WO2021080419A1 - Attractif pour hyperparasitoïde aphide - Google Patents

Attractif pour hyperparasitoïde aphide Download PDF

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Publication number
WO2021080419A1
WO2021080419A1 PCT/NL2020/050644 NL2020050644W WO2021080419A1 WO 2021080419 A1 WO2021080419 A1 WO 2021080419A1 NL 2020050644 W NL2020050644 W NL 2020050644W WO 2021080419 A1 WO2021080419 A1 WO 2021080419A1
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Prior art keywords
attractant
species
hyperparasitoid
aphid
group
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PCT/NL2020/050644
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English (en)
Inventor
Jetske Gudrun DE BOER
Tim GOELEN
Bart Lievens
Hans REDIERS
Hans JACQUEMYN
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Nederlands Instituut Voor Ecologie (Nioo-Knaw)
Katholieke Universiteit Leuven
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Publication of WO2021080419A1 publication Critical patent/WO2021080419A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N27/00—Biocides, pest repellants or attractants, or plant growth regulators containing hydrocarbons

Definitions

  • the invention relates to a use of an attractant to attract an aphid hyperparasitoid.
  • the invention further relates to an attractant for an aphid hyperparasitoid.
  • the invention further relates to a trap comprising the attractant.
  • Insect attractants are known in the art.
  • CN103210960 describes a flying insect pest attractant with an attracting effect to various flying insect pest, and simultaneously provides a flying insect pest attracting and controlling agent which has attracting and controlling effects to the various flying insect pest.
  • the flying insect pest attractant takes balsamic vinegar or attractant material containing balsamic vinegar as effective constituent.
  • the flying insect pest attracting and controlling agent includes the balsamic vinegar or attractant material containing the balsamic vinegar and pesticide.
  • Biological (pest) control may involve using a biological control agent, such as an insect, to reduce the population of an undesired organism via, for example, predation, parasitism and/or herbivory.
  • a biological control agent such as an insect
  • the undesired organism may be detrimental to the growth of (food) plants, and the biological control agent thus relieves the detrimental effect.
  • a biological control agent may especially be an organism that is used to control (the population of) an undesired organism.
  • the effectiveness of the biological control agent may be reduced by a number of factors, including biotic factors such as plants, (other) insects, micro-organisms, as well as abiotic factors such as pesticides, temperature and lighting.
  • the biological control agent may become exposed to a natural enemy of its own, thereby reducing its effectiveness in reducing the population of the undesired organism.
  • the wasp Aphid ins colemani is widely applied as a biological control agent as it may successfully parasitize a variety of (economically relevant) pest aphids detrimental to plant growth.
  • the hyperparasitoid Dendrocerm aphidum may parasitize A. colemani , thereby reducing the population of A. colemani and reducing its effectiveness as a biological control agent towards the pest aphids.
  • hyperparasitoid such as Dendrocerus aphidum
  • a biological control agent sensitive to the hyperparasitoid such as A. colemani to D. aphidum
  • biological pest control such as A. colemani to D. aphidum
  • the prior art may describe the use of pesticides to combat such a hyperparasitoid.
  • the use of pesticides may be undesirable, or even not permitted for organic and biodynamic agriculture, at least in part due to off-target effects against other organisms, including beneficial insects, especially against the biological control agent.
  • the prior art describes continuously providing more of the biological control agent to maintain sufficient population levels, which may be expensive and may create an environment for a hyperparasitoid to thrive in.
  • the prior art may describe alternating the biological control agent to create periods disadvantageous for a particular hyperparasitoid, which may imply not optimizing the use of the biological control agent against the primary pest. Further, the alternative biological control agents may typically be more expensive than the primary biological control agent.
  • the prior art may describe the use of attractants to attract an undesired organism, such as the hyperparasitoid, but these may be unspecific and may further attract the biological control agent. Trapping the biological control agent together with its hyperparasitoid may be particularly disadvantageous.
  • the present invention may have as object to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.
  • bacteria producing relatively high levels of monoterpenes and/or alkenes were attractive to the hyperparasitoid Dendrocerus aphidum.
  • these bacteria may produce relatively high levels of the monoterpenes limonene, linalool and/or geraniol, and/or the alkenes 1-decene, 9-methyl- 1-decene, and 1-tetradecene.
  • bacteria producing high levels of these monoterpenes and/or alkenes were also found to be repellent and/or neutral (not repellent nor attracting) to the often-used biological control agent Aphidius colemani , which is targeted by Dendrocerus aphidum.
  • the invention may provide a use of an attractant to attract an aphid hyperparasitoid, especially wherein the aphid hyperparasitoid species is a hymenopteran species.
  • the attractant may especially comprise a monoterpene selected from the group comprising limonene, linalool, and geraniol.
  • the attractant may especially comprise an alkene selected from the group comprising 1-decene, 9-methyl- 1-decene, and 1-tetradecene.
  • the use of the attractant according to the invention may enable attracting the undesired hyperparasitoid Dendrocerus aphidum , especially into a trap, while not affecting or repelling the desired biological control agent Aphidius colemani.
  • the attractant may essentially not affect the behavior of the biological control agent, i.e. neither attract nor repel the biological control agent, such that the attractant does not have a (detrimental) effect on the effectiveness of the biological control agent.
  • the biological control agent can continue to perform its intended functionalities, especially parasitizing on aphids, such as in an agricultural setting, whereas the threat presented by the hyperparasitoid can be removed with limited to no detrimental effect on the biological control agent.
  • the invention may provide a use of an attractant to attract an aphid hyperparasitoid.
  • the aphid hyperparasitoid may especially be a hymenopteran species, especially a Megaspilidae species, such as D. aphidum , or especially a Pteromalidae species, such as an Asaphes species or a Pachyneuron species, especially an Asaphes species selected from A. suspensus and A. vulgaris , or especially Pachyneuron aphidis.
  • the hymenopteran species may comprise a species selected from the genera Dendrocerus, Asaphes, Pachyneuron, Euneura, Coruna, and Syrphophagus, especially from the genera Dendrocerus, Asaphes, Pachyneuron, Euneura, and Coruna, more especially from the genera Dendrocerus, Asaphes, and Pachyneuron.
