Use of Fungal Organism Pythium Oligandrum Verwendung Des Pilzigen Organismus Pythium Oligandrum Utilisation Du Champignon Pythium Oligandrum

(19) TZZ Z¥ _T

(11) EP 2 503 892 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.: of the grant of the patent: A01N 63/04 (2006.01) A23B 7/155 (2006.01) 16.11.2016 Bulletin 2016/46 A23B 9/28 (2006.01)

(21) Application number: 09810837.6 (86) International application number: PCT/CZ2009/000152 (22) Date of filing: 11.12.2009 (87) International publication number: WO 2011/063771 (03.06.2011 Gazette 2011/22)

(54) USE OF FUNGAL ORGANISM PYTHIUM OLIGANDRUM VERWENDUNG DES PILZIGEN ORGANISMUS PYTHIUM OLIGANDRUM UTILISATION DU CHAMPIGNON PYTHIUM OLIGANDRUM

(84) Designated Contracting States: • DATABASE BIOSIS [Online] BIOSCIENCES AT BE BG CH CY CZ DE DK EE ES FI FR GB GR INFORMATION SERVICE, PHILADELPHIA, PA, HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL US; 1998, SVECOVA M ET AL: "The effect of PT RO SE SI SK SM TR Pythium oligandrum Drechsler mycoparasite of fungal species of the genus Ceratocystis s.l", (30) Priority: 27.11.2009 CZ 20090794 XP002631724, Database accession no. PREV199800516030& LESNICTVI (PRAGUE), vol. (43) Date of publication of application: 44, no. 9, 1998, pages 411-420, ISSN: 0024-1105 03.10.2012 Bulletin 2012/40 • DATABASECAB [Online]CAB INTERNATIONAL, WALLINGFORD, OXON, GB; 2001, A. Kunca & R. (73) Proprietor: Biopreparáty, spol. s r.o. Leontovyc: "Relationship between beech bark 252 62 Únetice (CZ) wounds and bark necrosis and possible methodfs to control bark necrosis", (72) Inventor: SUCHÁNEK, Martin XP002631725, retrieved from stn accession no. 160 00 Praha 6 (CZ) 2001:42943 Database accession no. 2001:42943 & A. KUNCA & R. LEONTOVYC: LESNICKY (74) Representative: Smrckova, Marie CASOPSIS (FORESTRY JOURNAL), vol. 45, no. Velflíkova 10 5/6, 1999, pages 317-324, Bratislava ISSN: 160 00 Praha 6 (CZ) 0323-1046 • S.A. K. LAING & J. W. DEACON: "Aggressiveness (56) References cited: and fungal host ranges of mycoparasitic Pythium WO-A1-98/16110 US-A- 4 259 317 species", SOIL BIOLOGY AND BIOCHEMISTRY, PERGAMON, OXFORD, GB, vol. 22, no. 7, 1 • J. BROZOVÁ: "Exploitation of the mycoparasitic January 1990 (1990-01-01), pages 905-911, fungus Pythium oligandrum in plant protection", XP023673307, ISSN: 0038-0717, DOI: PLANT PROTECTION SCIENCE, vol. 38, no. 1, DOI:10.1016/0038-0717(90)90128-M [retrieved on 2002, pages 29-35, XP002631723, 1990-01-01] • J. HYSEK, M. VACH & M. JAVUREK: "Biological protection of the main cereals against fungal specific diseases", COMM. AGRICULT. APPLIED BIOLOG. SCI., vol. 70, no. 3, 2005, pages 169-173, XP009146974, Ghent ISSN: 1379-1176

Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 503 892 B1

Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 2 503 892 B1

• ELZBIETA PATKOWSKA: ’EFFECTIVENESS OF Remarks: GRAPEFRUIT EXTRACT AND PYTHIUM Thefile contains technical information submitted after OLIGANDRUM IN THE CONTROL OF BEAN AND the application was filed and not included in this PEAS PATHOGENS’ JOURNAL OF PLANT specification PROTECTION RESEARCH, [Online] 01 January 2006, pages 15 - 28, XP055187654 Retrieved from the Internet: [retrieved on 2015-05-06] • HALINA KURZAWINSKA ET AL: ’The evaluation of Pythium oligandrum and chitosan in control of Phytophthorainfestans (Mont.) de Bary on potato plants’ FOLIA HORTICULTURAE vol. 21, no. 2, 25 October 2009, pages 13 - 23, XP055187641

