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(11) EP 2 313 488 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 1/14 (2006.01) A01N 63/04 (2006.01) 29.04.2015 Bulletin 2015/18 (86) International application number: (21) Application number: 09775812.2 PCT/CZ2009/000088

(22) Date of filing: 23.06.2009 (87) International publication number: WO 2010/006563 (21.01.2010 Gazette 2010/03)

(54) STRAIN OF ENTOMOPATHOGENIC FUMOSOROSEA CCM 8367 (CCEFO. 011.PFR) AND METHOD FOR CONTROLLING INSECT AND MITE PESTS STAMM DES ENTOMOPATHOGENEN PILZES ISARIA FUMOSOROSEA CCM 8367 (CCEFO. 011.PFR) UND VERFAHREN ZUR BEKÄMPFUNG VON INSEKTEN- UND MILBEN- SCHÄDLINGEN SOUCHE DU CHAMPIGNON ENTOMOPATHOGÈNE ISARIA FUMOSOROSEA CCM 8367 (CCEFO. 011.PFR) ET PROCÉDÉ POUR LUTTER CONTRE LES INSECTES ET LES ACARIENS NUISIBLES

(84) Designated Contracting States: • ZEMEK ROSTISLAV ET AL: "Perspectives for the AT BE BG CH CY CZ DE DK EE ES FI FR GB GR biological control of Cameraria ohridella." HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL COMMUNICATIONS IN AGRICULTURAL AND PT RO SE SI SK TR APPLIED BIOLOGICAL SCIENCES, vol. 72, no. 3, 2007, pages 521-526, XP008114865 ISSN: (30) Priority: 23.06.2008 CZ 20080394 1379-1176 • ZEMEK ROSTISLAV ET AL: "The first record of (43) Date of publication of application: 27.04.2011 Bulletin 2011/17 fumosoroseus(Deuteromycota :) on the hibernating pupae of Cameraria ohridella (73) Proprietor: Biology Centre AV CR, v.v.i. (Lepidoptera : Gracillariidae)" 370 05 Ceske Budejovice (CZ) ENTOMOLOGICAL RESEARCH, vol. 37, no. Suppl. 1, August 2007 (2007-08), pages A135- (72) Inventors: A136, XP008114913 ISSN: 1738-2297 • PRENEROVA, Eva • RICHTER DOREEN ET AL: "Pathogenicity of 398 43 Bernartice u Milevska (CZ) entomopathogenic fungi on hibernating pupae of • ZEMEK, Rostislav Cameraria ohridella Deschka & Dimic 1986 370 08 Stare Hodejovice (CZ) (Lepidoptera, Gracillariidae). Part 1: • VOLTER, Lubomír Pathogenicity against the naked pupa." 323 00 Plzen (CZ) COMMUNICATIONS IN AGRICULTURAL AND • WEYDA, Frantisek APPLIED BIOLOGICAL SCIENCES, vol. 72, no. 3, 370 05 Ceske Budejovice (CZ) 2007, pages 399-410, XP008114864 ISSN: 1379-1176 (74) Representative: Beetz & Partner mbB • RICHTER DOREEN ET AL: "Pathogenicity of Patentanwälte entomopathogenic fungi on hibernating pupae of Steinsdorfstraße 10 Cameraria ohridella Deschka & Dimic 1986 80538 München (DE) (Lepidoptera, Gracillariidae). Part 2: Efficacy of entomopathogenic fungi against pupa in her (56) References cited: pupal cell." COMMUNICATIONS IN EP-A- 0 494 592 WO-A-2007/059765 AGRICULTURAL AND APPLIED BIOLOGICAL SCIENCES, vol. 72, no. 3, 2007, pages 411-421, XP008114863 ISSN: 1379-1176