  • aphid hyperparasitoid may herein especially refer to an aphid hyperparasitoid that parasitizes on an aphid parasitoid, wherein the aphid parasitoid parasitizes on aphids.
  • the aphid hyperparasitoid may be a hyperparasitoid that uses a mummified aphid as host.
  • the hyperparasitoid D. aphidum may parasitize on the (primary) aphid parasitoid A. colemani, particularly targeting aphids mummified by A. colemani.
  • the biological control agent may be a primary parasitoid
  • the aphid hyperparasitoid may be a hyperparasitoid which, undesirably, parasitizes on the primary parasitoid.
  • the term “attractant” and similar terms may herein refer to an agent or substance that attracts a particular (type of) organism, especially an aphid hyperparasitoid, such as an aphid hyperparasitoid being a hymenopteran species, especially a Megaspilidae species.
  • the attractant may comprise one or more volatile compounds, and the target organism may be attracted to the one or more volatile compounds by olfaction. The attracted organism may especially be tempted to approach the (source of the) attractant.
  • the attractant is especially not (particularly) attractive for the biological control agent.
  • embodiments may comprise use of the attractant as a lure (or: “bait”).
  • Megaspilidae species and similar terms herein especially refer to a species belonging to the indicated genus/family/order, in this specific case a species belonging to the family of Megaspilidae.
  • the term Megaspilidae species may refer to a species belonging to any one of the genera Aetholagynodes , Archisynarsis, Conostigmus, Creator , Dendrocerus, Holophleps, Lagynodes, Megaspilus , Platyceraphron, Prolagynodes, Trassedia, Trichosteresis, and Typhlolagynodes , especially Dendrocerus.
  • the Megaspilidae species may be a hyperparasitoid.
  • the attractant may comprise an attractive compound, especially a terpene, more especially a monoterpene.
  • the term “attractive compound” may also refer to a plurality of attractive compounds.
  • the attractant may comprise a plurality of attractive compounds.
  • attraction may be provided by a plurality of compounds that together provide the attractive effect.
  • the attractant may further comprise a solvent, a carrier, or any other compound to aid in the storage and/or application of the attractant.
  • the attractant may comprise an agriculturally acceptable solvent, carrier, or other compound to aid in the storage and/or application of the attractant.
  • Terpenes are a large and diverse class of organic compounds, and comprise a wide variety of functional groups. Specifically, terpenes may be a class of hydrocarbons empirically regarded as consisting of (subunits) of isoprene (and/or isopentane), which has 5 carbon atoms.
  • a monoterpene may be a terpene empirically regarded as comprising two isoprene (and/or isopentane) subunits, and may thus comprise 10 carbon atoms.
  • Monoterpenes may comprise rings, but may also comprise an acyclic structure.
  • the term “terpene” may also refer to a terpenoid; a modified terpene with one or more additional functional groups, especially oxygen-comprising functional groups, such as a functional group selected from the group comprising a hydroxyl group, a ketone group, an aldehyde group, and a C-O-C ether bond.
  • the term “monoterpene” may herein refer to a monoterpenoid.
  • the terpene may comprise a terpenoid.
  • the monoterpene may comprise a monoterpenoid.
  • the terpene, especially the monoterpene may consist of C and H atoms.
  • the monoterpene may be selected from the group comprising limonene, linalool, and geraniol.
  • the monoterpene may comprise limonene, especially (R)- limonene, or especially (S)-limonene. In further embodiments, the monoterpene may comprise a mixture of (R)-limonene and (S)-limonene.
  • the monoterpene, especially the monoterpenoid may comprise linalool, especially (R)-linalool, or especially (S)-linalool. In further embodiments, the monoterpene may comprise a mixture of (R)-linalool and (S)-linalool.
  • the monoterpene especially the monoterpenoid, may comprise geraniol, especially trans-geraniol, or especially cis-geraniol (also known as “nerol”). In further embodiments, the monoterpene may comprise a mixture of trans-geraniol and cis- geraniol.
  • Two enantiomers and/or stereo-isomers of aterpene, especially of a monoterpene, may differ in properties, such as having different smells and effects on behavior. Hence, one of the enantiomers may be preferred over the other for the attractant.
  • the invention may provide a use of an attractant to attract an aphid hyperparasitoid, wherein the attractant comprises a monoterpene selected from the group comprising limonene, linalool, and geraniol and/or an alkene selected from the group comprising 1-decene, 9-methyl- 1-decene, and 1-tetradecene.
  • the attractant may comprise a monoterpene selected from the group comprising limonene, linalool, and geraniol.
  • the attractant may comprise a micro-organism, especially wherein the micro-organism produces, especially secretes (also: “emits”), an attractive compound (for an aphid hyperparasitoid, especially an aphid hyperparasitoid being a hymenopteran species, especially a Megaspilidae species), especially the monoterpene and/or the alkene.
  • the micro organism may especially comprise a prokaryote or a fungus, especially a prokaryote, such as a bacterium or an archaeum.
  • micro-organisms were identified to be attractive to Dendrocerm aphidum , while being repellent or at least unattractive to Aphidius colemani.
  • these micro-organisms may produce relatively high levels of monoterpenes, especially one or more of limonene, linalool and geraniol.
  • the phylogenetic affiliation of the micro-organisms was evaluated by amplifying and sequencing their 16S ribosomal RNA (rRNA) gene and comparing the sequence with type strains in the EzBiocloud 16S rRNA gene and whole-genome assembly database, which is described by Yoon et ah, “ Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies ”, International Journal of Systematic and Evolutionary Microbiology, 2017. The comparison to type strains based on 16S rRNA gene sequences may not provide a conclusive phylogenetic identification, but may provide a rough phylogenetic affiliation of the micro-organisms.
  • rRNA 16S ribosomal RNA
  • the micro-organism may have a 16S rRNA gene identity of at least 90%, such as at least 95%, especially at least 99%, such as at least 99.5%, with a species selected from a group of bacterial species comprising Staphylococcus spp. or with a species selected from a group of bacterial species comprising Curtobacterium spp, such as with a species selected from the group comprising Staphylococcus saprophyticus, or with a species selected from the group comprising Curtobacterium flaccumfaciens and Curtobacterium oceanosedimentum.
  • the micro-organism may comprise a species selected from the group comprising Staphylococcus spp.