2 1 EP 2 503 892 B1 2

Description also the US 5,961,971 /1999/, corresponding with WO 98/16110/1998/, Martin F. N.: "Biocontrol of fungal soil- Field of the Invention bome pathogens by Pythium oligandrum", which identifies 3 non-pathogenic isolates of the fungal organism Py- [0001] The invention relates to the use of the fungal 5 thium oligandrum efficient against damping-off of germi- organism Pythium oligandrum in connection with the ap- nating and growing plants of tomato and other vegetable plicable agens carrier, especially in the area of plant pro- species, which are caused by the phytopathogenic fungal duction. In particular it concerns the new application for organisms from the Pythium family. the elimination of originators of post-harvest losses [0004] There are also various scientific publications caused by fungal diseases, yeasts and bacteria. 10 about the topic of application of the fungal organism of Pythium oligandrum against damping-off of various Background of the Invention growing plants including vegetable species: for example successful application of Pythium oligandrum for the pro- [0002] After the chemical way of seed pickling against tectionof cucumberagainst damping-off, which is caused fungal diseases with fungicides, a new way of biological 15 by the phytopathogen Pythium splendens: Larsen H. protection started to be used in the seventies of the 20 th /1985/ entitled "Bekaempelse af Pythium med specielt century, which used living cells of antagonistic microor- henblik pa biologisk bekaempelse vha antagonister" ganism against the phytopathogens. It concerns e.g. (Thesis, Veterinary and Agriculture Univerzity, Copen- preparation stimulating, in case of application to the hagen, Denmark); also Thinggaard K. /1985/ with title seeds of sugar beet, the growth of growing plants and 20 "Interspecific antagonism: The use of Pythium oligan- protecting them against rotting pursuant to CS AO No. drum to control root rot of cucumber caused by Pythium 199 906 /1980 / of Vesel» D. entitled "Agent stimulating splendens" (6th Scandinavian postgraduate course in when applied on the sugar beet seeds the growth of the plant pathology. "Sene pathosystems",p.p. 21-28, No- descended plants and protecting them against scarlet vember 1985 in Sverige), and Thinggaard K., K. Larsen fever", as well as the CS AO No. 204 208 /1981/ Vesel » 25 and J. Hockenhull /1988/ with title "Antagonistic Pythium D., Hejdanek S. with title "Method of production of the against pathogenic Pythium on cucumber roots" (Bulletin preparation stimulating, in case of application to the OEPP/EPPO Bulletin 18, pp. 91-94). seeds of sugar beet", the growth of growing plants and [0005] In the scientific literature, Pythium oligandrum protecting them against scarlet fever", or CS AO No. is always identified in connection with diseases of roots 213659 /1986/, Vesel» D., Hejdánek S. "Preparation for 30 of growing plants and with mycoparazitism as the only the protection of sugar beet against rotting on the basis type of effect, refer to e.g. Vesel» D. /1987/ entitled "Bi- of oospora of the mycoparasitePythium ological control of damping-off pathogens by treating oligandrum." The latest said CS AO No. 213659 was pat- sugar-beet seed with a powdery preparation of the myc- ented in 12 other countries: USA 4259317, Canada oparasite Pythium oligandrum in large-scale field trials", 1132270, Federal Republic of Germany 2927224, Ger- 35 (pp. 445-449; In: Vancˇura V., Kunc F. /eds./ Interrelation- man Democratic Republic 157491, UK 2027448, France ships between microorganisms and plants in soil. Pro- 7917494, Japan 1337470, Denmark 156291, Belgium ceedings of an International Symposium Liblice, Czech- 877474, Austria 363734, Italy 1122032, Hungary 183040 oslovakia, June 22-27, 1987, Academia Publishing and it solves the protection against rotting of sugar beet House of the Czechoslovak Academy of Sciences, Else- / damping off, which are caused by the soil phytopatho- 40 vier Science Publishers, Amsterdam, The Netherlands, genicfungi. Thesame issues arealso solved withanother Prague 1989), identifies successful utilization of the bio- invention CS AO No. 247 378 /1986/, Stanek M., Vesel » preparation on the basis of P. oligandrum against damp- D., entitled "Preparation for protection of growing plants ing-off of beet plants. On the example of damping-off of of sugar beet against rotting", and also the utility model saplings of cucumber, Kratka J. Bergmanová, A. Kudelo- CZ No. 393 /1993/, Vesel», D. entitled "Preparation for 45 va /1994/ entitled "Effect of Pythium oligandrum and Py- support of growth of plants and/or their protection" (in thium ultimum on biochemical changes in cucumber Cu- Slovakia the corresponding utility model SK utility model cumis sativus L" (Ztschr. f. Pfl. Krankh. u. Pfl. Schutz, No.429), which solve also exclusively the protection 101 /4/, pp. 406-413) studied relationships between the against damping-off of germinating plants and with focus mycoparasite P. oligandrum and the plant treated with it, on forest nursery. 50 which is demonstrated e.g. by the stimulation of growth [0003] From other foreign patent literature, 2 patents of plants. were granted in the USA for the biological protection [0006] Al-Rawahi A. K., J. G. Hancock /1997/ entitled against damping-off of germinating plants. US 4,574,083 "Rhizosphere Competence of Pythium oligandrum", /1986/ Baker R., R. Lifshitz: "Isolates of Pythium species, (Phytopathology Vol., 87, 9; 951-959), studied the colo- which are antagonistic to Pythium ultimum", which deals 55 nization of rhizosphere and non-rhizosphere soil with the with the utilization of species of Pythium sp., which are said fungal organism on the roots of saplings. Benhamou antagonistic to the soil phytopathogenic fungus Pythium N., P. Rey, M. Chérif, J. Hockenhull and Y. Tirilly /1997/ ultimum, causing damping-off of germinating plants, and entitled " Treatment with the Mycoparasite Pythiumolig-

