarpii and M. ectocarpii ( Figure 3).
  • the endophyte strain AN596H obtained from the inner tissue of Ascophyllum nodosum was identified as belonging to Phaeosphaeria genus according to ITS sequences analysis.
  • a volume of 5 mL of fungal suspension (15 days pre-cultivated mycelium spread in sterilized artificial sea water) were added to 5 erlenmeyers flask containing each 600 mL of solid medium TUA+ASW+AN (for 1 L: 1 g yeast extract, 30 g monohydrated D-glucose, 1 g bactopeptone, 1 g potassium phosphate dibasic, 0.5 g magnesium sulfate, 0.01 g iron(II) sulfate, 5 g of dried and lyophilized powder of A.
  • nodosum 20 g agar dissolved in 8:2 ASW/disnHed water, pH 7-8).
  • Whole fungal cultures grown for 24 days at 18°C under natural light were then extracted three times by 5 L of ethyl acetate under mechanical agitation for 4 hours.
  • the pooled EtOAc fractions were dried over anhydrous magnesium sulfate, filtrated on filter paper N°l and concentrated under vacuum.
  • 2.3 g of dried crude extract were redissolved in a minimal volume of methanol and subjected to sephadex LH20 column (diameter 3 cm, length 1.10 m, containing crosslinked dextran gel) eluting with 100% methanol (Figure 6).
  • the fraction was then subjected to HPLC on a bonded silica Zorbax XDB-C18 column (21.2 mm x 150 mm, 5 ⁇ , Agilent) under 120 bar, flowrate of 10 mL/min and detection at 254 nm.
  • the elution occurred with buffer A (95:5 Water+0.5% TFA / ACN) and buffer B (5:95 Water+0.5% TFA / ACN) according the following gradient method: starting at 95/5 of A/B, 75/25 of A/B at 10 min, 50/50 of A/B at 20 min, 10/90 of A/B at 24 min and 95/5 of A/B at 27 min.
  • 11 sub-fractions were isolated as indicated on the chromatographic profile ( Figure 7).
  • the pure compounds MV-596-C5-1, MV-596-C5-3, MV-596-C5-8 and MV- 596-C5-11 were analyzed by NMR.
  • the sub fraction C5-5 was chromatographed on a gel column of Sephadex LH20 eluting with 100% methanol to provide two pure products, MV-596-C5-5-1 (1 mg) and MV-596-C5- 5-4 (1.8 mg) which were also analyzed by NMR.
  • the compound MV-596-C5-8 was isolated as a white amorphous solid.
  • the molecular formula was deduced from the protonated ions at m/z 211.0942 [M+H] + and at m/z 233.0792 [M+H] + in HR-ESI-MS.
  • This compound in solution in CD 3 OD was analysed by ID and 2D RMN and the data of this compound in CD 3 OD are shown in Table 1.
  • the 1 spectrum of this compound in CD 3 OD (400 MHz) showed sx 1.65 (2H, This compound is pyrenocine N.
  • the compound MV-596-C5-3 was isolated as a white amorphous solid.
  • the molecular formula was deduced from the protonated ions at m/z 209.0768 [M+H] + in HR-ESI-MS.
  • This compound in solution in CD 3 OD was analysed by ID and 2D RMN and the 1H and 13 C NMR data of this compound in CD 3 OD are shown in Table 2.
  • the 1H NMR spectrum of this compound in CD 3 OD (600 MHz) showed 12 protons: s
  • the compound MV-596-C5-1 was isolated as a white amorphous solid.
  • the molecular formula was deduced from the protonated ions at m/z 227.0890 [M+H] + in HR-ESI-MS.
  • This compound in solution in CD 3 OD was analysed by ID and 2D RMN and the 1H data of this compound in CD 3 OD are shown in Table 3.
  • the 1H NMR spectrum of this compound in CD 3 OD (400 MHz) showed 13 protons: s 5.50
  • the compound MV-596-C5-5-4 was isolated as a white amorphous solid.
  • the molecular formula was deduced from the protonated ions at m/z 241.1020 [M+H] + in HR-ESI-MS.
  • This compound in solution in CD 3 OD was analysed by ID and 2D RMN and the 1 H and 13 C NMR data of this compound in CD 3 OD are shown in Table 4.
  • the 1 H NMR spectrum of this compound in CD 3 OD (400 Mhz) showed 16 protons:
  • the compound MV-596-C5-11 was isolated as a white amorphous solid.
  • the molecular formula was deduced from the protonated ions at m/z 237.1089 [M+H] + and at m/z 259.0893 [M+H] + in HR-ESI-MS.
  • This compound in solution in CD 3 OD was analysed by ID and 2D RMN and the 1 H and 13 C NMR data of this compound in CD 3 OD are shown in Table 5.
  • the molecules characterized previously were tested against the algal pathogens according to the same protocol as described in Example 1 and the degree of infection was determined by microsopy and in some instances by qPCR as well.
  • the pyrenocines A and B showed an inhibition of the infection by the protists
  • Table 6 Activities of pyrenocine compounds against pathogens of the brown alga E. siliculosus.
  • Table 7 Activities of pyrenocine compounds and pyrenochaetic acid C towards pathogens of alga Pyropia yesoensi.
  • Pyrenocaetic acid C compound was also tested against the oomycete Pythium porphyrae, in alga Pyropia yesoensis, belonging to the division Rhodophyta, at the final concentration of 100 ⁇ g/ml.
  • pyrenochaetic acid C can stop the growth of the oomycete Pythium porphyrae mycelium in alga Pyropia yesoensis and induces the degeneration of Pythium porphyrae three days after treatment. No zoosporangium was formed after the treatment.
  • pyrenocines A, B, E and N did not show any critical changes in morphology, growth and reproduction on Pythium porphyrae, after observation by microscopy.

Abstract

The present invention relates to the use of a pyrenocine compound or a pyrenochaetic acid compound for the prevention or the treatment in algae of diseases induced by protistan pathogens. The present invention also relates to a new pyrenocine compound.