  • the micro-organism may be selected from the group comprising Staphylococcus spp. and Curtobacterium spp., especially from the group comprising S. saprophyticus, or the group comprising C. flaccumfaciens and C. oceanosedimentum.
  • Curtobacterium strains especially from the group comprising C. flaccumfaciens and C. oceanosedimentum , appear substantially and significantly attractive to I). aphidum , while evoking a neutral response on A. colemani.
  • the micro-organism may comprise a Staphylococcus species, especially the micro-organism may have a 16S rRNA gene identity of at least 90%, such as at least 95%, especially at least 99%, such as at least 99.5%, with S. saprophyticus. More especially the micro-organism may comprise S. saprophyticus , such as a S. saprophyticus strain isolated from Macrosiphum euphorbiae aphids. In particular, the micro-organism may comprise isolate ST18.16/160 (genbank accession number 16S ribosomal RNA gene sequence MK875120).
  • the micro-organism may comprise a Curtobacterium sp., especially the micro-organism may have a 16S rRNA gene identity of at least 90%, such as at least 95%, especially at least 99%, such as at least 99.5%, with C. flaccumfaciens and C. oceonasedimentum. More especially, the micro-organism may comprise one or more strains belonging to C. flaccumfaciens or C. oceanosedimentum. In further embodiments, the micro organism may comprise C. flaccumfaciens. In further embodiments, the micro-organism may comprise C. oceanosedimentum.
  • the micro-organism may comprise a Curtobacterium strain isolated from Myus persicae nicotianae aphids.
  • the micro organism may comprise isolate ST18.16/085 (genbank accession number 16S ribosomal RNA gene sequence MK875112).
  • ST18.16/085 has been deposited at the international depositary authority Belgian Coordinated Collections of Micro-organisms (BCCM), Laboratorium voor Microbiologie (LMG), in accordance with the Budapest treaty. ST18.16/085 has been assigned accession number LMG P-31537 by the depositary authority.
  • ST18.16/160 has been deposited at the international depositary authority Belgian Coordinated Collections of Micro-organisms (BCCM), Laboratorium voor Microbiologie (LMG), in accordance with the Budapest treaty. ST18.16/160 has been assigned accession number LMG P-31538 by the depositary authority.
  • the Megaspilidae species may comprise a Megaspilinae species, especially a Dendrocerus species, i.e., in embodiments the invention may provide a use of an attractant to attract a Megaspilinae species, especially a Dendrocerus species.
  • the Dendrocerus species may comprise Dendrocerus aphidum.
  • the attractant may comprise a repellent for a Braconidae species.
  • repellent and similar terms may refer to the opposite of an attractant, i.e., the repelled organism may especially be tempted to keep distance from the repellent.
  • the attractant may comprise an attractive compound (for a Megaspilidae species) and a repellent compound (for a Braconidae species).
  • the attractive compound and the repellent compound may be the same compound.
  • the attractant may comprise a plurality of compounds that (together) provide an attractive effect on a Megaspilidae species and a repellent effect on a Braconidae species.
  • the repellent compound for the Braconidae species may be selected from compounds not repellent for a Megaspilidae species, i.e., from the group comprising attractive or neutral compounds for a Megaspilidae species.
  • the attractant may be neutral to a Braconidae species, i.e., the attractant may comprise one or more compounds, wherein the one or more compounds (together) do not evoke a repelling or attracting effect on the Braconidae species.
  • the one or more compounds may comprise both compounds attractive to the Braconidae species and compounds repellent to the Braconidae species, such that the attractant as a whole is neutral to the Braconidae species.
  • the Braconidae species may comprise an Aphidius species, especially Aphidius colemani.
  • the use of the attractant may comprise attracting Dendrocerus aphidum and repelling or being neutral to Aphidius colemani , i.e., the use of the attractant may comprise exposing an object to the attractant to attract/ ) aphidum , and especially to repel or to be neutral to A. colemani.
  • the object may, for example, comprise a surface (exposed to the attractant, especially coated with the attractant) or may comprise a trap.
  • the attractant is repelling or neutral, especially neutral, may be particularly beneficial as then Aphidius colemani can carry out its role as biological control agent with reduced, or even no, interference from Dendrocerus aphidum.
  • the monoterpenes limonene, linalool, and geraniol attract Dendrocerus aphidum and repel or are neutral to Aphidius colemani.
  • the attractant may comprise a composition (“attractant composition”).
  • the composition may especially comprise two or more compounds.
  • the composition may comprise a monoterpene, especially a monoterpene selected from the group comprising limonene, linalool, and geraniol.
  • the composition may comprise an alkene, especially an alkene selected from the group comprising 1-decene, 9-methyl -1-decene, and 1-tetradecene.
  • the attractant may in particular be attractive if the attractive compound, especially the monoterpene, is within a specific concentration range.
  • a higher concentration of the attractive compound may, however, also provide a longer attraction range.
  • the person skilled in the art may select different concentrations of the attractive compound.
  • the attractant may comprise the monoterpene and/or the alkene in an amount (or concentration) suitable to attract the aphid hyperparasitoid. It will be clear to the person skilled in the art that such amount may vary depending on the exact (target) species and the selected (attracting) compound, as well as other (optional) components of the attractant (composition), such as a solvent, and may also vary dependent on, for example, environmental conditions such as other sources of attractants and/or repellents, wind, air conditioning systems, and on the desired range of attraction. The person skilled in the art will select a suitable amount in dependence of these conditions.
  • the attractant may comprise at least 1 ng of the monoterpene and/or the alkene, especially of the monoterpene, or especially of the alkene, such as at least 10 ng, especially at least 100 ng, such as at least 1 pg, especially at least 10 pg, such as at least 50 pg, especially at least 100 pg, such as at least 1 mg, especially at least 10 mg, such as at least 100 mg, especially at least 1 g, such as at least 10 g.
  • the attractant may especially comprise the monoterpene and/or the alkene in a dispenser, more especially in a tube, such as a tube having, for example, a volume of 4 ml.
  • the attractant may comprise at least 1 ng of limonene such as at least 10 ng, especially at least 100 ng, such as at least 1 pg, especially at least 10 pg, such as at least 50 pg, especially at least 100 pg, such as at least 1 mg, especially at least 10 mg, such as at least 100 mg, especially at least 1 g, such as at least 10 g.