3 3 EP 2 503 892 B1 4 andrum Triggers Induction of Defence-related reactions [0009] From the review of Broˇzová J. /2002/ entitled in Tomato Roots When Challenged with Fusarium ox- "Exploitation of the mycoparasitic fungus Pythium olig- ysporum f. sp. radicis-lycopersici" (Phytopathology, Vol. andrum in plant protection" (Plant Protection Science, 87, 1: 108-122) submitted proofs thatPythium oligan- vol. 38 (1) 29-35), is known exploitation of the mycopar- drum initiates defence responses in the tomato roots 5 asitic fungus Pythium oligandrum in plant protection for against the phytopathogenic fungus Fusarium oxyspo- the growth stimulation of plants and for biological control rum f. sp. radicis-lycopersici as the complementary tape on a wide spectrum of crop plants. It does not attack their of effect of the mycoparazitism. Mísˇa D. /1997/ entitled tissue but occurs on the root surface, predominantly in "Efficiency of biological preparations against grey mould the regions of hypocotyl - taproot, together with plant lBofrytis cinerea Pers. et Fries /on grapevine" (Proceed- 10 pathogenic fungi. ings of the XIV Slovak and Czech Plant Protection Con- [0010] H»sek J., Vach M., Javu°rek M. /2005/ with title ference in Nitra 3rd-4th September 1997, pp. 294-296), "Biological protection of the main cereals against fungal tested the efficiency of the preparation with Pythium oli- specific diseases" (Commun. Agricult. Applied Biolog. gandrum as the contact fungicide against the grey mould Sci. Ghent University, vol. 70 (3) 169-173), also disclos- on grapes of the grapevine. Vesel » D. /1998/ entitled "Py- 15 es, that application of Pythium oligandrum as seed treat- thium oligandrum as a biological agent stimulating seed- ment or as spray in a composition further containing fer- potato germination and suppressing potato late blight pri- tilizers results in the control of various phytopathogenetic mary infection" (5.2. 98, 7th InternationalCongress of microorganism as well as in an enhanced yield of cereal Plant Pathology, Edinburgh) by the treatment of the seed- crops winter wheat and barley. ling tubers of potatoes, growth of sprouts and rootlets 20 [0011] Sˇ vecová M., Cˇíz ˇková D., Vesel» D. /1998/ en- was stimulated and the development of symptoms of the titled "The effect of Pythium oligandrum Drechsler myc- primary infection of potato late blight was slowed down. oparasite of fungal species of the genus Ceratocystis s.l." [0007] Benhamou N.,P. Rey, K. Picard,Y. Tirilly /1999/ (Lesnictvi Prague, vol. 44.(9) 411-420), disclose the ad- entitled "Ultrastructural andCytochemical Aspects of the vantageous properties of Pythium oligandrum in the con- Interaction Between the Mycoparasite Pythium oligan- 25 trol of wood affecting fungi namely the possibility to use drum and Soilbome Plant Pathogens" (Phytopathology, the mycoparasite Pythium oligandrum to affect the oc- Vol. 89 6: 506-517), studied the interactions between the currence of Ophiostomatales in the integrated control of oomycetes Pythium oligandrum and various soil oomyc- forest - trees. etes and plants pathogens P. ultimum, P. aphaniderma- [0012] Another document US 4, 259,317 Vesel » D, He- tum, F. oxysporum f. sp. radicis-lycopersici, Verticillium 30 jdánek S. /1981/ with title "Preparation for the protection alboatrum, Rhizoctonia solani and Phytophthora of emerging sugar beets against damping-off, and meth- megasperma. They found out that at least two different ods of its production" relates to a dry powdered prepa- mechanisms seemed to be responsible in the process of ration to be applied onto sugar beet seeds, the prepara- the attack of the oomycete P. oligandrum on the patho- tion containing, as its active constituent, organs of repro- gens: mycoparasitism 35 duction of Pythium oligandrum Drechsler, particularly the and antibiosis. Vesel» D. /2000/ entitled "Polyversum as oospores, in a sufficiently high concentration of at least an effective seed dresser inwheat" (p. 120, Proceedings about 1,000,000 oospores per gram. Such concentration of 13th International EFOAM Scientific conference /Alf make it possible to apply about 100, 000 oospores or Oldi T., W. Lockeretz, V. Niggli /eds./ 28-31 August 2000, more onto every seed, which proved sufficient to secure Basel), stated that Pythium oligandrum as the seed 40 protection of emerging sugar beet plants against "damp- dresser improved health conditions of roots of winter ing off" or "black leg" disease. wheat, whereby it also increased yields. [0013] Kunca A., Leontovyc R.. /1999/ entitled "Rela- [0008] In addition to the aforesaid author’s certifica- tionship between beech bark wounds and bark necrosis tions and patents of the inventions, two scientific publi- and possible methods to control bark necrosis" (Lesnick » cations deal with the type of production of the preparation 45 cˇasopis 45 (5/6) 317-324), also disclose the advanta- based on the application of the fungal organism Pythium geous properties of Pythium oligandrum in the control of oligandrum: Vesel» D., S. Hejdánek /1984/ entitled "Mi- wood affecting fungi as well as the relationship between crobial relations of Pythiumoligandrum and Problems in beech bark wounds and bark necrosis and possible the Use of This Organism for the Biological Control of methods to control bark necrosis. Damping-off in Sugar-beet" (Zbl. Mikrobiol. 139, pp.50 [0014] According to Laing S.A.K. Deacon J.W. /1990/ 257-265) compare the advantages and problems of sta- entitled "Aggressiveness and fungal host ranges of myc- tionary surface cultivation and submersion cultivations. oparasitic Pythium species" (Soil Biol Biochem 22 (7) Mc Quilken M. P., J. M. Whipps and R. C. Cooke /1990/ 905-911), is also known that Pythium oligandrum is ag- entitled "Oospores of the biocontrol agent Pythium olig- gressive mycoparasite organism as evidenced by its abil- andrum bulk produced in liquid culture" (Mycol. Res. 94, 55 ity to reduce cell activities of host fungi on cellulose film 5, pp. 613-616) developed the method of production of or filter paper. oospores of P. oligandrum in static and aerated cultiva- [0015] Kurzawinska H., Mazur S. /2009/ with title "The tion in liquid medium. evaluation of Pythium oligandrum and chitosan in control