Description

PREVENTION OR TREATMENT IN ALGAE OF DISEASES INDUCED BY
PROTISTAN PATHOGENS
The present invention relates to the use of a pyrenocine compound or a pyrenochaetic acid compound for the prevention or the treatment in algae of diseases induced by a broad spectrum of protistan pathogens. The present invention also relates to a new pyrenocine compound.
Algae, in particular marine algae belonging to the class Phaeophyceae or the division Rhodophyta play a fundamental role in coastal ecosystems and form the first link in the food chain. These two classes of algae are complex multicellular photosynthetic eukaryotes.
Agars, carrageenan and alginates obtained from Phaeophyceae or Rhodophyta are hydrocolloids, which are present in a variety of consumer products. 55 000 tones thereof are produced annually. The market value is estimated at US $ 585 million. Alginate and other cell wall polymers are used in pharmaceutical, cosmetic and food products. The market value is estimated at US $ 213 million. In medicine, the alginates are used in the manufacture of biomaterials and implants. They are also components of toothpaste, ointment for treatment of wounds and burns, compresses and dental implants. Mannitol obtained from Phaeophyceae is a polyol used in solution for treating renal failure, increased intracranial pressure and for determining the hematocrit of a patient or for diagnosing a vasodilatation. Phloroghicinol, extracted from brown algae has antispasmodic and analgesic effects and is used in the treatment of digestive, urinary and gynecological spasmodic pain. Laminarin, obtained by extraction of the alga Laminaria digitata, belonging to the genus Laminaria belonging to the class of Phaeophyceae is used in priming plant defenses against diseases, for example the treatment of septoria in wheat and in the treatment of mildew in barley.
Red algae belonging mainly to the genus Pyropia and Porphyra (laver, also known as Nori in Japan) of the division of Rhodophyta is an important marine crop, extensively cultivated in Japan, Korea, and other oriental countries, with a market currently worth over US $ 1 billion per year and which accounts for 60% of the entire seaweed industry. Pyropia yezoensis (formerly known as Porphyra yezoensis) is the most economically valuable species of Pyropia due to its high quality.
Comparable with other living organisms, these marine algae are subject to several diseases caused by fungi, protists, bacteria or viruses and the recent development of intensive and dense marine culture practices has enabled some new diseases to spread much faster than before (Kim et al., 2014). As aquaculture continues to rise worldwide, pathogens of algae are becoming a significant economic burden (Gachon etal., 2010).
The exploration of pathogens in Phaeophyceae and Rhodophyta is steadily growing.
Protists are a polyphyletic group encompassing all eukaryotes, to the exclusion of animals, plants and fungi. Protists are eukaryotic organisms with typically unicellular, filamentous or parenchymatous organization. They have little or no vegetative tissue differentiation, except for reproduction (Adl et al, 2007).
Protists include the clade SAR. The name SAR derives from the acronym of the three groups composing this clade: Stramenopiles, Alveolata and Rhizaria. The Stramenopiles comprise oomycete and hyphochytrid micro-organisms and Rhizaria comprises phytomyxean micro-organisms (e.g. Plasmodiophora micro-organisms) (Adl et al, 2012).
Oomycetes are among the pathogens responsible of the algal diseases previously cited. Oomycetes are fungus-like organisms widely present in the phycosphere (i.e., the physicochemical environment in which an alga lives) and constitute a major cause of disease in phycoculture and against which no treatment is truly effective. For example, serious damage is caused by the oomycete pathogens, Pythium porphyrae and Olpidiopsis spp.fsubspecies), which decrease the productivity of Pyropia and Porphyra sea farms. In Seocheon sea farms, an outbreak of Olpidiopsis spp. disease resulted in approximately US $ 1.6 million in loss, representing approximately 24.5% of total sales during the 2012-2013 season (Kim et al, 2014). Olpidiopsis disease also concerns brown algae, for example Undaria pinnatifida (Akiyama et al, 1977).
In laboratory cultures, the oomycete Eurychasma dicksonii infects virtually all species of brown algae tested. Phylogenetically, E. dicksonii represents the most basal member of the oomycete lineage. It is frequently found infecting brown algae in the field, throughout the world. In particular, E. dicksonii infects the genome model seaweed Ectocarpus siliculosus (Gachon et al., 2009).
Oomycetes propagate and infect their host through flagellated zoospores, that are typically biflagellated. Marine genera such as Eurychasma and Olpidiopsis are obligate biotrophs, that penetrate their host and develop intracellularly. The penetration mechanism has been well described in the oomycete Eurychasma dicksonii, which cannot live without its host. The infection mechanism comprises: adhesion of a flagellate zoospore to the host cell surface, encystment on the surface, formation of an appressorial pad, germination of the cyst and penetration into the host cell. The thallus grows intracellularly, undergoes synchronised nuclear divisions to form a syncytium, and typical of holocarpic pathogens, ultimately converts itself into a sporangium that releases new propagules. (Uppalapatti et al., 1999; Tsigogoti et al, 2015; Sekimoto et al, 2008a). Likewise, Olpidiopsis has an intracellular, development cycle, well understood on the pathogens of Pyropia and Porphyra, Olpidiopsis pyropiae and Olpidiopsis porphyrae (Sekimoto et al., 2008b; Klochkova et al., 2015). Olpidiopsis pyropiae infects Pyropia yezoensis, and is responsible for the red rot disease.
In contrast, Pythium is an oomycete genus containing over a hundred species with contrasting lifestyle ranging from saprophytes to parasites of plants, mammals, and marine algae (Levesque et al, 2004). Pythium porphyrae, which belongs to the same species complex as Pythium chondricola, form a germ tubes and appressoria, and colonise the thallus of their host with hyphae (Kazama et al, 1977). In the same Pythium genus, P. marinum also grows on Pyropia and closely related red algae.
The host specificity of oomycetes which infect marine algae is poorly studied, primarily because extensive culture collections of algae and pathogens are necessary to carry out such investigations (West et al., 2006). Due to the lack of such biological resources, the taxonomic affiliation of many protistan pathogens of algae remains imperfectly known, with only scarce, decade- or century-old records based on morphological observations. In particular thanks to the rapid expansion of aquaculture worldwide and the diversification of farmed algal species, novel pathogen species keep being described at a very high rate.
Hyphochytriales share many similar morphological features with obligate biotrophic oomycetes such as Olpidiopsis oomycetes, but are characterised by a single anterior flagellum.
In laboratory cultures, Anisolpidium ectocarpti, a hyphochytrid pathogen originally isolated from Ectocarpus sp., infects over 20 brown algae species, including the kelp Macrocystis pyrifera.
The brown algae belonging to the class of Phaeophyceae are also infected by protistan pathogens that are only distantly or unrelated to oomycetes. For example, large kelps such as Durvillea antartica (Goecke et al., 2012) were infected by phytomyxea (Phytomyxea, Rhizaria (Adl et al, 2012)) pathogens, closely related to another pathogen of Ectocarpus, namely Maullinia ectocarpi (Maier et al, 2000).
In laboratory cultures, Maullinia ectocarpii is also able to infect multiple brown algal species belonging to the class Phaeophyceae, including the kelp Macrocystis pyrifera.
Phytomyxea are also biotrophic parasites that alternate the production of two types of intracellular Plasmodia (sporogenic and sporangia!). These plasmodia result from the development of biflagellated primary and secondary zoospores. Hie Plasmodium derived from a secondary zoospore forms resting spores.
No cure or treatment has been described to control or eradicate infections caused by phytomyxea in marine algae.
In aquaculture, exposure to air and acid wash are a common treatment to eliminate pathogens from cultivated red algae. Depending on the causative agent, they are at best partially effective. In particular, acid washing is only partially effective against Pythium porphyrae and ineffective against the Olpidiopsis diseases of laver. In Korea, acid washing is banned because of its negative environmental impact (Kim etal., 2014).
It remains a need to provide new, effective and environmentally- friendly compounds and methods for the prevention or the treatment in algae of diseases induced by protistan pathogens (microorganisms), in particular in brown algae belonging to the class Phaeophyceae and red algae belonging to the division Rhodophyta.
Pyrenocines and pyrenochaetic acids are mycotoxins produced by fungi isolated from plants or algae. Pyrenocine compounds are pyran-2-one derivatives, substituted at position 4 by a methoxy group, at position 5 by a saturated or unsaturated, substituted or unsubstituted butyryl group and said pyran-2-one derivatives being further substituted at position 6 by a substituted or unsubstituted alkyl chain (such as a methyl group). Pyrenochaetic acid compounds are benzoic acid derivatives, substituted at position 3 by a methoxy group, at position 4 by a saturated or unsaturated, substituted or unsubstituted butyryl group and said benzoic acid derivatives being further substituted at position 5 by a methyl group. Some of these compounds are reported to have antibacterial, antifungal or algicidal properties. Pyrenocines A and B and pyrenochaetic acids A, B and C have been isolated from culture filtrates of Pyrenochaeta terrestris, the causal fungus of onion pink root disease (Sato et al., 1981). Pyrenocines A, B, D and £ are produced by a strain of Penicillium waksmanii Zaleski OUPS-N133 separated from the brown alga Sargassum ringgoldianum (Amagata et al, 1998). More recently, pyrenocines J, K, L and M have been isolated from the fungus Pkomopsis sp., from the plant Cistus salvifolius internal strain 7852 (Hidayat et al, 2012). Methods for obtaining these pyrenocine and pyrenochaetic acid are described in Sato et al, 1981, Amagata et al., 1998 and Hidayat et al., 2012. Pyrenocines A, B and pyrenochaetic acid A have also been obtained by chemical synthesis (Ichihara et al., 1981 and Ichihara et al., 1983). The biological activity of these pyrenocine compounds has already been described against fungal and bacterial pathogens. For example, pyrenocine A inhibits the mycelium growth of Fusarium oxysporum, the spores germination of F. oxysporum forma specialis Cepae (F. oxysporum f. sp. Cepae), F, solani f. sp. pisi, Mucor hiemalis and Rhizopus stolonifer. Pyrenocine A also inhibits the growth of positive- gram bacteria such as Bacillus subtilis, Staphylococcus aureus and Escherichia coli (Sparace et al., 1987). The cytotoxicity of pyrenocines A, B and E has also been tested in a lymphocytic leukemia test system (Amagata et al, 1998). The biological activities of pyrenocines J, K, L and M have been assessed against bacteria, plant fungi, plant fungi-like and algae (Hidayat et al, 2012). However, to the knowledge of the inventors, the anti-oomycete, anti-hypho chytrid and anti -phytomyxean activities of pyrenocine compounds and pyrenochaetic acids has never been reported in algae.