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• KALMUS M ET AL: "Efficacy of • FARGUES ET AL: "Genetic variability among entomopathogenic fungi against eggs and larvae Paecilomyces fumosoroseus isolates from of the horse chestnut leafminer Cameraria various geographical and host insect origins ohridella" JOURNAL OF PLANT DISEASES AND based on the rDNA- ITS regions" MYCOLOGICAL PROTECTION, vol. 114, no. 2, April 2007 RESEARCH, vol. 106, no. 9, 1 September 2002 (2007-04), page 92, XP001537307 ISSN: 1861-3829 (2002-09-01), pages 1066-1074, XP022474272 • CANTONE ET AL: "Intraspecific diversity in ISSN: 0953-7562 Paecilomyces fumosoroseus" MYCOLOGICAL RESEARCH, ELSEVIER, GB, vol. 102, no. 2, 1 February 1998 (1998-02-01), pages 209-215, XP022451572 ISSN: 0953-7562

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Description in modem applied entomology and their development reached the stage of commercial bioinsecticides in many Technical field countries. [0006] Entomopathogenic fungus Isaria fumosorosea [0001] Mycoinsecticid which is usable as a considerate 5 (syn. Paecilomyces fumosoroseus) (WIZE) Brown & biological mean of plant and wood protection against in- Smith is in comparison to other species of entomopath- sect and mite pests. ogenic Deuteromycet ( , , Verticillium lecanii etc.) a less studied spe- Background art cies. Fungus Isaria fumosorosea (syn. Paecilomyces fu- 10 mosoroseus), often reported under abbreviation PFR, is [0002] Plant protection against insect pests is per- a cosmopolite polyphagous entomopathogenic species. formed largely using conventional chemical insecticides. Most records of the isolation of this fungus from insects Their application is, however, problematic from nature (under natural conditions) frequently report species of conservation and human health points of view. Insects the orders Lepidoptera, Diptera and Coleoptera as the become more and more resistant to them which in turn 15 hosts. The PFR was for the first time reported as naturally leads to the increase of dosage or needs to develop other occurring pathogen in the Trialeurodes( vapo- more effective insecticides. The opportunities in this di- rariorum) populations in Peking in 1983, where strong rection are approaching the limits. More rigorous legis- local epizootics occurred, which temporarily eradicated lative also restrains this way of pest control. this pest. This strain of PFR was isolated and as highly [0003] The horse chestnut leaf miner,Cameraria 20 virulent against greenhouse whitefly was assigned as a ohridella (Deschka et Dimi ) (Lepidoptera: Gracillarii- subspecies Paecilomyces fumosoroseus var. beijingen- dae) has favourable conditions for its development in the sis. Czech Republic. This pest was discovered for the first [0007] Substantial increase of interest in this pathogen time at Ohrid lake in Macedonia between Albania and happened when harmfulness of the whitefly Bemisia ta- Greece in 1984. This important pest of the horse chestnut 25 baci increased in field agroecosystems in Southern USA. started to spread invasively through whole Europe where Since 1987 regular spontaneous epizootics of Paecilo- it has good conditions for its development (for the lack myces fumosoroseus in Bemisia tabaci populations on of natural enemies) while