4 5 EP 2 503 892 B1 6 of Phytophthora infestans (Mont.) de Bary on potato family Fusarium (aflatoxins) in agricultural commod- plants" (Folia Holticulturae, Vol. 21 (2) 12-23), deter- ities. Nowadays, the presence of mycotoxins in the mined the effect of tuber dressing and plant spraying with food chain has been a real health risk to the con- Pythium oligandrum on the top leaves and tubers of "Ibis" sumer. potatoes infested by Phytophthora infestans observing 5 2. During the application of the preparation, the effi- a significant inhibition of top leaf and tuber infestation. cient substance of which is Pythium oligandrum, on [0016] Patkowska E. /2006/ "Effectiveness of grape- the walls of warehouses and surfaces of the storage fruit extract and Pythium oligandrum in the control of bean technology (e.g. silos, warehouses of foodstuffs with and peas pathogens" (Journal of Plant Protection Re- controlled as well as uncontrolled atmosphere, racks search, Vol. 46 (1) 15-28), reported the effectiveness of 10 and stackers), cellars, in the food-processing facili- grapefruit extract and Pythium oligandrum in protection ties (e.g. digester houses, preparation rooms, pack- of common bean, runner bean, and pea from soil-borne ing rooms), surface of packing for storage of food- pathogenic fungi when dressing the seeds of particular stuffs and semi-products (e.g. storage transport box- plant species with these means. es and cases), on the surface of transport packing [0017] Since 1997, the bio-fungicide preparation with 15 (e.g. cases for fruit, boxes for bananas), on the sur- the growth-stimulating effect, the efficient substance of face of sales racks and on both internal and external which is the fungal organismPythium oligandrum surfaces of sales packing, pathogenic microflora, Drechsler, has been manufactured and commercially which caused rot decay, was demonstrably sup- distributedby the company Biopreparáty, s.r.o.in approx- pressed. The said applications considerably extend- imately fifteen states of the world with various trade20 ed the storage period of stored agricultural commod- names. One of the names is bio-fungicide Polyversum. ities. 3. It is possible to apply the fungal organism Pythium Summary of the Invention oligandrum on paper cellulose materials, which are used directly or as a semi-product for the production [0018] The presented invention solves the problem of 25 of packing materials (e.g. fine paper packages of occurrence of the originators of microbial diseases caus- piece fruit and vegetables, bags and carrier bags for ing post-harvest losses. of cultural plants. fruit and vegetables). [0019] The nature of the invention is the use of the fungal organism Pythium oligandrum as a strong myco- Examples parasite and as efficient agens, with the applicable inor- 30 ganic and/or organic carrier in the form of the preparation [0021] Pythium oligandrum may be used for the afore- for prevention and elimination of occurrence of the orig- said purposes for the application in aqueous solutions, inators of microbial diseases causing post-harvest losses namely in the form of dry mixtures or liquid cultures, in by application after harvest or during storage of products powder or in granules. of cultural plants. 35 [0020] Surprisingly, the advantages has been found The application of Pythium oligandrum as efficient sub- out that: stance in aqueous solutions in the form of dry mixtures.