The inventors have found that pyrenocine compounds and pyrenochaetic acid C compounds have a broad-spectrum anti-protistan activity, in particular an anti- oomycete, anti-hyphochytrid and/or anti-phytomyxean activity (i.e., has properties useful for preventing or treating diseases induced by oomycete pathogens, hyphochytrid pathogens or phytomyxean pathogens), in algae, in particular, brown algae belonging to the class Phaeophyceae and red algae belonging to the division Rhodophyta. The inventors have also isolated a new compound belonging to the pyrenocine family, called pyrenocine N having an anti-oomycete activity.
Accordingly, the present invention provides the use of a pyrenocine compound or a pyrenochaetic acid compound for the prevention or the treatment in an alga, preferably in a brown alga belonging to the class Phaeophyceae or a red alga belonging to the division Rhodophyta, of a disease induced by a protistan pathogen (microorganism).
The present invention also provides a method for preventing or treating a disease induced by a protistan pathogen (microorganism) in an alga, preferably in a brown alga belonging to the class Phaeophyceae or a red alga belonging to the division Rhodophyta, comprising contacting an effective amount of a pyrenocine compound or a pyrenochaetic acid compound with said alga or portion thereof.
In a particular embodiment of the present invention, the alga is in a culture pool. "An effective amount" refers to an amount of a pyrenocine compound or a pyrenochaetic acid compound which is effective in preventing or treating in an alga a disease induced by a protistan pathogen. One skilled in the art can determine what an effective amount is appropriate for preventing or treating a disease induced by a protistan pathogen.
The term "treating" refers to the inhibition of the infection by a protistan pathogen in an alga, by slowing down or stopping the growth of the pathogen, and/or inducing the death of the pathogen. The term "preventing" refers to the induction of the resistance to an infection by a protistan pathogen in an alga (i.e. inhibition of the appearance of an infection).
"A pyrenocine compound" is a pyran-2-one derivative, substituted at position 4 by a methoxy group, at position 5 by a saturated or unsaturated, substituted or unsubstituted butyryl group and said pyran-2-one derivative being further substituted at position 6 by substituted or unsubstituted alkyl chain. Said alkyl chain is preferably a methyl group. It includes pyrenocine A, B, D, E, F, G, H, J, K, L, M and N.
"A pyrenochaetic acid compound" is a benzoic acid derivative, substituted at position 3 by a methoxy group, at position 4 by a saturated or unsaturated, substituted or unsubstituted butyryl group and said benzoic acid derivative being further substituted at position 5 by a methyl group. It includes pyrenochaetic acid A, B and C.
Examples of pyrenocine compounds and pyrenochaetic acid compounds are illustrated in Sato et al, 1981, Amagata et al., 1998, and Hidayat et al, 2012.
In a preferred embodiment the pyrenocine compound is selected from the group consisting of pyrenocine A, pyrenocine B, pyrenocine E and pyrenocine N.
In another preferred embodiment the pyrenochaetic acid compound is pyrenochaetic acid C.
Preferred pyrenocine and pyrenochaetic acid compounds of the present invention have the following formula:
Figure imgf000007_0001
Figure imgf000008_0001
A pyrenocine can be used alone or in combination with one or more other pyrenocine and/or pyrenochaetic acid. In a same way, pyrenochaetic acid, in particular pyrenochaetic acid C, can be used alone or in combination with one or more pyrenocine.
Advantageously, these compounds are isolated from a Phaeosphaeria sp. strain in particular the strain Phaeosphaeria sp. AN596H, which is an endophyte fungus, isolated from Ascophyllum nodosum, a brown alga belonging to the class Phaeophyceae.
The strain Phaeosphaeria sp. AN596H was deposited according to the Budapest Treaty at CNCM (Collection Nationale de Culture de Microorganismes, 25 rue du Docteur Roux, 75724 Paris Cedex 15) on January 14, 2016, under the number CNCM 1-5041.
Diseases induced by a protistan pathogen include red rot disease, "chytrid blight" disease, preferably red rot disease.
Protist responsible of a disease in a red alga and/or a brown alga include:
Olpidiopsis subspecies {Olpidiopsis spp.), Eurychasmopsis spp., Petersenia spp., Pleotrachelus spp., Labyrinthomyxa sauvageaui, Olpidium spp., Sirolpidium spp., Phagomyxa algarum, Phagomyxa bellerocheae, Phagomyxa odontellae, Maullinia ectocarpii and closely related, incompletely described, species of phytomyxea, Anisolpidium ectocarpii, Anisolpidium sphacellarum, Anisolpidium joklianum and Anisolpidium rosenvingii.
The brown alga belonging to the class Phaeophyceae includes the alga of the following orders: Laminariales, Fucales, Ectocarpales, Ascoseirales, Desmarestiales, Dictyotales, Nemodermatales, Ralfsiales, Scytothamnales, Sphacelariales, Sporochnales, Syringodermatales, Tilopteri dales and Disco sporangiales.
The order Laminariales comprises the following families: Laminariaceae, Lessoniaceae and Alariaceae. The order Fucales comprises the family Fucaceae, Hormosiraceae and Durvilleaceae.
The order Ecto carpal es comprises the family Ectocarpaceae.
The order Ectocaipales and more precisely the species Ectocarpus siliculosus is phylogenetically close to the species belonging to the order Laminariales.
The family Laminariaceae comprises the following genera: Laminaria, Saccharina, Macrocystis and Nereocystis.
The family Lessoniaceae comprises the following genera: Ecklonia and Eisenia. The family Alariaceae comprises the following genera: Undaria and Alaria. The family Fucaceae comprises the following genera: Ascophyllum and
Pelvetia.
The family Hormosiraceae comprises the genus Hormosira.
The family Durvilleaceae comprises the genus Durvillea.
The family Ectocarpaceae comprises the genus Ectocarpus.
The genus Laminaria comprises the following species: Laminaria digitata,
Laminaria hyperborea and Laminaria longissima.
The genus Saccharina comprises the following species: Saccharina japonica and Saccharina latissima.
The genus Macrocystis comprises the following species: Macrocystis pyrifera and Macrocystis pomifera.
An example of Macrocystis pyrifera is the strain deposited at CCAP (The Culture Collection of Algae and Protozoa, Scottish Marine institute, OBAN, Argyll PA37 1QA Scotland), under the number CCAP 1323/1.
The genus Nereocystis comprises the species Nereocystis luetkeana.
The genus EcMonia comprises the following species: Ecklonia kurome and
Ecklonia radiata.
The genus Eisenia comprises the species Eisenia bicyclis {Ecklonia bicyclis). The genus Undaria comprises the following species: Undaria pinnatifida, Undaria crenata, Undaria undarioides and Undaria peterseniana.
The genus Alaria comprises the species Alaria esculenta.
The genus Ascophyllum comprises the species Ascophyllum nodosum.
The genus Pelvetia comprises the species Pelvetia canaliculata.
The genus Durvillea comprises the following species: Durvillea antartica and Durvillea poha.
The genus Ectocarpus comprises the following species: Ectocarpus siliculosus,
Ectocarpus fasciculatus, Ectocarpus crouaniorum, Ectocarpus subulatus and Ectocarpus mitchellae. An example of Ectocarpus siliculosus is the strain deposited at the CCAP, under the number CCAP 1310/4.
The brown alga is preferably selected from the genus Laminaria, Saccharina, Ascophyllum, Pelvetia and Ectocarpus, more preferably selected from the species Laminaria digitata, Saccharina latissima, Ascophyllum nodosum, Pelvetia canaliculata and Ectocarpus siliculosus.
The red alga belonging to the division Rhodophyta includes the alga of the following orders: Bangiales, Palmariales, Gelidiales, Gracilariales, Nemaliades and Gigartinales.
The order Bangiales comprises the family Bangiaceae.
The order Palmariales comprises the family Palmariaceae.
The order Gelidiales comprises the family Gelidiaceae.
The order Gracilariales comprises the family Gracilariaceae.
The order Nemaliades comprises the family Bonnemaisoniaceae.
The order Gigartinales comprises the family Solieriaceae.
The family Bangiaceae comprises the following genera: Pyropia and Porphyra.
The family Palmariaceae comprises the genus Palmaria.
The family Gelidiaceae comprises the genus Gelidium.
The family Gracilariaceae comprises the following genera: Gracilaria and Gracilariopsis.
The family Bonnemaisoniaceae comprises the genus Asparagopsis.
The family Solieriaceae comprises the following genera: Kappaphycus and Eucheuma.
The genus Pyropia comprises the following species: Pyropia abbottiae, Pyropia acanthopohora, Pyropia aeodis, Pyropia bajacaliforniensis, Pyropia brumalis, Pyropia cinnamomea, Pyropia columbiensis, Pyropia columbina, Pyropia conwayae, Pyropia crassa, Pyropia dentate, Pyropia denticulate, Pyropia elongate, Pyropia endiviifolia, Pyropia fallax, Pyropia francissii, Pyropia fiicicola, Pyropia gardneri, Pyropia haitanensis, Pyropia hiberna, Pyropia hollenbergii, Pyropia ishigecola, Pyropia kanakaensis, Pyropia katadae, Pyropia kinositae, Pyropia koreana, Pyropia kuniedae, Pyropia kurogii, Pyropia lacerate, Pyropia lanceolate, Pyropia leucosticta, Pyropia montereyensis, Pyropia moriensis, Pyropia nereocystis, Pyropia nitida, Pyropia njordis, Pyrpopia onoi, Pyropia orbicularis, Pyropia parva, Pyropia peggicovensis, Pyropia pendula, Pyropia perforate, Pyropia plicata, Pyropia protolanceolata, Pyropia pseudolinearis, Pyropia pulchella, Pyropia rahura, Pyropia raulaguilarii, Pyropia saldanhae, Pyropia seriata, Pyropia smithii, Pyropia spiralis, Pyropia suborbiculata, Pyropia tanegashimensis, Pyropia tenera, Pyropia tenuipedalis, Pyropia thuaea, Pyropia thuretii, Pyropia torta, Pyropia vietnamensis, Pyropia virididentata and Pyropia yezoensis .