  • the attractant may comprise at least 1 ng of linalool such as at least 10 ng, especially at least 100 ng, such as at least 1 pg, especially at least 10 pg, such as at least 50 pg, especially at least 100 pg, such as at least 1 mg, especially at least 10 mg, such as at least 100 mg, especially at least 1 g, such as at least 10 g.
  • the attractant may comprise at least 1 ng of geraniol such as at least 10 ng, especially at least 100 ng, such as at least 1 pg, especially at least 10 pg, such as at least 50 pg, especially at least 100 pg, such as at least 1 mg, especially at least 10 mg, such as at least 100 mg, especially at least 1 g, such as at least 10 g.
  • the attractant may comprise at least 1 ng of 1-decene such as at least 10 ng, especially at least 100 ng, such as at least 1 pg, especially at least 10 pg, such as at least 50 pg, especially at least 100 pg, such as at least 1 mg, especially at least 10 mg, such as at least 100 mg, especially at least 1 g, such as at least 10 g.
  • the attractant may comprise at least 1 ng of 9-methyl- 1-decene such as at least 10 ng, especially at least 100 ng, such as at least 1 pg, especially at least 10 pg, such as at least 50 pg, especially at least 100 pg, such as at least 1 mg, especially at least 10 mg, such as at least 100 mg, especially at least 1 g, such as at least 10 g.
  • the attractant may comprise at least 1 ng of 1-tetradecene such as at least 10 ng, especially at least 100 ng, such as at least 1 pg, especially at least 10 pg, such as at least 50 pg, especially at least 100 pg, such as at least 1 mg, especially at least 10 mg, such as at least 100 mg, especially at least 1 g, such as at least 10 g.
  • the attractant may (further) comprise at least 1 ng of 6-m ethyl-5 -hepten-2-one and/or l-octen-3-ol, especially of 6-methyl-5-hepten-2-one, or especially of l-octen-3-ol such as at least 10 ng, especially at least 100 ng, such as at least 1 pg, especially at least 10 pg, such as at least 50 pg, especially at least 100 pg, such as at least 1 g, especially at least 10 g.
  • the attractant may comprise the monoterpene and/or the alkene, especially the monoterpene, or especially the alkene, in a concentration of at least 10 ng/ml, such as at least 100 ng/ml, especially at least 1 pg/ml, such as at least 2 pg/ml, especially at least 5 pg/ml, such as at least 10 pg/ml
  • the attractant may comprise limonene in a concentration of at least 10 ng/ml, such as at least 100 ng/ml, especially at least 1 pg/ml, such as at least 2 pg/ml, especially at least 5 pg/ml, such as at least 10 pg/ml.
  • the attractant may comprise linalool in a concentration of at least 10 ng/ml, such as at least 100 ng/ml, especially at least 1 pg/ml, such as at least 2 pg/ml, especially at least 5 pg/ml, such as at least 10 pg/ml.
  • the attractant may comprise geraniol in a concentration of at least 10 ng/ml, such as at least 100 ng/ml, especially at least 1 pg/ml , such as at least 2 pg/ml, especially at least 5 pg/ml, such as at least 10 pg/ml.
  • the attractant may comprise 1-decene in a concentration of at least 10 ng/ml, such as at least 100 ng/ml, especially at least 1 pg/ml, such as at least 2 pg/ml, especially at least 5 pg/ml, such as at least 10 pg/ml.
  • the attractant may comprise 9-methyl- 1-decene in a concentration of at least 10 ng/ml, such as at least 100 ng/ml, especially at least 1 pg/ml, such as at least 2 pg/ml, especially at least 5 pg/ml, such as at least 10 pg/ml.
  • the attractant may comprise 1-tetradecene in a concentration of at least 10 ng/ml, such as at least 100 ng/ml, especially at least 1 pg/ml, such as at least 2 pg/ml, especially at least 5 pg/ml, such as at least 10 pg/ml.
  • the attractant may comprise 6-m ethyl-5 -hepten-2-one and/or l-octen-3-ol, especially 6-m ethyl-5 -hepten-2-one, or especially l-octen-3-ol, in a concentration of at least 10 ng/ml, such as at least 100 ng/ml, especially at least 1 pg/ml, such as at least 2 pg/ml, especially at least 5 pg/ml, such as at least 10 pg/ml.
  • the micro-organisms that were attractive to Dendrocerus aphidum produced substantially more of several (attractive) compounds.
  • One or more of these compounds may thus contribute to attracting an aphid hyperparasitoid, especially wherein the aphid hyperparasitoid is a hymenopteran species, especially a Megaspilidae species.
  • Compounds which may contribute to attracting an aphid hyperparasitoid were selected by identifying compounds that are more prevalent in the samples from the attractive microbes, including both ST18.16/085 and ST18.16/160 for this analysis, and for which a two- sided t-test with equal variance indicated a probability ⁇ 0.1 that the “attractive” values and the “non-attractive” values (i.e., related to the repelling or neutral microbes) originate from the same distribution; the measurements corresponding to the blank medium were not considered for these t-tests.
  • the attractant may comprise one or more attractive compounds selected from the group comprising 2-methyl-propanoic acid, n-octane, acetic acid, alpha-methyl-cyclohexanepropanol, 4-cyclohexyl-2-butanone, ammonium acetate, nonan-2-ol, 1-decene, 4-methyl-2-propyl-l-pentanol, 9-decen-l-ol, 2-ethyl-hexanoic acid, 9-methyl-l- decene, 2-methyl-5-(l-methylethyl)-pyrazine, acetophenone, tridecan-2-one, tetradecane, butyl- formate, 3-methylthio-propionaldehyde, butyric acid, 4-m ethyl- l-(l-m ethylethyl)-3-cy cl ohexen-
  • attractive compounds selected from the group comprising 2-methyl-propa
  • the attractant may comprise the attractive compound in a concentration of at least 1 ng/ml, such as at least 10 ng/ml, especially at least 100 ng/ml, such as at least 1 pg/ml, such as at least 2 pg/ml, especially at least 5 pg/ml, such as at least 10 pg/ml, such as when offered in a solution.