1. There are new applications of preparations, the [0022] For the application of Pythium oligandrum as efficient substance of which is Pythium oligandrum, 40 efficient substance in form of aqueous solutions in the for the prevention of occurrence of originators of mi- form of dry mixtures (powders) as wettable powders in- crobial diseases causing post-harvest losses before tended for application in the form of suspension after dis- and during storage of products and seeds of cultural persion in water, both mineral and organic materials may plants and all other commodities of both plant and be used as the inert filling of such powders. As suitable animal agricultural production. 45 filling materials for such application, e.g. mineral and syn- Surprisingly it has been demonstrated that the ap- thetic filling materials produced from silica, natural clay, plication of the preparation, the efficient substance vermiculite and various silicate materials can be used. of which is the fungal organism Pythium oligandrum, As other suitable filling materials, we can also identify significantly suppresses the originators of fungal, sulphates (calcium sulphate), silicates (magnesium sili- yeast and bacterial diseases of stored products of 50 cate). As surface active substance in these wetting the agricultural production (e.g. vegetables, fruit, agents, there are two types of tensides: anion (anion- nuts, cereals, feeding mixtures for animals and other active, anionic) and non-ion (non-ionogen, non-ionic). agricultural commodities) and semi-products and fi- Anion tensides are especially of the type of alkyl sul- nal products before the end application by the con- phates, alkyl-or alken sulphonan and alkyl benzene sul- sumer (people, animals). The application of the fun- 55 phonan. Non-ion tensides are especially products of gal organism Pythium oligandrum after the harvest higher alcohols but also alkyl phenols and aliphatic acids (e.g. in case of cereals) results in a decreased oc- with alkylene oxide (i.e. either with ethylene oxide or, as currence of residua of fungi (mycotoxins) of e.g. the the case may be, propylene oxide). The most preferred