The genus Porphyra comprises the following species: Porphyra akasakae, Porphyra angusta, Porphyra argentinensis, Porphyra atropurpurea, Porphyra augustinae, Porphyra autumnalis, Porphyra bangiaeformis, Porphyra bulbopes, Porphyra capensis, Porphyra cornea, Porphyra ceylanica, Porphyra chauhanii, Porphyra conwayae, Porphyra corallicola, Porphyra cordata, Porphyra cucullata, Porphyra cuneiformis, Porphyra delicatula, Porphyra dentimarginata, Porphyra diocia, Porphyra drachii, Porphyra fiijianensis, Porphyra gardneri, Porphyra grateloupicola, Porphyra grayana, Porphyra guangdongensis, Porphyra hospitans, Porphyra inaequicrassa, Porphyra indica, Porphyra ionae, Porphyra irregularis, Porphyra kanyakumariensis, Porphyra kinositae, Porphyra lanceolate, Porphyra ledermannii, Porphyra linearis, Porphyra lucasii, Porphyra maculosa, Porphyra malvanensis, Porphyra marcosii, Porphyra marginata, Porphyra martensiana, Porphyra microphylla, Porphyra minima, Porphyra minor, Porphyra monosporangia, Porphyra mumfordii, Porphyra njordii, Porphyra nobilis, Porphyra ochotensis, Porphyra okamurae, Porphyra okhaensis, Porphyra oligospermatangia, Porphyra perforate, Porphyra plocamiestris, Porphyra pseudocrassa, Porphyra pudica, Porphyra pujalsiae, Porphyra pulchra, Porphyra punctate, Porphyra purpurea, Porphyra quingdaoensis, Porphyra ramosissima, Porphyra reniformis, Porphyra rizzinii, Porphyra roseana,Porphyra schistothallus, Porphyra segregate, Porphyra subtumentus, Porphyra tanakae, Porphyra tenius, Porphyra tenuissima, Porphyra tristanensis, Porphyra umbilicalis, Porphyra umbiliculata, Porphyra violacea, Porphyra vulgaris and Porphyra woolhouseae.
The genus Palmaria comprises the following species: Palmaria callophylloides,
Palmaria centrocarpa, Palmaria decipiens, Palmaria georgica, Palmaria hecatensis, Palmaria integrifolia, Palmaria marginicrassa, Palmaria mollis, Palmaria moniliformis, Palmaria palmata and Palmaria stenogona.
The genus Gelidium comprises the following species: Gelidium amansii, Gelidium robustum, Gelidium chilense, Gelidium sesquipedale, Gelidium pristoides and Gelidium canariense.
The genus Gracilaria comprises the following species: Gracilaria chilensis, Gracilaria gracilis, Gracilaria gigas, Gracilaria tenuisti, Gracilaria edulis, Gracilaria multipartita and Gracilaria bursa-pastoris,
The genus Gracilariopsis comprises the following species: Gracilariopsis lemaneiformis, Gracilariopsis longissimi, Gracilariopis andersonii and Gracilaiopsis sjoestedtii. The genus Asparagopsis comprises the following species: Asparagopsis armata, Asparagopsis svedelii and Asparagopsis taxiformis.
The genus Kappaphycus comprises the following species: Kappaphycus alvarezii, Kappaphycus cottonii, Kappaphycus inermis, Kappaphycus malesianus, Kappaphycus procrusteanus and Kappaphycus striatus.
The genus Eucheuma comprises the following species: Eucheuma adhaerens, Eucheuma amakusaense, Eucheuma arnoldii, Eucheuma cartilagineum, Eucheuma cervicorne, Eucheuma chondriforme, Eucheuma crassum, Eucheuma crustiforma, Eucheuma deformans, Eucheuma denticulatum, Eucheuma dichotonum, Eucheuma edula, Eucheuma horizontals Eucheuma horridum, Eucheuma isiforme, Eucheuma johnstonii, Eucheuma jugata, Eucheuma kraftianum, Eucheuma leeuwenii, Eucheuma nodulosum, Eucheuma nudum, Eucheuma odontophorum, Eucheuma perplexum, Eucheuma platycladum, Eucheuma serra, Eucheuma simplex, Eucheuma sonderi, Eucheuma vermiculare and Eucheuma xishaensis.
In a preferred embodiment the red alga is Pyropia yezoensis or Porphyra purpurea.
The red and the brown alga of the present invention can be a natural or artificial hybrid between 2 species. For example such a hybrid can be a cultivar obtained from the crossing between Laminaria longissima x Saccharina japonica (Zhang et al., 2007) or obtained after a protoplast fusion between Pyropia yezoensis and Pyropia haitanensis (Dai etal, 1993).
The oomycete pathogen comprises:
- the following orders: Eurychasmales, Olpidiopsidales, Pythiales, Peronosporales and Myzocytiopsidales and,
- the following genera: Eurychasmidium, Petersenia, Pontisma, and
Sirolpidium, that are suspected to be closely related or indeed overlap with the aforementioned orders.
The order Eurychasmales comprises the genus Eurychasma.
The order Olpidiopsidiales comprises the genus Olpidiopsis.
The order Pythiales comprises the genus Pythium.
The order Peronosporales comprises the following species: Pythium porphyrae, Pythium chondricola and Pythium marinum.
The order Myzocytiopsidales comprises the following genera: Pontisma, Petersenia and Sirolpidium.
The genus Eurychasmidium comprises the following species: Eurychasmidium joycei, Eurychasmidium sacculus and Eurychasmidium tumefaciens. The genus Petersenia comprises the following species: Petersenia lobota, Petersenia pollagaster and Petersenia palmariae.
The genus Pontisma comprises the following species: Pontisma antithamnionis, Pontisma dangeardii, Pontisma feldmanii, Pontisma inhabilis, Pontisma lagenidioles and Pontisma magnusii.
The genus Sirolpidium comprises the species Sirolpidium ectocarpii.
The genus Eurychasma comprises the species Eurychasma dicksonii.
Examples of Eurychasma dicksonii are the strains CCAP 4018/1 and the CCAP 4018/3.
The genus Olpidiopsis comprises the following species: Olpidiopsis bostrychiae, Olpidiopsis feldmannii, Olpidiopsis porphyrae, Olpidiopsis pyropiae, Olpidiopsis antithamnionis, Olpidiopsis dangeardii, Olpidiopsis magnusii, and Olpidiopsis tumefaciens.
The genus Pythium comprises the species Pythium porphyrae.
The oomycete pathogen (microorganism) is preferably selected from the species
Eurychasma dicksonii and Olpidiopsis pyropiae.
Eurychasma dicksonii infects a brown alga belonging to the class Phaeophyceae, more particularly, belonging to the genus Laminaria, Saccharina, Macrocystis, and Ectocarpus. Eurychasma dicksonii infects the species such as Macrocystis pyrifera, Laminaria digitata, Saccharina latissima, and Ectocarpus siliculosus, preferably, Laminaria digitata, Saccharina latissima, and Ectocarpus siliculosus, more preferably Ectocarpus siliculosus.
Olpidiopsis pyropiae infects a red alga belonging to the division Rhodophyta, preferably the family Bangiaceae, more preferably the species such as Pyropia yezoensis and Pyropia haitanensis, and even more preferably Pyropia yezoensis.
The hyphochytriale comprises the family Anisolpidiaceae (Adl et al, 2012). The family Anisolpidiaceae comprises the genus Anisolpidium.
The genus Anisolpidium comprises the following species: Anisolpidium ectocarpii, Anisolpidium joklianum, Anisolpidium rosenvingii, Anisolpidium minutum, Anisolpidium olpidium and Anisolpidium sphacellarum.
An example of Anisolpidium ectocarpii is the strain Anisolpidium ectocarpii CCAP 4001/1.
Anisolpidium ectocarpii infects a brown alga belonging to the class Phaeophyceae, more particularly, belonging to the genus Laminaria, Saccharina, and Ectocarpus. Anisolpidium ectocarpii infects the species such as Laminaria digitata, Saccharina latissima, Ectocarpus mitchellae and Ectocarpus siliculosus, preferably, Laminaria digitata, Saccharina latissima, Ascophyllum nodosum, Pelvetia canaliculate and Ectocarpus siliculosus, more preferably Ectocarpus siliculosus.
The hyphochytrid pathogen (microorganism) is preferably the species Anisolpidium ectocarpii.
The phytomyxea includes the following genera: Maullinia, Phagomyxa and
Labyrinthula.
The genus Maullinia comprises the species Maullinia ectocarpii.
An example of Maullinia ectocarpii is the strain Maullinia ectocarpii CCAP 1538/1.
The genus Phagomyxa comprises the species Phagomyxa algarum
The genus Labyrinthula comprises the following species: Labyrinthula pohlia and Labyrinthula roscoffiensis.
Maullinia ectocarpii infects a brown alga belonging to the class Phaeophyceae, more particularly, belonging to the genus Laminaria, Saccharina, and Ectocarpus. Maullinia ectocarpii infects the species such as Laminaria digitata, Saccharina latissima, and Ectocarpus siliculosus, preferably, Laminaria digitata, Saccharina latissima, or Ectocarpus siliculosus, more preferably Ectocarpus siliculosus.
The phytomyxean pathogen (microorganism) is preferably the species Maullina ectocarpii.
In an advantageous embodiment of the present invention, the pyrenocine compound is selected from the group consisting of pyrenocine A and pyrenocine B and the protistan pathogen is selected from the group consisting of the genus Eurychasma and Anisolpidium, preferably selected from the species Eurychasma dicksonii and Anisolpidium ectocarpii. Preferably, the pyrerocine compound is the pyrenocine A and the protistan pathogen is selected from the species Eurychasma dicksonii and Anisolpidium ectocarpii.
In another advantageous embodiment of the present invention, the pyrenocine compound is selected from the group consisting of pyrenocine A, pyrenocine B and pyrenocine E and the protistan pathogen is of the genus Maullinia, preferably the species Maullinia ectocarpii. Preferably, the pyrerocine compound is the pyrenocine A and the protistan pathogen is the species Maullinia ectocarpii.
In another advantageous embodiment of the present invention, the pyrenocine or pyrenochaetic acid compound is selected from the group of pyrenocine A, pyrenocine N and pyrenochaetic acid C and the protistan pathogen is of the genus Olpidiopsiale, preferably the species Olpidiopsis pyropiae. Preferably, the pyrenocine compound is selected from pyrenocine A and pyrenochaetic acid C and the protistan pathogen is the species Olpidiopsis pyropiae. More preferably, the pyrenocine compound is the pyrenocine A and the protistan pathogen is the species Olpidiopsis pyropiae.
In another advantageous embodiment of the present invention, the pyrenochaetic acid compound is pyrenochaetic acid C and the protistan pathogen is of the genus Pythium, preferably the species Pythium porphyrae.
In one aspect of the present invention, pyrenocine A has an anti-protistan activity (i.e., has properties useful for preventing or treating, preferably treating, a disease induced by a protistan pathogen) against the following species of a protistan pathogen: Eurychasma dicksonii, Anisolpidium ectocarpii and Maullinia ectocarpii, in a brown alga belonging to the class Phaeophyceae, preferably in Ectocarpus siliculosus.
In another aspect of the present invention, pyrenocine A has an anti-oomycete activity against Olpidiopsis pyropiae, in a red alga belonging to the division Rhodophyta, preferably in Pyropia yezoensis.
In another aspect of the present invention, pyrenocine B has an anti-protistan activity against the following species of a protistan pathogen: Eurychasma dicksonii, Anisolpidium ectocarpii, Maullinia ectocarpii, in a brown alga belonging to the class Phaeophyceae, preferably in Ectocarpus siliculosus.