  • the attractant may comprise the attractive compound in an amount of at least 1 ng, such as at least 10 ng, especially at least 100 ng, such as at least 1 pg, especially at least 10 pg, such as at least 50 pg, especially when offered as a pure compound, such as released from a point-source.
  • the attractant may comprise 2-methyl-propanoic acid. In further embodiments, the attractant may comprise n-octane. In further embodiments, the attractant may comprise acetic acid. In further embodiments, the attractant may comprise alpha- methyl-cyclohexanepropanol. In further embodiments, the attractant may comprise 4-cyclohexyl-
  • the attractant may comprise ammonium acetate.
  • the attractant may comprise nonan-2-ol.
  • the attractant may comprise 1-decene.
  • the attractant may comprise 4-methyl-2-propyl-
  • the attractant may comprise 9-decen-l-ol. In further embodiments, the attractant may comprise 2-ethyl-hexanoic acid. In further embodiments, the attractant may comprise 9-methyl- 1-decene. In further embodiments, the attractant may comprise
  • the attractant may comprise acetophenone. In further embodiments, the attractant may comprise tridecan-2-one. In further embodiments, the attractant may comprise tetradecane. In further embodiments, the attractant may comprise butyl-formate. In further embodiments, the attractant may comprise 3-methylthio- propionaldehyde. In further embodiments, the attractant may comprise butyric acid. In further embodiments, the attractant may comprise 4-m ethyl- l-(l-methylethyl)-3-cyclohexen-l-ol. In further embodiments, the attractant may comprise benzyl alcohol.
  • the attractant may comprise benzothi azole. In further embodiments, the attractant may comprise 3,5- dimethyl-benzaldehyde. In further embodiments, the attractant may comprise 3 -methyl-butanoic acid. In further embodiments, the attractant may comprise benzene.
  • compound classes were observed to be produced more by the micro organisms that were attractive to Dendrocerus aphidum. Hence, these compound classes may contribute to attracting an aphid hyperparasitoid, especially an aphid hyperparasitoid being a hymenopteran species, especially a Megaspilidae species.
  • the attractant especially the composition, may comprise an alkene selected from the group comprising 1-decene, 9-methyl- 1-decene, and 1 -tetradecene, especially selected from the group comprising 1-decene and 9-methyl -1-decene, more especially 9-methyl- 1-decene.
  • the micro-organism may produce the alkene.
  • micro-organisms attractive to D. aphidum may produce more monoterpenes and/or alkenes.
  • several other classes of compounds may be produced more by micro-organisms attractive to D. aphidum.
  • the attractant especially the composition, may comprise an ester selected from the group comprising isobutyl-formate, butyl-formate, ethyl butanoate, butyl acetate, ethyl-3 -methyl butanoate butyl propanoate, butyl -isobutyrate, butyl- butanoate, butyl 2-methyl butanoate, butyl isovalerate, o-tert-butyl cyclohexyl acetate, butyl- isobutyl-phthalate, especially butyl-formate.
  • an ester selected from the group comprising isobutyl-formate, butyl-formate, ethyl butanoate, butyl acetate, ethyl-3 -methyl butanoate butyl propanoate, butyl -isobutyrate, butyl- butanoate, butyl 2-methyl butanoate, butyl isovalerate,
  • the attractant may comprise the ester in an amount of at least 1 ng, such as at least 10 ng, especially at least 100 ng, such as at least 1 pg, especially at least 10 pg. In further embodiments, the attractant may comprise the ester in a concentration of at least 1 ng/ml, especially at least 10 ng/ml, such as at least 100 ng/ml, especially at least 1 pg/ml, such as at least 10 pg/ml.
  • the micro-organism may produce the ester.
  • the attractant especially the composition, may (further) comprise an organic acid selected from the group comprising acetic acid, 2-methyl-propanoic acid, butyric acid, 3 -methyl-pyruvic acid, 3 -methyl-butanoic acid, 2-methyl-butanoic acid, n- heptanoic acid, 2-ethyl-hexanoic acid, 3,3-dimethyl-heptanoic acid, octanoic acid, nonanoic acid, isobornyl acrylate, methyl-ethyl-adipate, especially selected from the group comprising acetic acid, 2-methyl-propanoic acid, butyric acid, 3 -methyl-butanoic acid, 2-ethyl-hexanoic acid.
  • an organic acid selected from the group comprising acetic acid, 2-methyl-propanoic acid, butyric acid, 3 -methyl-pyruvic acid, 3 -methyl-butanoic acid, 2-methyl-butanoic
  • the attractant may comprise the organic acid in an amount of at least 1 ng, such as at least 10 ng, especially at least 100 ng, such as at least 1 pg, especially at least 10 pg. In further embodiments, the attractant may comprise the organic acid in a concentration of at least 1 ng/ml, especially at least 10 ng/ml, such as at least 100 ng/ml, especially at least 1 pg/ml, such as at least 10 pg/ml.
  • the micro-organism may produce the organic acid.
  • the attractant especially the composition, may (further) comprise a compound selected from the group comprising 6-methyl-5-hepten-2-one and 1-octen- 3-ol, especially 6-methyl-5-hepten-2-one, or especially l-octen-3-ol.
  • the aphid hyperparasitoid comprises D. aphidum
  • the compound selected from the group comprising 6-methyl-5-hepten-2-one and l-octen-3-ol may especially be l-octen-3-ol as 6- methyl-5-hepten-2-one has been found also to attract A. colemani. These compounds were identified to be attractive to an aphid hyperparasitoid using a Y-tube olfactometer bioassay (see below).
  • the use may be for plant protection, especially crop protection, such as in agriculture, especially such as in farming, especially one or more of biological farming, horticulture, gardening, floriculture, and greenhouse cultivation.
  • crop protection such as in agriculture
  • farming especially one or more of biological farming, horticulture, gardening, floriculture, and greenhouse cultivation.
  • traps may be used (see further below).
  • the use may especially comprise facilitating protecting a plant protected by a hyperparasitoid as a biological control agent.
  • the use may especially comprise facilitating protecting a plant selected from the group comprising sweet pepper, eggplant, tomato, cucumber, and melon.
  • the use may comprise luring the aphid hyperparasitoid, into a trap, i.e., the attractant may be used to lure the aphid hyperparasitoid into a trap, especially wherein the attractant is enclosed in the trap.