5 7 EP 2 503 892 B1 8 are wetting agents of alkyl-benzene and alkyl-naphtha- Application of Pythium oligandrum as efficient substance lene sulphonates, sulphates, aliphatic alcohols, amines in wetting agents or amides of acid, long chains of esters of sodium isethionate, esters of 45 sodium succinate, sulphates or [0028] Such wetting agents are used as suitable wet- sulphonated of vegetable oils and ditertiary non-saturat- 5 ting agents that are of an anion or non-ion type. ed with glycols. The preferred dispergators are methyl cellulose, polyvinyl alcohol, lignin sulphonate, polymeric Type of preparation of Pythium oligandrum alkyl naphthalene sulphonates, sodium naphthalene sulphonates, polymethylene bisnaphthalene sulphonate [0029] The fungal organism Pythium oligandrum is and natrium-50-N-methyl-N-(long chain of the acid). 10 grown in common fermenters on corn media without sup- [0023] Such wettable powders are represented usually plying further nutrition. After the removal of the superna- in the concentration from 0.5 percentage by weight to 99 tant by centrifugal operation or in the evaporator, the sed- percentage by weight. If required, a part may be replaced iment with the production of oospora is dried. It is possible with inhibitor of corrosion or anti-foaming or both. to separate oospora from the supernatant in the spray 15 drier in an advantageous way. The advantage of this way Application of Pythium oligandrum as efficient substance is obtaining a clean concentrate of oospora, virtually within liquid cultures out presence of the used nutrient medium and also myc- elium that is destroyed in the aforesaid handling / drying [0024] For the application of Pythium oligandrum as / separation. Such obtained the concentrate, which can efficient substance_in liquid cultures, the most preferred 20 be homogenized at any time with the applicable inorganic wetting agents are alkyl benzene and alkyl naphthalene or organic carrier of the efficient agens /internal filling sulphonates, sulphated aliphatic alcohols, amines or material/. amides of acids, long chains of esters of sodium isethionate, esters of 45 sodium sulphosuccinate, sulphated or Testing of biological efficiency of the preparation of Py- sulphonated vegetable oils. 25 thium oligandrum according to the invention