In another aspect of the present invention, pyrenocine E has an anti- phytomyxean activity against Maullinia ectocarpii, in a brown alga belonging to the class Phaeophyceae, preferably in Ectocarpus siliculosus.
In another aspect of the present invention, pyrenochaetic acid C has an anti- oomycete activity against a following species of oomycete pathogen: Olpidiopsis pyropiae and Pythium porphyrae, in a red alga belonging to the division Rhodophyta, preferably Pyropia yezoensis.
The pyrenocine compound or pyrenochaetic acid compound used for the prevention or the treatment, in a brown alga belonging to the class Phaeophyceae or in a red alga belonging to the division Rhodophyta, of a disease induced by a protistan pathogen can be formulated in a liquid, emulsion, foam, paste, powder or gel form.
The pyrenocine or pyrenochaetic acid compound as defined above can be included into a cyclodextrine. Methods of encapsulation of a compound into cyclodextrines are well-known in the art Munin et al, 2011 describes a method for encapsulating polyphenolic compounds into cyclodextins. Such method can apply for pyrenocine and pyrenochaetic acid compounds.
The pyrenocine or pyrenochaetic acid compound as defined above can be formulated as stable water-dispersible nanoassemblies of homogeneous size, as described in the art for derivatives of curcumin (Cheikh-Ali et al, 2015). The pyrenocine or pyrenochaetic acid compound as defined above can be in solution with a polar aprotic solvent or a polar protic solvent. The polar aprotic solvent is preferably selected from the group consisting of acetone, N,N- dimethylforrnamide (DMF), acetonitrile and dimethylsulfoxide (DMSO), more preferably dimethylsulfoxide (DMSO). The polar protic solvent is preferably selected from the group consisting of methanol, ethanol and butanol, more preferably methanol.
The concentration of a pyrenocine compound or a pyrenochaetic acid compound, used for the prevention or the treatment, in a brown alga belonging to the class Phaeophyceae, of a disease induced by a protistan pathogen (microorganism) is advantageously
Figure imgf000016_0003
preferably between
Figure imgf000016_0002
and 100 μ^ιηΐ, more preferably between 1 and 10 ug/ml.
The concentration of a pyrenocine compound or a pyrenochaetic acid compound, used for the prevention or the treatment, in a red alga belonging to the division Rhodophyta, of a disease induced by a protistan pathogen is advantageously between
Figure imgf000016_0004
and 200 μg/ml, preferably between 5 and more
Figure imgf000016_0006
preferably between 10 and
Figure imgf000016_0005
The pyrenocine compound or the pyrenochaetic compound can be applied once a day, for one week, every day, to repeat every month for 6 months.
The present invention also provides the isolated compound pyrenocine N, of formula:
Figure imgf000016_0001
The present invention also provides a composition, such as an anti -protistan composition, comprising the compound pyrenocine N.
Said composition can comprise a polar protic or aprotic solvent as described above.
Pyrenocine N can be formulated as stable water-dispersible nanoassemblies of homogeneous size as described above.
Pyrenocine N can also be included into a cyclodextrine as described above.
Pyrenocine N is obtainable by the method described below. The present invention also provides a method for obtaining the compound pyrenocine N, comprising the steps of:
a) provision of a culture of a strain of Phaeosphaeria, preferably the strain Phaeosphaeria sp. AN596H (CNCM 1-5041);
b) extraction of the metabolites from the culture by a solvent ;
c) separation of metabolites by chromatography;
d) isolation of the fraction corresponding to pyrenocine N, which is eluted between the pyrenocine A and the pyrenocine C. Preferably, the elution occurred with buffer A (95:5 Water+0.5% TFA / ACN) and buffer B (5:95 Water+0.5% TFA (Trifluoroacetic acid) / (ACN)) according the following gradient method: starting at 95/5 of A/B, 75/25 of A/B at 10 min, 50/50 of A/B at 20 min, 10/90 of A/B at 24 min and 95/5 of A/B at 27 min. In these conditions, the collected fraction at 18.3 min leads to the isolation of Pyrenocine N.
In step a) the strain of Phaeosphaeria can be cultured in a solid medium, such as the culture medium TUA+ASW+AN (described in Example 2 below).
In step b), the culture can be extracted with a solvent optionally under mechanical agitation. The solvent can be an organic polar aprotic solvent, such as, ethyl acetate and acetonitrile, preferably ethyl acetate or an organic polar prone solvent, preferably, methanol.
In step c) the chromatography can include an exclusion chromatography and/or
HPLC, preferably an exclusion chromatography followed by a HPLC. Preferably, the exclusion chromatography is performed on a column made of a gel filtration resin and elution with methanol. The column can be a silica gel column or a reverse-phase CI 8 silica gel column. For example, the column is made of Sephadex* G, Sephadex* LH, Bio-Gel* P, Bio-Gel* A, Sephacryl*, Sepharosee or Fractogel* TSK and more preferably the column is made of Sephadex* LH20. Preferably, the HPLC is performed on a stationary phase CI 8 column and elution with buffer A (95:5 Water+0.5% Trifluoroacetic acid (TFA) / Acetonitrile (ACN)) and buffer B (5:95 Water+0.5% TFA / ACN). For example, the stationary phase is a bonded silica, zirconium oxide, titan oxide or aluminum oxide CI 8 phases with polar, non polar endcapping, di-isobutyl side chains or reverse phases with phenyl, phenyl-hexyl or pentafluorophenyl and more preferably the stationary phase is a bonded silica Zorbax XBD CI 8 column. The method for obtaining the compound pyrenocine N is described in details in Example 2 below.
Pyrenocine N could also be obtained from pyrenocine B by hydrogenation.
The present invention also provides an isolated extract of a Phaeosphaeria strain comprising the compound pyrenocine N. Preferably, the Phaeosphaeria strain is isolated from Ascophyllum nodosum, in particular the strain Phaeosphaeria sp. AN596H (CNCM 1-5041).
The present invention also provides a Phaeosphaeria strain wherein it is the strain Phaeosphaeria sp. AN596H CNCM 1-5041, deposited according to the Budapest Treaty at CNCM on January 14, 2016.
In addition to the above features, the invention further comprises other features which will emerge from the following description, which refers to an example illustrating the present invention, as well as to the appended figures.
Figure 1 shows the experimental setup of test of a brown alga resistance against a protist pathogen.
Figure 2 shows the infection score assigned with the light microscopy observations.
Figure 3 shows the average scores of infection of Ectocarpus siliculosus obtained during tests of biological assessments of endophytic extract AN596H at 10
Figure imgf000018_0001
against E. dicksonii CCAP4018/1 (point), E. dicksonii CCAP4018/3 (grey), Maullinia ectocarpii (hatching), Anisolpidium ectocarpii (black) after counting by optical microscopy. Extracts from endophytic strains isolated and identified and the positive and negative controls are represented on the ordinate. The score scale is represented on the abscissa.
Figure 4 shows the DNA quantification using qPCR. (A) Amplification curves.
(B) Melting curves. On the ordinate is expressed the measure of the fluorescence (arbitrary units) and on the abscissa is expressed the number of selected amplification cycle.
Figure 5 shows the linear correlation between the values of ACt (ordinate) obtained by qPCR and microscopic scores (abscissa) obtained by optical microscopy observations of quantification of infection by E. dicksonii CCAP4018/1 and CCAP4018/3.
Figure 6 shows the steps of the protocol of isolation of the active compounds against a protist pathogen.
Figure 7 shows the chromatographic profile of fraction C5 comprising pyrenocine compounds and acid pyrenochaetic C, obtained by HPLC.
Figure 8 shows the average of scores obtained by microscopy scores of infection of Ectocarpus siliculosus by the different pathogens: E. dicksonii CCAP4018/1 (point), E. dicksonii CCAP4018/3 (grey), Maullinia ectocarpii (hatching), Anisolpidium ectocarpii (black) in the presence of purified pyrenocine compounds at 1 μg/ml. Figure 9 shows the average of ACt values obtained by quantification of E. dicksonii infection in E. siliculosus in the presence of pyrenocine A at a final concentration of 1 μg/ml.
EXAMPLE 1: EFFECT OF COMPOUNDS ON ALGAL PATHOGENIC PROTISTS
Materials and Methods
i. Cultivation of pathogens
Cultivation of the pathogen Eurychasma dicksonii was performed as previously described (Gachon et al, 2009). The pathogens Maullinia ectocarpii and Anisolpidium ectocarpii are maintained in an identical manner.
ii. Co-incubation with extracts
An extract of the endophytic isolated Phaeosphaeria sp. ANS96H (CNCM I- 5041) obtained from the inner tissue of Ascophyllum nodosum holdfast was diluted in DMSO at a concentration of 10 mg/ml and tested at a final concentration of
Tests were performed in 6 -well plates, in which 7 ml of culture medium and 7 μΐ of the endophyte extract ANS96H (final concentration: 10 μg/ml ) were added to each well (Figure 1). Subsequently, Ectocarpus siliculosus CCAP 1310/4 was added to each well. The inoculum Macrocystis pyrifera CCAP 1323/1 infected by the pathogen Eurychasma dicksonii CCAP 4018/1 was placed in a cell strainer with a pore size of 70 μιη and put into the well. Positive controls (omitting the addition of endophyte extract) and negative controls (omitting the pathogen-infected inoculum) were also added. Each sample was run in triplicates and the experiments were performed three times independently. The plates were sealed with parafilm and incubated at 15°C with a photoperiod of 12 hours (light intensity: for
Figure imgf000019_0001
15 to 20 days in order to develop infection symptoms.
The co-incubation with the pathogens E. dicksonii CCAP 4018/3, M. ectocarpii CCAP 1538/1 and A. ectocarpii CCAP 4001/1 was performed as described for E. dicksonii CCAP 4018/1. In case of A, ectocarpii the incubation time was 4 days.
iii. Determination of the degree of infection with a score scale
Following incubation with the pathogen, E. siliculosus was transferred to a microscope slide and analysed via microscopy (Zeiss Axiovert 2, DIC III apochromatic).
The degree of infection was determined by applying an arbitrary score scale based on the number of infected algal cells. Thus, a score of 0 was assigned if the algal culture was not infected (no infected algal cell), a score of 1 for low infection (1- 10 infected cells), a score of 2 for moderate infection (11-100 algal cells infected) and a score of 3 for a dense infection (> 101 algal cells infected) (Figure 2). iv. Quantification of the degree of infection by quantitative PCR (qPCR)