  • the invention may provide the attractant as such, i.e., the invention may provide an attractant for attracting an aphid hyperparasitoid.
  • the attractant may especially comprise a monoterpene selected from the group comprising limonene, linalool, and geraniol.
  • the attractant may comprise an alkene selected from the group comprising 1-decene, 9-methyl- 1-decene, and 1-tetradecene.
  • the attractant comprises at least a monoterpene and at least one of the afore mentioned alkenes.
  • the attractant may further comprise other compounds or substances in the composition in number and concentration to which they do not detract from the attractant property of the composition as a whole for hyperparasitoids.
  • the attractant may comprise another compound selected from the group comprising carriers, stabilizers, and insecticide.
  • the attractant may comprise an agriculturally acceptable carrier.
  • the attractant may comprise an agriculturally acceptable stabilizer.
  • the attractant may comprise an agriculturally acceptable insecticide.
  • the term “comprise” may, particularly with regards to the attractant, especially the composition, also refer to “further comprise”.
  • the invention may provide an attractant composition comprising an attractant, wherein the attractant comprises one or more of (i) a monoterpene selected from the group comprising limonene, linalool, and geraniol, and (ii) an alkene selected from the group comprising 1-decene, 9-methyl- 1-decene, and 1-tetradecene.
  • the invention may provide the trap as such, i.e., the invention may provide a trap for trapping an aphid hyperparasitoid, the trap comprising, especially enclosing, the attractant or the attractant composition according to the invention, especially the attractant, or especially the attractant composition.
  • the trap may comprise a physical trap, i.e., a trap in which the trapped organism becomes physically trapped (it cannot fly or crawl out).
  • the trap may comprise a (removeable) glue, i.e., the trapped organism becomes attached to a surface on or within the trap.
  • the trap may comprise a toxin suitable to kill the aphid hyperparasitoid, especially wherein the aphid hyperparasitoid is a hymenopteran species, especially a Megaspilidae species.
  • an embodiment describing the use with respect to compound concentrations may, for example, also apply to the attractant.
  • an embodiment of the attractant relating to composition, such as the organic acid may, for example, further apply to the trap.
  • the invention may herein, as an example, primarily be described with regards to embodiments pertaining to an attractant for a Dendrocerus species, especially Dendrocerus aphidum. However, it will be clear to the person skilled in the art that the invention is not limited to such embodiments.
  • the invention may, for example, further pertain to a Pteromalidae species, such as to Asaphes vulgaris, or such as Asaphes suspensus.
  • Fig. 1A-B depict experimental data regarding embodiments of the invention.
  • Fig. 2A-K depict experimental data regarding the attractiveness of different compound classes to a Megaspilidae species.
  • Fig. 3 depicts experimental data regarding the attractiveness of different compounds to an aphid hyperparasitoid.
  • the schematic drawings are not necessarily on scale.
  • Experiment 1 Evaluating attractive and repellent effects of micro-organisms on a Megaspilidae species and a Braconidae species. Experiments were performed using adult females of the primary parasitoid A. colemani as Braconidae species and its hyperparasitoid D. aphidum as aphid hyperparasitoid. Aphidius colemani may be a generalist aphid parasitoid; D. aphidum may be a generalist, secondary idiobiont ectoparasitoid attacking pre-pupal and pupal stages of hymenopteran primary parasitoids such as Aphidius spp. inside aphid mummies.
  • mummies were placed inside a nylon insect cage (20 cm x 20 cm x 20 cm, BugDorm, MegaView Science Co., Ltd., Taichung, Taiwan) and kept under controlled conditions (22°C, 70% relative humidity and a 16:8-h light: dark photoperiod) until parasitoid emergence. All experiments were performed with ⁇ 24-h- old, food and water-starved females.
  • bacterial strains were used to assess the olfactory response of A. colemani and D. aphidum to their mVOCs (microbial Volatile Organic Compounds).
  • the bacterial strains were isolated from the habitat of A. colemani , including aphids and honeydew, as well as from Aphidius and Dendrocerus parasitoids.
  • Bacterial isolates were tentatively identified by amplifying and sequencing the 16S ribosomal RNA (rRNA) gene. All isolates were kept in trypticsoy broth (TSB; Oxoid, Hampshire, UK) containing 25% glycerol at -80°C until further use.
  • TTB trypticsoy broth
  • the phylogenetic affiliation was determined based on 16S rRNA gene sequencing and identification using the EZBiocloud database in November 2018.
  • the indicated matches are the closest matches to type strains.
  • the samples were collected in agricultural settings, except when indicated with an asterisk; these samples were obtained from lab cultures.
  • bacterial stock cultures were plated on tryptic soy agar
  • TLB tissue culture
  • cells were washed twice in sterile physiological water (0.9% NaCl) and diluted in sterile physiological water until an optical density (OD 600 nm) of 1 was reached.
  • 1.5 ml of this cell suspension was used to inoculate a 250 ml Erlenmeyer flask containing 150 ml GYP25 medium prepared by filter- sterilizing (pore size 0.22 pm) a medium of 5% w/v glucose, 0.5% w/v peptone (BactoTM Peptone, BD Biosciences, San Jose, USA) and 0.25% w/v yeast extract (Sigma-Aldrich, Saint Louis, USA).
  • a glass Y-tube olfactometer was placed on a table that was homogeneously illuminated by four high frequency 24W T5 TL-fluore scent tubes (16 x 549 mm, 1350 Lumen, 5500K; True-Light®, Naturalite Benelux, Ansen, The Netherlands) with a 96% color representation of true daylight at a height of 0.45 m. To reduce, especially eliminate, visual cues that could affect (hyper)parasitoid responses, the olfactometer was fully enclosed with white curtains.
  • the olfactometer was positioned at a 20° incline (angle between Y-tube and horizontal plane), known to stimulate movement of the insects towards the bifurcation.
  • 150 pL of the cell-free cultivation medium was loaded on a 37 mm-diameter filter paper (Macherey- Nagel, Diiren, Germany) and placed in one of the odor chambers, whereas in the second chamber another filter paper was placed on which 150 pL blank medium was added as a control.