Application of Pythium oligandrum as efficient substance [0030] All tests were performed by the company Bio- in emulsified oils preparáty spol. s. r. o. in the research laboratory. The applications extended the storage life; thanks to the elim- [0025] Emulsified oils are usually solutions or suspen- 30 ination of rot and mould processes, the storage and pres- sions of the active material in waterless mixable solvents ervation possibilities of crops and foodstuffs have im- together with surface-active substances and/or emulsifi- proved rapidly. ers. Emulsified oil may be mixed with the active substance, with an organic solvent. The following are suita- Accreditation and certification ble: chlorinated solvents, water non-miscible ethers, es- 35 ters, ketones or in a mixture with aromatic hydrocarbons. [0031] The tests concerning the content of the active [0026] The most suitable emulsifiers for emulsified oil substance Pythium oligandrum were performed. Further are long chains of alkyl or mercaptan polyethoxy alco- to that, the development and progress of efficiency on hols, alkyl acryl polyethoxy alcohols, sorbitan-aliphatic moulds and phytoparasites and their mutual interaction, acids, esters, ethers poly sorbitan with esters of aliphatic 40 which was monitored, were detected. The company Bi- acids, polyethylene glycol with esters of aliphatic acids opreparáty spol. s. r. o. ensures cultivation and research of colophony, aliphatic alkylol amides. Condensates of of new cultivars of the microorganismPythium oligan- calcium and amine salt of aliphatic alcohols of sulphates, drum. Thanks to the application of various selective soils, oil sulphonates dissoluble in oil, or preferably mixtures the microorganism Pythium oligandrum is cultivated for of the said components should form approximately from 45 certain applications so that their impact is as efficient as 1 to 90 percent by weight from the total composition. possible. Another process, which is performed by this company, is the actual incubation and fermentation of Application of Pythium oligandrum as efficient substance the cultures of Pythium oligandrum. in granules 50 Field of Application [0027] Granules are physically stable, particles of the composition including propagules of the efficient sub- [0032] The invention can be used as a new application stance Pythium oligandrum with an organic or inorganic method in the prevention of occurrence of originators of carrier. For the purpose of washing out of the efficient microbial diseases during the storage of products and substance from the granule, surface active substances 55 seeds and other commodities of plant can be added.

6 9 EP 2 503 892 B1 10

Claims

1. A use of the fungal organism Pythium oligandrum, as efficient agens, with the applicable inorganic and/or organic carrier in the form of the preparation 5 for prevention and elimination of occurrence of the originators of microbial diseases causing post-harvest losses by application after harvest or during storage of products of cultural plants. 10 2. The use according to Claim 1, that products of cultural plants are namely fruits, vegetables, nuts, cereals and other crops.

15 Patentansprüche

1. Verwendung des pilzigen Organismus Pythium oligandrum als Wirkstoff mit einem applizierbaren an- organischen und/oder organischen Träger in der20 Form eines Mittels für die Prävention und Eliminie- rung des Auftretens der Erreger mikrobieller Erkran- kungen, die Verluste nach der Ernte verursachen, durch Anwendung nach der Ente oder während der Lagerung von Produkten von Kulturpflanzen. 25

2. Verwendung nach Anspruch 1, wobei die Produkte von Kulturpflanzen insbesondere Obst, Gemüse, Nüsse, Getreide und andere Früchte sind. 30

Revendications

1. Utilisation du Champignon Pythium oligandrum en tant que substance active, avec support applicable 35 inorganique et/ou organique sous forme de produit pour la prévention et l’élimination de la présence de facteurs de maladies microbiennes, entraînant des pertes d’après-moisson par une application après la moisson ou pendant l’entreposage de produits vé- 40 gétaux pour la culture.

2. Utilisation selon la revendication 1, où les produits végétaux pour la culture sont avant tout des fruits, légumes, noix, céréales et autres plantes. 45

7 EP 2 503 892 B1

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

• US 45740831986 A [0003] • WO 98161101998 A [0003] • US 59619711999 A [0003] • US 4259317 A [0012]