not being enough regulated by various host plants/localities were reported in Florida natural mechanisms. Damage inflicted to horse chestnut where a new strain ofPaecilomyces fumosoroseus leaves cause serious weakening of most of the infested 30 (PFR) was isolated in 1987. The isolated strain was as- trees. Contemporary pest control methods are based ei- signed as PFR 97 - strain Apopka (Apopka - name of the ther on the application of non-selective insecticides or region in Florida. The pure culture was identified in 1988 composting or burning of decaying (fallen) leaves. All and deposited at the American Type Culture Collection these methods, however, also kill beneficial organisms (strain ATCC 20874). Strain PFR 97 turned out to be able including natural enemies of Cameraria ohridella. 35 to cause large epizootics in Bemisia tabaci populations [0004] An application of biological control, i.e. methods both in greenhouses and in the field crops. Further ex- based on natural antagonistic interactions between or- periments with this strain proved the polyphagous feature ganisms for suppression of pest population is safer to of this strain and a high virulence against , thrips, natural balance and stability of agricultural and forests larvae of certain moth species, larvae and pupae of leaf- ecosystems. Most of these biocontrol agents are harm- 40 miners (Diptera) and even a high activity against two- less for non-target organisms including man and, contra- spotted spider mite Tetranychus( urticae). Contrary to ry to chemical insecticides, do not posses any ecological the fungus Verticillium lecanii, PFR is able to cause in- risk for environment and thus contribute to biodiversity fection also in the eggs of and spider mites. conservation.For this reason,biocontrol agents are more [0008] R. Zemek et al. (Comm. Appl. Biol. Sci., Vol. and more preferred as alternative means of insect pest 45 72, No. 3, 2007, p. 521) have studied the impact of Pae- control at the global scale. cilomyces fumosoroseus, Paecilomyces farinosus and [0005] Mycoinsecticides are such means of biological Beauveria bassiana on the horse chestnut leaf-miner control. They are based on entomopathogenic fungi. For (Cameraria ohridella) and discuss the perspectives of practical pest control representatives of several key Deu- fungal bioagents in control of C. ohridella. In a short com- teromycet (Hyphomycetes: Moniliales) are of dominant 50 munication (Entomological Research, Vol. 37, Suppl. 1, importance.The best known are entomopathogenicfungi August 2007 (2007-08), pages A135-A136) R. Zemek et belonging to the genera Beauveria, Hirsutella, Metarhizi- al. provide a first record of the effect of entomopathogenic um, Nomuraea, Isaria (Paecilomyces), Tolypocladium fungus Paecilomyces fumosoroseus (Deuteromycota: and Verticillium (Lecanicillium). Among many species of Hyphomydetes) on the hibernating pupae of Cameraria these genera about 25 species are contemporarily used 55 ohridella (Lepidoptera: Gracillariidae). in the form of standard biopesticides. Application of my- [0009] D. Richter et al. (Comm. Appl. Biol. Sci., Vol. coinsecticides in pest control is one of the approaches 72, No. 3, 2007 pages 399-410) have examined strains from Paecilomyces fumosoroseus and other ento-