  • PI Preference Index
  • the P-values indicate the probability of the null hypothesis being true as determined via a variance type III Wald chi-square test under the null hypothesis that the insects show no preference for any (odor) source.
  • (hyper)parasitoid olfactory response was analyzed using a Generalized Linear Mixed Model (GLMM) based on a binomial with a logit link function (logistic regression) using bacterial treatment as fixed factor (performed in R with the glmer function from the lme4 package).
  • GLMM Generalized Linear Mixed Model
  • the attractant 110 especially comprises a micro organism.
  • Fig. la further depicts several comparative examples also comprising a micro organism.
  • the tested micro-organisms are (also see table above): ST18.16/150 (Bi), ST18.16/133 (B 2 ), ST18.16/043 (B 3 ), ST18.16/085 (B 4 ), ST18.16/160 (B s ), ST18.17/028 (Be) and ST18.17/002 (B 7 ), and negative control (N c ) comprising blank growth medium,
  • the micro-organism may comprise a Curtobacterium sp., especially Curtobacterium flaccumfaciens or Curtobacterium oceanosedimentum , more especially ST.18.16/085 (B 4 ).
  • the micro-organism may comprise a Staphylococcus sp., especially Staphylococcus saprophyticus, more especially ST.18.16/160 (B 5 ).
  • B 4 and B 5 appear to be substantially attractive to D. aphidum , especially B 4 is substantially and significantly attractive to D. aphidum.
  • the non- inoculated, sterile medium was used as a negative control.
  • GC-MS analyses were performed with a Thermo Trace 1300 system (Thermo Fisher Scientific, Watham, USA) fitted with a MXT-5 column (30 m length x 0.18 mm inner diameter x 0.18 pm film thickness; Restek, Bellefonte, USA) and a ISQ mass spectrometer (Thermo Fisher Scientific, Waltham, USA).
  • a programmed gas flow was used whereby the helium gas flow was set at 2.7 ml/min for 0.1 min, followed by a decrease in flow of 20 ml/min 2 to the normal 0.9 ml/min.
  • the GC oven was programmed as follows: the temperature was initiated at 30°C, held for 3 min and then raised to 80°C at 7°C/min. Next, the temperature was raised to 125°C at 2°C/min and finally the temperature was raised to 270°C at 8°C/min.
  • Mass spectra were recorded in centroid mode using a mass acquisition range of 33 to 550 atomic mass units, a scan rate of 5 scans/s and an electron impact ionization energy of 70 eV.
  • a mix of linear n-alkanes (from C7 to C40, Supelco, Bellefonte, USA) were injected into the GC-MS under identical conditions to serve as external retention index markers. Volatile compounds were identified and quantified by analyzing the chromatograms with AMDIS v2.71 to deconvolute overlapping peaks. The empirical spectra were manually identified using the NIST MS Search v2.0g software, using the NIST2011, FFNSC and Adams libraries. This allowed for identification of compounds from which the spectrum profiles were used as covariates for the elution profiles extraction. The extraction was performed for every compound in every chromatogram over a time restricted window using weighted non-negative least square analysis. Finally, the peak areas were computed from the extracted profiles.
  • the compounds of table 2 were divided into the following compound classes to assess whether certain compound classes invoked an insect response. Specifically, the compounds were divided into the compound classes including alcohol, aldehyde, ester, ketone, alkane, cycloalkane, alkene, aromatic, organic acid, terpene, and miscellaneous.
  • the compound class alcohol comprises ethanol, 3-methyl-4-penten-2-ol, isopentyl alcohol, 2-methyl- 1 -butanol, 3-methyl -2 -buten-l-ol, 2,3-butanediol, 2-methyl-mercaptoethanol, pinacol, n-hexanol, 4-heptanol, 1-butoxy -2 -propanol, 3 -(methylthio)-l -propanol, 2 -m ethyl-2 - octen-4-ol, 4-methyl-2-propyl-l-pentanol, 2-methyl-6-methylene-2-octanol, n-octanol, nonan-2- ol, 2-phenylethanol, isoborneol, 4-m ethyl- l-(l-methylethyl)-3-cy cl ohexen-l-ol, alpha-methyl- cyclohexanepropano
  • the attractant may comprise one or more compounds selected from the compound class alcohol.
  • the compound class aldehyde comprises acetaldehyde, butanal, 3-methylthio- propionaldehyde, phenylacetaldehyde, and 3,5-dimethyl-benzaldehyde.
  • the attractant may comprise one or more compounds selected from the compound class aldehyde.
  • the compound class ester comprises isobutyl-formate, butyl-formate, ethyl butanoate, butyl acetate, ethyl-3 -methyl butanoate, butyl propanoate, butyl -isobutyrate, butyl- butanoate, butyl 2-methyl butanoate, butyl isovalerate, o-tert-butyl cyclohexyl acetate, butyl-and isobutyl-phthalate.
  • the attractant may comprise one or more compounds selected from the compound class ester.
  • the compound class ketone comprises 2,3-butanedione, 2-butanone, 1-hydroxy- 2-propanone, 2,3-pentandione, acetoin, 4-methyl-pentan-2-one, 2-hexanone, 4-methyl-2- heptanone, 5,5-dimethyl-2,4-hexanedione, acetophenone, 4-cyclohexyl-2-butanone, undecan-2- one, and tridecan-2-one.
  • the attractant may comprise one or more compounds selected from the compound class ketone.
  • the compound class alkane comprises n-hexane, 2,2,4-trimethyl-pentane, n- octane, nonane, and tetradecane.
  • the attractant may comprise one or more compounds selected from the compound class alkane.
  • the compound class cycloalkane comprises 1,3-dimethyl-cyclopentane and 1,5- dimethyl-6-oxa-bicyclo[3,l,0]hexane.
  • the attractant may comprise one or more compounds selected from the compound class cycloalkane.
  • the compound class alkene comprises 1-decene, 9-methyl- 1-decene, and 1- tetradecene.
  • the attractant may comprise one or more compounds selected from the compound class alkene.
  • the compound class aromatic comprises benzene, 1,3 -dimethyl-benzene, 1-ethyl- 2-methyl-benzene, cyclopropyl-benzene, benzyl alcohol, ortho-cymene, para-cymenene, and indole.