Non-patent literature cited in the description

• Bekaempelse af Pythium med specielt henblik pa bi- • VESEL® D. Pythium oligandrum as a biological agent ologisk bekaempelse vha antagonister. LARSEN H. stimulating seed-potato germination and suppress- Thesis. Veterinary and Agriculture Univerzity, 1985 ing potato late blight primary infection. 7th Interna- [0004] tional Congress of Plant Pathology, 1998 [0006] • THINGGAARD K. Interspecific antagonism: The use • BENHAMOU N. ; P. REY ; K. PICARD ; Y. TIRILLY. of Pythium oligandrum to control root rot of cucumber Ultrastructural and Cytochemical Aspects of the In- caused by Pythium splendens.6th Scandinavian teraction Between the Mycoparasite Pythium oligan- postgraduate course in plant pathology. ’’Sene pa- drum and Soilbome Plant Pathogens. Phytopatholo- thosystems, 1985, 21-28, November 1985 [0004] gy, 1999, vol. 89 (6), 506-517 [0007] • THINGGAARDK. ; K.LARSEN ; J. HOCKENHULL. • Polyversum as an effective seed dresser in wheat. Antagonistic Pythium against pathogenic Pythium on VESEL® D. Proceedings of 13th International cucumber roots. Bulletin OEPP/EPPO Bulletin, 1988, EFOAM Scientific conference /Alf Oldi T., W. Lock- vol. 18, 91-94 [0004] eretz, V. Niggli /eds./ 28-31 August 2000. 2000, 120 • Biological control of damping-off pathogens by treat- [0007] ing sugar-beet seed with a powdery preparation of • VESEL® D. ; S. HEJDÁNEK. Microbial relations of the mycoparasite. VESEL® D. Pythium oligandrum Pythium oligandrum and Problems in the Use of This in large-scale field trials. 1987, 445-449 [0005] Organism for the Biological Control of Damping-off • Proceedings of an International Symposium Liblice, in Sugar-beet. Zbl. Mikrobiol., 1984, vol. 139, Czechoslovakia, June 22-27, 1987. Academia Pub- 257-265 [0008] lishing House of the Czechoslovak Academy of Sci- • MC QUILKEN M. P. ; J. M. WHIPPS ; R. C. COOKE. ences, Elsevier Science Publishers, 1989 [0005] Oospores of the biocontrol agent Pythium oligandrum • KRATKA J. BERGMANOVÁ ; A. KUDELOVA. Ef- bulk produced in liquid culture.Mycol. Res., 1990, fect of Pythium oligandrum and Pythium ultimum on vol. 94 (5), 613-616 [0008] biochemical changes in cucumber. Cucumis sativus • BROZˇ OVÁ J. Exploitation of the mycoparasitic fun- L, 1994, vol. 101 /4, 406-413 [0005] gus Pythium oligandrum in plant protection.Plant • AL-RAWAHI A. K. ; J. G. HANCOCK. Rhizosphere Protection Science, 2002, vol. 38 (1), 29-35 [0009] Competence of Pythium oligandrum. Phytopatholo- • Biological protection of the main cereals against fun- gy, 1997, vol. 87 (9), 951-959 [0006] gal specific diseases. H®SEK J. ; VACH M. ; JAVU° - • BENHAMOU N. ; P. REY ; M. CHÉRIF ; J. REK M. Commun. Agricult. Applied Biolog. Sci. Gh- HOCKENHULL ; Y. TIRILLY. Treatment with the ent University, 2005, vol. 70, 169-173 [0010] Mycoparasite Pythium oligandrum Triggers Induction • Sˇ VECOVÁ M. ; CˇÍZ ˇ KOVÁ D. ; VESEL® D. The effect of Defence-related reactions in Tomato Roots When of Pythium oligandrum Drechsler mycoparasite of Challenged with Fusarium oxysporum f. sp. radi- fungal species of the genus Ceratocystis s.l.Les- cis-lycopersici. Phytopathology, 1997, vol. 87 (1), nictvi, 1998, vol. 44 (9), 411-420 [0011] 108-122 [0006] • KUNCA A. ; LEONTOVYC R. Relationship between •MÍSˇ A D. Efficiency of biological preparations against beech bark wounds and bark necrosis and possible grey mould lBofrytis cinerea Pers. et Fries /on grape- methods to control bark necrosis. Lesnick» cˇasopis, vine. Proceedings of the XIV Slovak and Czech Plant 1999, vol. 45 (5/6), 317-324 [0013] Protection Conference in Nitra 3rd-4th September • LAING S.A.K. ; DEACON J.W. Aggressiveness and 1997, 1997, 294-296 [0006] fungal host ranges of mycoparasitic Pythium species. Soil Biol Biochem, 1990, vol. 22 (7), 905-911 [0014]

8 EP 2 503 892 B1

• KURZAWINSKA H. ; MAZUR S. The evaluation of • PATKOWSKA E. Effectiveness of grapefruit extract Pythium oligandrum and chitosan in control of Phy- and Pythium oligandrum in the control of bean and tophthora infestans (Mont.) de Bary on potato plants. peas pathogens. Journal of Plant Protection Re- Folia Holticulturae, 2009, vol. 21 (2), 12-23 [0015] search, 2006, vol. 46 (1), 15-28 [0016]