Ectocarpus material that was previously for microscopy scoring was then harvested, frozen in RNAlater® (Thermo Fisher) at - 80 °C buffer for analysis by qPCR.
The extraction of genomic DNA and qPCR was performed as previously described (Gachon et αί, 2009).
The Ct value ("threshold cycle"), which is the intersection between the amplification curve and a given fluorescence threshold line, allows measuring the relative concentration of amplicons of the target DNA.
The specific amplification of 18S rRNA genes from E. siliculosus and SSU rDNA of E. dicksonii was carried out using the primer pairs CG64 / CG65 and CG60 / CG61 respectively (Gachon et al, 2009).
The qPCR reactions were performed in triplicates on cyclers Quantica (technetium Barloworld, UK) and LightCycler® (Roche 96SW1.1, UK). The qPCR reaction consisted of 10 μΐ qPCR buffer (qPCR MasterMix Mesagreen Plus for SYBR Assay, Eurogentec), 1 μΐ of each sense and anti-sense primer at a final concentration of 300 nM and 8 μΐ of DNA in a final volume of 20 μΐ. The amplification program was as follows: denaturation for 10 min at 95°C and then 45 amplification cycles of 15 s at 95°C followed by incubation at 60°C for 1 min, with the reading of fluorescence to monitor the PCR reaction in real time.
The melting curve characteristics were obtained with a final cycle defined by an increase of temperature of 70 to 95°C (± 1°C every 20 seconds). The infection was quantified for each sample by calculating a ACt value (Eurychasma Ct - Ectocarpus Ct) obtained from the amplification of algal and pathogen DNA (Figure 4).
Results
Endophyte extract AN596H completely inhibits the infection of E. siliculosus CCAP 1310/4 by E. dicksonii CCAP 4018/1, at
Figure imgf000020_0001
in all experiments preformed as assessed by microscopy. Subsequent quantification of infection via qPCR confirmed the visual scoring via microscopy (Figure 3). Thus, the ACt value obtained by the molecular quantification allows obtaining very similar results to those obtained by the determination of the infection score as shown in Figure 5.
The value of the ACt is weak (<20) when infection of the alga is high and this value is high (> 20) when there is a weak infection or no infection
This extract was then evaluated on another strain of Eurychasma dicksonii CCAP 4018/3 and on Anisolpidium ectocarpii and Maullinia ectocarpii.
It has thus been demonstrated that endophyte extract AN596H had inhibitory activity in a wide range since it prevents infection of E. siliculosus by four different pathogen strains belonging to the oomycete, hyphochytriale and phytomyxea, i.e., the two E. dicksonii, A. ectocarpii and M. ectocarpii (Figure 3).
EXAMPLE 2; ISOLATION AND CARACTERISATION OF COMPOUNDS HAVING AN ANTI-PROTISTAN ACTIVITY
Materials and Methods
The endophyte strain AN596H obtained from the inner tissue of Ascophyllum nodosum was identified as belonging to Phaeosphaeria genus according to ITS sequences analysis. A volume of 5 mL of fungal suspension (15 days pre-cultivated mycelium spread in sterilized artificial sea water) were added to 5 erlenmeyers flask containing each 600 mL of solid medium TUA+ASW+AN (for 1 L: 1 g yeast extract, 30 g monohydrated D-glucose, 1 g bactopeptone, 1 g potassium phosphate dibasic, 0.5 g magnesium sulfate, 0.01 g iron(II) sulfate, 5 g of dried and lyophilized powder of A. nodosum, 20 g agar dissolved in 8:2 ASW/disnHed water, pH 7-8). Whole fungal cultures grown for 24 days at 18°C under natural light were then extracted three times by 5 L of ethyl acetate under mechanical agitation for 4 hours. The pooled EtOAc fractions were dried over anhydrous magnesium sulfate, filtrated on filter paper N°l and concentrated under vacuum. 2.3 g of dried crude extract were redissolved in a minimal volume of methanol and subjected to sephadex LH20 column (diameter 3 cm, length 1.10 m, containing crosslinked dextran gel) eluting with 100% methanol (Figure 6). It resulted in 72 fractions which were analyzed with TLC chromatography on Silicate plate Si60 F254 (Merck & Machery Nagel) with 1:1 cyclohexane/ethyl acetate eluting and revelation with sulfide vanillin (5mL H2S04, lOOmL ethanol 96°) at 200°C. The sub-fractions 34 to 41 displayed identical TLC profiles and were combined to yield a fraction of 82 mg which was dissolved in 300 \iL of methanol. The fraction was then subjected to HPLC on a bonded silica Zorbax XDB-C18 column (21.2 mm x 150 mm, 5 μιη, Agilent) under 120 bar, flowrate of 10 mL/min and detection at 254 nm. The elution occurred with buffer A (95:5 Water+0.5% TFA / ACN) and buffer B (5:95 Water+0.5% TFA / ACN) according the following gradient method: starting at 95/5 of A/B, 75/25 of A/B at 10 min, 50/50 of A/B at 20 min, 10/90 of A/B at 24 min and 95/5 of A/B at 27 min. 11 sub-fractions were isolated as indicated on the chromatographic profile (Figure 7).
The pure compounds MV-596-C5-1, MV-596-C5-3, MV-596-C5-8 and MV- 596-C5-11 were analyzed by NMR. The sub fraction C5-5 was chromatographed on a gel column of Sephadex LH20 eluting with 100% methanol to provide two pure products, MV-596-C5-5-1 (1 mg) and MV-596-C5- 5-4 (1.8 mg) which were also analyzed by NMR.
Results After elution, the collected fraction at 18.3 rnin lead to the isolation of new pyrenocine N (ra = 1.0 nig) characterized with spectroscopic technics.
The compound MV-596-C5-8 was isolated as a white amorphous solid. The molecular formula was deduced from the protonated ions at m/z 211.0942 [M+H]+ and at m/z 233.0792 [M+H]+ in HR-ESI-MS. This compound in solution in CD3OD was analysed by ID and 2D RMN and the
Figure imgf000022_0005
data of this compound in CD3OD are shown in Table 1. The1
Figure imgf000022_0004
spectrum of this compound in CD3OD (400 MHz) showed
Figure imgf000022_0003
sx 1.65 (2H,
Figure imgf000022_0002
This compound is pyrenocine N.
Table 1. 1H and 13C NMR data of pyrenocine N in CD3OD (1H 400.13 MHz, 13C 150 MHz, 298 K).
Figure imgf000022_0001
The compound MV-596-C5-3 was isolated as a white amorphous solid. The molecular formula was deduced from the protonated ions at m/z 209.0768 [M+H]+ in HR-ESI-MS. This compound in solution in CD3OD was analysed by ID and 2D RMN and the 1H and 13C NMR data of this compound in CD3OD are shown in Table 2. The 1H NMR spectrum of this compound in CD3OD (600 MHz) showed 12 protons: s
Figure imgf000022_0006
This
Figure imgf000022_0007
compound is pyrenocine A.
Table 2. 1H and 13C NMR data of pyrenocine A in CD3OD (Ή 600 MHz, ,3C 150 MHz, 298 K).
Figure imgf000023_0001
The compound MV-596-C5-1 was isolated as a white amorphous solid. The molecular formula was deduced from the protonated ions at m/z 227.0890 [M+H]+ in HR-ESI-MS. This compound in solution in CD3OD was analysed by ID and 2D RMN and the 1H data of this compound in CD3OD are shown in Table 3. The 1H NMR spectrum of this compound in CD3OD (400 MHz) showed 13 protons: s 5.50
Figure imgf000023_0003
(3H). This compound is pyrenocine B.
Table 3. 1H NMR data of pyrenocine B in
Figure imgf000023_0004
Figure imgf000023_0002
The compound MV-596-C5-5-4 was isolated as a white amorphous solid. The molecular formula was deduced from the protonated ions at m/z 241.1020 [M+H]+ in HR-ESI-MS. This compound in solution in CD3OD was analysed by ID and 2D RMN and the 1H and 13C NMR data of this compound in CD3OD are shown in Table 4. The 1H NMR spectrum of this compound in CD3OD (400 Mhz) showed 16 protons:
Figure imgf000024_0003
Figure imgf000024_0002
s 3.83 (3H), s 2.24 (3H), s 3.25 (3H). This compound is pyrenocine E.
Table 4. 1H and I3C NMR data of pyrenocine E in CD3OD (1H 600 MHz, 13C 150 MHz, 298 K).
Figure imgf000024_0001
The compound MV-596-C5-11 was isolated as a white amorphous solid. The molecular formula was deduced from the protonated ions at m/z 237.1089 [M+H]+ and at m/z 259.0893 [M+H]+ in HR-ESI-MS. This compound in solution in CD3OD was analysed by ID and 2D RMN and the 1H and 13C NMR data of this compound in CD3OD are shown in Table 5. The 1H NMR spectrum of this compound in CD3OD (400 Mhz) showed 15 protons: s 7.43 (1H), s 7.55 (1H), t 2.73 (2H), sx 1.70 (2H), t 0.99 (3H), s 3.86 (3H), s 2.26 (3H). This compound is pyrenocaetic acid C.
Table 5. 1H and l3C NMR data of pyrenochaetic acid C in CD3OD (1H 600 MHz, 13C 150 MHz, 298 K).
Figure imgf000025_0001
EXAMPLE 3: BIOLOGICAL ASSESSMENT OF MOLECULES HAVING
ANTI-PROTISTAN ACTIVITIES
Materials and Methods
The molecules characterized previously were tested against the algal pathogens according to the same protocol as described in Example 1 and the degree of infection was determined by microsopy and in some instances by qPCR as well.
Results
The pyrenocines A and B showed an inhibition of the infection by the protists
Ectocarpus dicksonii, Anisolpidium ectocarpii and Maullinia ectocarpii in all independent experiments at concentration of
Figure imgf000025_0002
An infection score of 0 close to that of the negative control (no pathogen) was observed for both compounds (Figure 8, Table 6). For Ectocarpus dicksonii, the scores obtained from incubation with Pyrenocine A were confirmed via qPCR (Figure 9).
Furthermore Pyrenocine E showed an inhibition of infection by Maullinia ectocarpii (Table 6).
These experiments have been tested against other protists: Anisolpidium ectocarpii and Maullinia ectocarpii. The results obtained are shown in Table 6.
Table 6: Activities of pyrenocine compounds against pathogens of the brown alga E. siliculosus.
Figure imgf000026_0001
These pyrenocine compounds and pyrenocaetic acid C compound were also tested against the oomycete Olpidiopsis pyropiae, in alga Pyropia yesoensis, belonging to the division Rhodophyta, at the final concentration of 100 μg/ml. The results obtained are shown in Table 7.
Table 7: Activities of pyrenocine compounds and pyrenochaetic acid C towards pathogens of alga Pyropia yesoensi.
Figure imgf000026_0002
Pyrenocaetic acid C compound was also tested against the oomycete Pythium porphyrae, in alga Pyropia yesoensis, belonging to the division Rhodophyta, at the final concentration of 100 μg/ml.
It has been also observed that pyrenochaetic acid C can stop the growth of the oomycete Pythium porphyrae mycelium in alga Pyropia yesoensis and induces the degeneration of Pythium porphyrae three days after treatment. No zoosporangium was formed after the treatment.
In the same conditions, pyrenocines A, B, E and N did not show any critical changes in morphology, growth and reproduction on Pythium porphyrae, after observation by microscopy.
Figure imgf000027_0001
Sparace S.A. et al., Canadian Journal of Microbiology. 1987. 33:327-330. Tsirigoti A. et al., Protoplasma. 2015. 252:845-56
Uppalapatti, S.R., etal, J. Phycol. 1999. 36:359-366.
West, J.A. etal., Phycological Research. 2006. 54:72-85.
Zhang Q.S. et al, J. Appl Phycol. 2007. 19:303-311.

