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:

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 preferably between 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 and 200 μg/ml, preferably between 5 and more

preferably between 10 and

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:

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*, Sepharose ^e 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 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

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 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 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.

Table 1. 1H and ¹³ C NMR data of pyrenocine N in CD ₃ OD (1H 400.13 MHz, ¹³ C 150 MHz, 298 K).

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

This

compound is pyrenocine A.

Table 2. 1H and ¹³ C NMR data of pyrenocine A in CD ₃ OD (Ή 600 MHz, ^,3 C 150 MHz, 298 K).

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

(3H). This compound is pyrenocine B.

Table 3. 1H NMR data of pyrenocine B in

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:

s 3.83 (3H), s 2.24 (3H), s 3.25 (3H). This compound is pyrenocine E.

Table 4. 1H and ^I3 C NMR data of pyrenocine E in CD ₃ OD (1H 600 MHz, ¹³ C 150 MHz, 298 K).

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 ¹ H NMR spectrum of this compound in CD ₃ OD (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 ^l3 C NMR data of pyrenochaetic acid C in CD ₃ OD (1H 600 MHz, ¹³ C 150 MHz, 298 K).

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 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.

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.

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.

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