3 3 EP 2 313 488 B1 4 mopathogenic fungi in a standardized biotest to control Figure the horse-chestnut leaf miner in her pupal stage in winter. Results of the efficacy of these fungi against pupae of C. [0013] ohridella and on naked pupa of C. ohridella at different temperatures and different soil humidity are presented. 5 Figure 1: Cumulative mortality of Cameraria ohridel- In a further communication of D. Richter et al. (Comm. la pupae after the treatment with conidiospores or Appl. Biol. Sci., Vol. 72, No. 3, 2007, pages 411-421) blastospores of CCM 8367 strain of Isaria fumoso- similar experiments are carried out to examine the path- rosea or with distilled water (control). ogenicity of entomopathogenic fungi against the naked pupa of C. ohridella. 10 Examples [0010] M. Kalmus and C. Büttner (Journal of Plant Dis- eases and Protection, Vol. 114, No. 2, April 2007 Example 1 (2007-4), page 92) examine the efficacy of entomopath- ogenic fungi against eggs and larvae of the horse chest- Application of conidiospores of CCM 8367 strain of Isaria nut leafminer Cameraria ohridella.Hatching ratesof eggs 15 fumosorosea against pupae of the horse chestnut leaf in a tray experiments and mortality of larvae in the seed- miner ling experiment are presented. [0014] Conidiospores were collected from the fungus Disclosure of the invention Isariafumosorosea grown on agar dish (Sabouraud agar) 20 and put into 50 ml of sterile distilled water with 10 ml of [0011] Research and development of new means of the soaking agent Tween. Homogenous suspension pre- plant protection based on influencing physiological proc- pared by a hand homogenizer was diluted to the concen- esses in a pest organism (so called biorational insecti- tration of 5x107 conidiospores/ml. cides, i.e. means based on natural models), or on prin- One hundred of hibernating pupae of the horse chestnut ciples of biological control when natural enemies of pests 25 leafminer was collected from fallen leaves of horse chest- like predators, parasitoids or pathogenic organisms are nut trees. Each pupa was immersed (dipped) into the used, is a solution for modem plant protection. Biological prepared suspension for one second. 100 pupae im- control methods utilise natural antagonistic interactions mersed to distilled water with Tween only served as a between organisms for the suppression of pest popula- control. Pupae were then placed on filter paper to remove tion without disturbing natural balance so that they con- 30 excess suspension or water, put into dishes with high tribute to the stability of ecosystems in man-affected en- humidity and incubated in dark climatized box at 23°C. vironment. The biocontrol agents proved to be harmless Survival of pupae was checked using dissection micro- for the non-target organisms including man and, contrary scope by mechanical stimulation at 12-hours intervals. to chemical insecticides, do not posses any ecological Resulting mortality of pupae treated by conidiospores or risk for the environment and thus contribute to biodiver- 35 water only is shown in Fig. 1. sity conservation. For this reason, biocontrol agents are more and more preferred as alternative means of insect Example 2 pest control at the global scale. Our strain of entomopath- ogenic fungus Isaria fumosorosea with high virulence is Application of blastospores of CCM 8367 strain of Isaria a prospective biocontrol agent against insect and mite 40 fumosorosea against pupae of the horse chestnut leaf pests. The Isaria fumosorosea strain is deposited under miner number CCM 8367 (CCEFO.011.PFR) as patent culture in CCM collection (Czech Collection of Microorganisms) [0015] The strain of Isaria fumosorosea was cultivated in Brno. Spores of this microorganism attack insect pests using orbital shaker at 140 cycles per minute and tem- and mites and kill them. 45 perature 23°C using grow media of the following compo- [0012] A preparation based on the spores of Isaria fu- sition: 2% glucose, 2% maltose, 2% peptone for bacte- mosorosea strain CCM 8367 for control of insect pests riology, pH=5.5. The cultivation was stopped after 120 (belonging to the orders Lepidoptera, Coleoptera, Hy- hours and the biomass of blastospores was isolated from menoptera and Homoptera) and mites is made by sub- the grow media by centrifugation at 2500 rotations/min. merged or surface cultivation. For application, homoge- 50 for 20 minutes. A suspension of blastospores was pre- nous suspension of spores is prepared from a biomass pared from the biomass by diluting with distilled water so concentrate or the spores are applied in the mixture with that the concentration was 5 3107 blastospores/ml. One an inert carrier. After the application the fungus grows hundred of hibernating pupae of the horse chestnut leaf and infects insect pests and mites which then die. The miner was collected from fallen leaves of the horse chest- advantage is that the fungus is a microorganism naturally 55 nut trees. Each pupa was immersed into the prepared occurring in the environment, and thus no disturbance of suspension of blastospores for one second. 100 pupae the ecosystem is made. immersed to distilled water with Tween only served as a negative control. The pupae were then placed on filter