Claims

1. Use of a pyrenocine compound or a pyrenochaetic acid compound for the prevention or the treatment in an alga of a disease induced by a protistan pathogen.
2. A method for preventing or treating a disease induced by a protistan pathogen in an alga comprising contacting an effective amount of a pyrenocine compound or a pyrenochaetic acid compound with said alga or portion thereof.
3.
Use according to claim 1 or the method according to claim 2, wherein the pyrenocine compound is selected from the group consisting of pyrenocine A, pyrenocine B, pyrenocine E, pyrenocine N and the pyrenochaetic acid compound is pyrenochaetic acid C.
4. Use or method according to any one of claims 1 to 3, wherein the alga is a brown alga belonging to the class Phaeophyceae or a red alga belonging to the division Khodophyta.
5. Use or method according to any one of claims 1 to 4, wherein the brown alga is selected from the group consisting of the genera Laminaria, Saccharina, Macrocystis and Ectocarpus, preferably the species Laminaria digitata and Ectocarpus siliculosus and the red alga is of the genera Pyropia and Porphyra, preferably the species Pyropia yezoensis and Porphyra purpurea.
6. Use or method according to any one of claims 1 to 5, wherein the protistan pathogen is selected from the group consisting of oomycete, hyphochytrid and phytomyxean pathogens.
7. Use or method according to any one of claims 1 to 6, wherein the protistan pathogen is selected from the group consisting of the following genera: Eurychasma, Olpidiopsis, Anisolpidium, Pythium and Maullinia.
8. Use or method according to any one of claims 1 to 7, wherein the protistan pathogen is selected from the group consisting of the following species:, Eurychasma dicksonii, Olpidiopsis bostrychiae, Olpidiopsis feldmannii, Olpidiopsis porphyrae, Olpidiopsis pyropiae, Pythium porphyrae, Pythium chondricola, Pythium marinum, Anisolpidium ectocarpii, Anisolpidium joklianum, Anisolpidium rosenvingii, Anisolpidium sphacellarum and Maullinia ectocarpii.
9. Use or method according to any one of claims 6 to 8, wherein the oomycete pathogen is from the group consisting of the genus Eurychasma and Olpidiopsis, preferably the species Eurychasma dicksonii and Olpidiopsis pyropiae, wherein the hyphochytrid pathogen is from the genus Anisolpidium, preferably the species Anisolpidium ectocarpii, and wherein the phytomyxean pathogen is the species Maullinia ectocarpii .
10. Use or method according to any one of claims 1 to 9, wherein the pyrenocine compound is selected from the group consisting of pyrenocine A and pyrenocine B and the protistan pathogen is selected from the group consisting of a Eurychasma and Anisolpidium pathogen, preferably selected from
Eurychasma dicksonii and Anisolpidium ectocarpii.
11. Use or method according to any one of claims 1 to 9, wherein the pyrenocine compound is selected from the group consisting of pyrenocine A, pyrenocine B and pyrenocine £ and the protistan pathogen is Maullinia ectocarpii.
12. Use or method according to claims 10 or 11, wherein the alga is a brown alga, preferably an alga from the genus Ectocarpus, more preferably Ectocarpus siliculosus.
13. Use or method according to any one of claims 1 to 9, wherein the pyrenocine or pyrenochaetic acid compound is selected from the group consisting of pyrenocine A, pyrenocine N and pyrenochaetic acid C and the protistan pathogen is an Olpidiopsis pathogen, preferably Olpidiopsis pyropiae.
14. Use or method according to any one of claims 1 to 9, wherein the pyrenochaetic acid compound is pyrenochaetic acid C and and the protistan pathogen is a Pythium pathogen, preferably Pythium Porphyrae.
15. Use or method according to any one of claims 1 to 14, wherein the pyrenocine or pyrenochaetic acid compound is included into a cyclodextrine.
16. Use or method according to any one of claims 1 to 14, wherein the pyrenocine or pyrenochaetic acid compound is in solution with a polar aprotic solvent, preferably dimethyl sulfoxide (DMSO) or a polar protic solvent, preferably methanol.
17. The pyrenocine N of formula :
Figure imgf000031_0001
18. An anti-protistan composition comprising the compound pyrenocine N of claim 17.
19. A method for obtaining the compound pyrenocine N of claim 17, comprising the steps of :
a) provision of a culture of a strain of Phaeosphaeria; b) extraction of the metabolites from the culture by a solvent; c) separation of metabolites by chromatography;
d) isolation of the fraction corresponding to pyrenocine N, eluted between the pyrenocine A and the pyrenocine C.
20. An isolated extract of a Phaeosphaeria strain, preferably from the strain Phaeosphaeria sp. AN596H CNCM 1-5041 comprising the compound pyrenocine N of claim 17.
PCT/IB2016/000151 2016-01-22 2016-01-22 Prevention or treatment in algae of diseases induced by protistan pathogens WO2017125775A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/IB2016/000151 WO2017125775A1 (en) 2016-01-22 2016-01-22 Prevention or treatment in algae of diseases induced by protistan pathogens