  • the attractant may comprise one or more compounds selected from the compound class aromatic.
  • the compound class organic acid comprises acetic acid, 2-methyl-propanoic acid, butyric acid, 3 -methyl-pyruvic acid, 3 -methyl-butanoic acid, 2-methyl-butanoic acid, n-heptanoic acid, 2-ethyl-hexanoic acid, 3,3-dimethyl-heptanoic acid, octanoic acid, nonanoic acid, isobornyl acrylate, and methyl-ethyl-adipate.
  • the attractant may comprise one or more compounds selected from the compound class organic acid.
  • the compound class terpene comprises linalool, limonene, and geraniol.
  • the attractant may comprise one or more compounds selected from the compound class terpene.
  • the compound class miscellaneous comprises azetidine, 1-butanamine, ammonium acetate, methyl pyrazine, 2-propyl-l,3-dioxolane, dihydro-2-methyl-thiophen-3-one, 2-methyl-5-(l-methylethyl)-pyrazine, tetramethyl-pyrazine, and benzothi azole.
  • the attractant may comprise one or more compounds selected from the compound class miscellaneous.
  • Fig. 2A-J depict the observed linkage between the volatile composition of the cell- free bacterial cultivation media and the olfactory response of the Megaspilidae species, specifically Dendrocerus aphidum. Results are shown for the compound classes alcohol (Ci), aldehydes (C2), esters (C3), ketones (C4), alkanes (C5), cycloalkanes (Ce), alkenes (C7), aromatics (Cs), organic acids (C 9 ), terpenes (C10), and miscellaneous (Cn).
  • Alcohol Ci
  • aldehydes C2
  • esters C3
  • ketones C4
  • alkanes C5
  • cycloalkanes Ce
  • alkenes C7
  • aromatics Cs
  • organic acids C 9
  • terpenes C10
  • miscellaneous Cn
  • the attractant may comprise alcohols and/or ketones in a concentration below the threshold concentration.
  • the attractiveness of several candidate (synthetic) attractive compounds to the aphid hyperparsite D. aphidum was evaluated in a Y-tube olfactometer.
  • the olfactometer consisted of two glass jars (30 1) that were connected with the two arms of a glass Y-shaped tube (diameter 1 cm, arm length 8 cm) with PTFE tubing. Charcoal-filtered and moisturized air was led into each glass jar at a rate of 150 ml/min.
  • the Y-tube was positioned at an angle of approximately 20° to the table surface to stimulate hyperparasitoids to respond by negative geotaxis and positive phototaxis.
  • Candidate compounds were offered in 4 ml LDPE vials containing 10 m ⁇ pure compound unless specified otherwise.
  • One lure vial was placed at the bottom of one glass jar and an empty LDPE vial at the bottom of the other glass jar as a control.
  • Dendrocerus aphidum was reared on mummies of Myzus persicae nicotianae parasitized by Aphidius colemani on sweet pepper.
  • Females between 3-8 days old were used for experiments.
  • Females were considered to have mated and were fed honey ad libitum but had not been allowed to parasitize aphid mummies (naive with respect to hosts).
  • a separate set of experiments was performed with females that had been allowed to parasitize aphid mummies on a pepper plant for 24h prior to the experiment (experienced with respect to hosts).
  • Fig. 3 depicts the experimental results. In particular, it depicts the behavioral responses of D. aphidum as average ( ⁇ standard error) preference index (PI), calculated as the difference between the number of D.
  • PI standard error preference index
  • lures are: (R)-(-)- linalool (Li), (+/-)-linalool (L 2 ), nerol (L 3 ), geraniol (L 4 ), l-octen-3-ol (L 5 ), methyl salicylate (Eb), (E)-B-farnesene (L 7 ), l-octen-3-ol (Lx), 6-methyl-5-hepten-2-one (L 9 ), 6-methyl-5-hepten- 2-one as 1 pi pure compound (L 10 ).
  • Lures L 1 -L 5 were evaluated using naive/) aphidum females, whereas lures L6-L10 were tested using experienced D. aphidum females.
  • the statistical analysis comprised a two-sided binomial test per candidate lure to evaluate if the choice of D. aphidum differed from a 50:50 choice for treatment and control odors, using the total numbers of hyperparasitoids that chose for each odor source (i.e. summed over experimental days).
  • the terms “substantially” or “essentially” herein, and similar terms, will be understood by the person skilled in the art.
  • the terms “substantially” or “essentially” may also include embodiments with “entirely”, “completely”, “all”, etc.
  • the adjective substantially or essentially may also be removed.
  • the term “substantially” or the term “essentially” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%.
  • the terms ’’about” and “approximately” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%.
  • a phrase “item 1 and/or item 2” and similar phrases may relate to one or more of item 1 and item 2.
  • the term “comprising” may in an embodiment refer to "consisting of but may in another embodiment also refer to "containing at least the defined species and optionally one or more other species”.
  • the invention may be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer.
  • a device claim, or an apparatus claim, or a system claim enumerating several means, several of these means may be embodied by one and the same item of hardware.
  • the mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.
  • the invention also provides a control system that may control the device, apparatus, or system, or that may execute the herein described method or process. Yet further, the invention also provides a computer program product, when running on a computer which is functionally coupled to or comprised by the device, apparatus, or system, controls one or more controllable elements of such device, apparatus, or system.
  • the invention further applies to a device, apparatus, or system comprising one or more of the characterizing features described in the description and/or shown in the attached drawings.
  • the invention further pertains to a method or process comprising one or more of the characterizing features described in the description and/or shown in the attached drawings.
  • a method or an embodiment of the method is described being executed in a device, apparatus, or system, it will be understood that the device, apparatus, or system is suitable for or configured for (executing) the method or the embodiment of the method respectively.

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Abstract

L'invention concerne l'utilisation d'un attractif (110) pour attirer un hyperparasitoïde aphide, l'attractif (110) comprenant un monoterpène choisi dans le groupe comprenant le limonène, le linalol et le géraniol et/ou un alcène choisi dans le groupe comprenant le 1-décène, le 9-méthyl-1-décène et le 1-tétradécène.
PCT/NL2020/050644 2019-10-21 2020-10-20 Attractif pour hyperparasitoïde aphide WO2021080419A1 (fr)

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