4 5 EP 2 313 488 B1 6 paper to remove excess suspension or water, put into pension or water the leaves were placed into dishes with dishes with high humidity and incubated in dark clima- high humidity and incubated in dark climatized box at tized box at 23°C. Survival of pupae was checked using 23°C. Mortality of the eggs was assessed one week later. dissection microscope by mechanical stimulation at 12- Totally, 143 eggs were treated with the fungus, 314 hours intervals. Resulting mortality of pupae treated by 5 served as a negative control. Results showed that the blastospores or water only is shown in Fig. 1. mortality of eggs treated with the suspension of blast- ospores was 88.81%, by 76% higher compared to that Example 3 of negative control.

Application of conidiospores of CCM 8367 strain of Isaria 10 Industrial applicability fumosorosea against the eggs of the horse chestnut leaf miner [0018] Biological protection of plants, woods and other organic substrates against insect and mite pests. [0016] Conidiospores were collected from fungus Isar- ia fumosorosea grown on agar dish (Sabouraud agar) 15 and put into 100 ml of sterile distilled water with 20m l Claims soaking agent Tween. Homogenous suspension pre- pared by a hand homogenizer was diluted to the concen- 1. The strain of the entomopathogenic fungusIsaria tration of 53107 conidiospores/ml. A leaf of the horse fumosorosea CCM 8367 (CCEFO.011.PFR) depos- chestnut tree, Aesculus hippocastanum, with deposited 20 ited in the collection CCM (Czech Collection of Mi- eggs of Cameraria ohridella (Deschka et Dimi ) (Lepi- croorganisms) in Brno, Czech Republic. doptera: Gracillariidae) was immersed into the prepared suspension for one second. Another leaf with the eggs 2. The method for controlling insect and mite pests us- immersed into distilled water with Tween only served as ing entomopathogenic fungus Isaria fumosorosea a negative control. After removing excess suspension or 25 CCM 8367 (CCEFO.011.PFR) according to claim 1, water the leaves were placed into dishes with high hu- wherein infested plants and woods or substrate of midity and incubated in a dark climatized box at 23°C. organic origin are treated by its spores. Mortality of the eggs was assessed one week later. To- tally, 102 eggs were treated with the fungus, 314 eggs 3. The method for controlling insect and mite pests ac- served as a negative control. Results showed that the 30 cording to claim 2, wherein the spores are in liquid mortality of eggs treated with the suspension of conid- or solid inert carrier. iospores was 52.94%, by 40% higher compared to that of negative control. Patentansprüche Example 4 35 1. Stamm des insektenpathogenen Pilzes Isaria fumo- Application of blastospores of CCM 8367 strain of Isaria sorosea CCM 8367 (CCEFO.011.PFR), Bestandteil fumosorosea against the eggs of the horse chestnut leaf der Tschechischen Sammlung von Mikroorganis- miner men (Czech Collection of Microorganisms, CCM) in 40 Brno, Tschechische Republik. [0017] The strain of Isaria fumosorosea was cultivated using orbital shaker at 140 cycles per minute and tem- 2. Verfahren zur Bekämpfung von schädlichen Insek- perature 23°C using grow media of the following compo- ten und Milben mit Hilfe des insektenpathogenen Pil- sition: 2% glucose, 2% maltose, 2% peptone for bacte- zes Isaria fumosorosea CCM 8367 (CCE- riology, pH=5.5. The cultivation was stopped after 120 45 FO.011.PFR) gemäß Anspruch 1, wobei die befal- hours and the biomass of blastospores was isolated from lenen Pflanzen, Gehölze oder Substrate organi- grow media by centrifugation at 2500 rotations/min. for schen Ursprungs mit den Pilzsporen behandelt wer- 20 minutes. A suspension of blastospores was prepared den. from the biomass by diluting in 100 ml of distilled water with 20 ml of the soaking agent Tween so that the con- 50 3. Verfahren zur Bekämpfung von schädlichen Insek- centration was 53107 blastospores/ml. A leaf of the ten undMilben gemäß Anspruch 2, wobei dieSporen horse chestnut tree, Aesculus hippocastanum, with de- in einem flüssigen oder festen inerten Träger vorlie- gen. posited eggs of Cameraria ohridella (Deschka et Dimi ) (Lepidoptera: Gracillariidae) was immersed into pre- 55 pared suspension for one second. Another leaf with the Revendications eggs immersed into distilled water with Tween only served as a negative control. After removing excess sus- 1. Souche du champignon entomopathogène Isaria fu-

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mosorosea CCM 8367 (CCEFO.011.PFR) déposée à la Collection tchèque de microorganismes (Czech Collection of Microorganisms, CCM) à Brno, Répu- blique tchèque. 5 2. Procédé d’élimination des insectes et des acariens nuisibles à l’aide du champignon entomopathogène Isaria fumosorosea CCM 8367 (CCEFO.011.PFR) selon la revendication 1, dans lequel les bois, les plantes ou les substrats d’origine biologique infestés 10 sont traités avec des spores de la souche susmen- tionnée.

3. Procédé d’élimination des insectes et des acariens nuisibles selon la revendication 2, dans lequel les 15 spores sont fixées à un support inerte solide ou li- quide.

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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.

Non-patent literature cited in the description

• R. ZEMEK et al. Comm. Appl. Biol. Sci., 2007, vol. • D. RICHTER et al. Comm. Appl. Biol. Sci., 2007, vol. 72 (3), 521 [0008] 72 (3), 411-421 [0009] •R.ZEMEK. Entomological Research, August 2007, • M. KALMUS ; C. BÜTTNER. Journal of Plant Dis- vol. 37 (1), A135-A136 [0008] eases and Protection, April 2007, vol. 114 (2), 92 • D. RICHTER et al. Comm. Appl. Biol. Sci., 2007, vol. [0010] 72 (3), 399-410 [0009]

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