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/IB2016/000151 WO2017125775A1 (en) 2016-01-22 2016-01-22 Prevention or treatment in algae of diseases induced by protistan pathogens

Publications (1)

Publication Number Publication Date
WO2017125775A1 true WO2017125775A1 (en) 2017-07-27

Family

ID=55442836

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2016/000151 WO2017125775A1 (en) 2016-01-22 2016-01-22 Prevention or treatment in algae of diseases induced by protistan pathogens

Country Status (1)

Country Link
WO (1) WO2017125775A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108239607A (en) * 2018-04-10 2018-07-03 邹丹丹 A kind of method cultivated, collect red rot disease bacterium mycelia
WO2019119082A1 (en) * 2017-12-20 2019-06-27 Universidade Estadual De Campinas - Unicamp Process for obtaining the fungus phaeosphaeria sp. and fermentation broth thereof and uses thereof

Non-Patent Citations (29)

* Cited by examiner, † Cited by third party
Title
ADL S.N. ET AL., EUKARYOTIC MICROBIOLOGY, vol. 59, 2012, pages 429 - 493
ADL S.N. ET AL., SYSTEMATIC BIOLOGY, vol. 56, 2007, pages 684 - 689
AKIYAMA, K. ET AL., BULLETIN OF TOHOKU REGIONAL FISHERIES RESEARCH LABORATORY, vol. 37, 1977, pages 43 - 49
AMAGATA T. ET AL., THE JOURNAL OF ANTIBIOTICS, vol. 51, 1998, pages 432 - 434
BLOIN N.A. ET AL., TRENDS PLANT SCI., vol. 16, 2011, pages 29 - 37
CHEIKH-ALI Z. ET AL., CHEMMEDCHEM., vol. 10, 2015, pages 411 - 418
DAI J ET AL., AQUACULTURE, vol. 11, 1993, pages 139 - 145
GACHON C.M.M. ET AL., APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 75, 2009, pages 322 - 328
GACHON C.M.M. ET AL., TRENDS IN PLANT SCIENCE, vol. 15, 2010, pages 633 - 640
GOECKE F. ET AL., PLOS ONE, vol. 7, no. 9, 2012, pages E45358
HIDAYAT H. ET AL., FITOTERAPIA, vol. 83, 2012, pages 523 - 526
HIDAYAT H. ET AL.: "Pyrenocines J-M: Four new pyrenocines from the endophytic fungus, Phomopsis sp.", FITOTERAPIA, vol. 83, no. 3, 2012, pages 523 - 526, XP028917388 *
ICHIHARA A. ET AL., AGRIC. BIOL. CHEM., vol. 48, 1983, pages 833 - 834
ICHIHARA A. ET AL., TETRAHEDRON LETTERS, 1981, pages 224005 - 4006
KAZAMA F.Y. ET AL., MYCOLOGIA, vol. 69, 1977, pages 246 - 54
KIM G.H., ALGAE, vol. 29, 2014, pages 249 - 265
KIM S.K. ET AL.: "Marine Algae Extracts: Processes, Products, and Applications", 2015, WILEY
KLOCHKOVA TA ET AL., JOURNAL OF APPLIED PHYCOLOGY, vol. 24, 2015, pages 135 - 144
LEVESQUE C.A. ET AL., MYCOL RES., vol. 108, 2004, pages 1363 - 1383
MAIER 1. ET AL., PROTIST., vol. 151, 2000, pages 225 - 238
MUNIN A. ET AL., PHARMACEUTICS, vol. 3, 2011, pages 793 - 829
SATO H. ET AL., AGRIC. BIOL. CHEM., vol. 45, 1981, pages 1675 - 1679
SEKIMOTO S. ET AL., MYCOLOGICAL RESEARCH, vol. 112, 2008, pages 361 - 374
SEKIMOTO S. ET AL., PROTIST., vol. 159, 2008, pages 299 - 318
SPARACE S.A. ET AL., CANADIAN JOURNAL OF MICROBIOLOGY, vol. 33, 1987, pages 327 - 330
TSIRIGOTI A. ET AL., PROTOPLASMA, vol. 252, 2015, pages 845 - 56
UPPALAPATTI, S.R. ET AL., J. PHYCOL., vol. 36, 1999, pages 359 - 366
WEST, J.A. ET AL., PHYCOLOGICAL RESEARCH, vol. 54, 2006, pages 72 - 85
ZHANG Q.S. ET AL., J. APPL PHYCOL., vol. 19, 2007, pages 303 - 311

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019119082A1 (en) * 2017-12-20 2019-06-27 Universidade Estadual De Campinas - Unicamp Process for obtaining the fungus phaeosphaeria sp. and fermentation broth thereof and uses thereof
CN108239607A (en) * 2018-04-10 2018-07-03 邹丹丹 A kind of method cultivated, collect red rot disease bacterium mycelia
CN108239607B (en) * 2018-04-10 2021-07-27 邹丹丹 Method for culturing and collecting mycelia of saprophyta purpurea

Similar Documents

Publication Publication Date Title
Crone et al. Survival of Phytophthora cinnamomi as oospores, stromata, and thick-walled chlamydospores in roots of symptomatic and asymptomatic annual and herbaceous perennial plant species
Kumar et al. Antimicrobial compounds from marine halophytes for silkworm disease treatment
Sudheep et al. Non-mycorrhizal fungal endophytes in two orchids of Kaiga forest (Western Ghats), India
Mansuya et al. Antibacterial activity and qualitative phytochemical analysis of selected seaweeds from Gulf of Mannar region
Bajpai Antimicrobial bioactive compounds from marine algae: A mini review
Tong et al. Antimicrobial activities of endophytic fungal isolates from medicinal herb Orthosiphon stamineus Benth
Dhanya et al. Antimicrobial activity of Ulva reticulata and its endophytes
Murate et al. Activity of secondary bacterial metabolites in the control of citrus canker.
Das et al. Antibacterial properties of selected freshwater microalgae against pathogenic bacteria
Kim et al. Antimicrobial activity of ethanol extracts of Laminaria japonica against oral microorganisms
Dixit et al. Paenibacillus lentimorbus induces autophagy for protecting tomato from Sclerotium rolfsii infection
Saleh et al. Antimicrobial activity of the marine algal extracts against selected pathogens
Safavi et al. Biological activity of methanol extract from Nostoc sp. N42 and Fischerella sp. S29 isolated from aquatic and terrestrial ecosystems
Wan Harun et al. Effect of Piper betle and Brucea javanica on the differential expression of hyphal wall protein (HWP1) in non-Candida albicans Candida (NCAC) species
Sofy et al. Potential impacts of seed bacterization or salix extract in faba bean for enhancing protection against bean yellow mosaic disease
Bratchkova et al. Microalgae as producers of biologically active compounds with antibacterial, antiviral, antifungal, antialgal, antiprotozoal, antiparasitic and anticancer activity
Liu et al. An endophytic strain of genus Paenibacillus isolated from the fruits of Noni (Morinda citrifolia L.) has antagonistic activity against a Noni's pathogenic strain of genus Aspergillus
RAZAFSHA et al. Inhibitory effects of aqueous onion and garlic extracts on growth and keratinase activity in Trichophyton mentagrophytes
WO2017125775A1 (en) Prevention or treatment in algae of diseases induced by protistan pathogens
Yoshida et al. Mannosylerythritol lipids secreted by phyllosphere yeast Pseudozyma antarctica is associated with its filamentous growth and propagation on plant surfaces
Chae et al. Rapid direct identification ofCryptococcus neoformans from pigeon droppings by nested PCR usingCNLAC1 gene
Gacheva et al. Suboptimal growth temperatures enhance the biological activity of cultured cyanobacterium Gloeocapsa sp.
Messahli et al. Anticandidal activity of dichloromethane extract obtained from the red algae A. armata of the Algerian coast
Al-Dossary et al. Molecular identification of five species of family Chaetomiaceae (Sordariomycetes, Ascomycota) from Iraqi soil
KR20180001716A (en) Antimicrobial composition for preventing bacterial blight containing extracts of Bacillus subtilis culture medium

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16706678

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16706678

Country of ref document: EP

Kind code of ref document: A1