WO2013034599A1 - Limonene-containing mating disruptant and mating disruption method using the same - Google Patents

Abstract

Provided is a mating disruptant having an improved mating disruption effect against pest insects. Specifically, provided is a mating disruptant composition for the control of codiing moth (C. pomoneila) populations whose main ingredient is the synthetic sex pheromone codlemone, the disruptant comprising the said pheromone and the plant volatile Iimonene, wherein an amount of the codlemone component is at 5 to 20% by weight relative to an amount of the Iimonene; and a mating disruption method using the mating disruptant where the codlemone and the Iimonene compounds can be dispersed together from the same source or separately from adjacent sources.

Description

Limonene-containing mating disruptant and mating disruption method using the same

Field of the invention

The present invention relates to a composition comprising the sex pheromone, E8,E10-dodecadien-l-ol (codlemone) and limonene for combating the codling moth (Cydia pomonella) in crops or fruit orchards.

A further object of the present invention is to provide an efficient method for combating codling moth in crops or fruit orchards by the use of said composition.

Background of the Invention

Biological techniques of pest control aim to keep infestation within manageable limits in an ecologically harmonious manner without using chemical pesticides (Birch AN et al, 2011 ).

Pheromones are species-specific scent substances emitted by animals, such as insects to trigger certain behaviors or a biological activity in their population. The activity of pheromones is directed solely towards the insect species intended, thus enabling species-specific pest control approaches (Witzgall P et al, 2010, review). Sex pheromones produced by female moths attract conspecific males and are intended to facilitate female-male encounters and to allow mating. These sex pheromones are emitted as species-specific multi-component blends (Tillman JA et al, 1999, review; Matsumoto S, 2010, review).

Sex pheromones do not give rise to resistance problems. They are active at small concentrations, biodegradable and not toxic to the environment and most can be chemically synthesized for widespread use.

The codling moth Cydia pomonella (Lepidoptera, Tortricidae) is a member of the lepidopteran family of butterflies. It feeds mainly on apples, pears, walnuts and apricots (http://www.aqf.qov.bc.ca/cropprot/tfipm/codlinqmoth.htm). It is considered a major worldwide agricultural pest and has grown resistant to most insecticides used against it (Stara J et al, 2006).

SUBSTITUTE SHEET (RULE 26) Mating disruption is an environmentally safe insecticide-free pest management strategy that uses synthetically-produced pheromones to confuse males (see http://www.omafra.qov.on.ca/enqlish/crops/facts/03-079.htm for review). By releasing synthetic species-specific pheromones in the air in sufficiently high quantities in infested crops or orchards, the males are unable to orient to natural sources of sex pheromone (females) and fail to locate the calling female, insemination reproduction is prevented and the infestation level decreases.

The mating disruption technique has already been tested and commercially used (Witzgall P et al, 2010). However, it needs to become more efficient, economic and reliable, especially at high insect population densities.

The main sex pheromone compound of codling moth, (£,E)-8,10-dodecadienol (E8,E10-12:OH; codlemone), was identified by Roelofs and coworkers (Roelofs et al, 1971 ). Other minor compounds in female sex pheromone glands, such as dodecan- 1-ol (120H) and tetradecanol (140H) were subsequently identified (Arn H et al, 1985). The identification of codlemone has opened the way to mating disruption protocols for controlling codling moth populations (Witzgall P et al, 2008).

Mating disruption exhibits limits in efficacy, especially at a high density of pest populations. Synergist compounds are sought to increase the , efficacy of the main pheromone (Witzgall P et al, 2010). Numerous experiments showed that secondary pheromone components of females such as dodecan-1-ol (Arn H et al, 985) or (E,E)-8, 10-dodecadienyl acetate (Witzgall P et al, 2001 ) enhanced attraction of codling moth males to codlemone.

The non-toxic, behaviour-modifying family of chemicals (semiochemicals) comprise not only pheromones but also chemicals which mediate interactions between different species, named kairomones. Those kairomones include host plant volatiles that affect the behaviours of plant-feeding insects like the codling moth (Dudareva N et al, 2006, review). Pheromones and kairomones are mostly perceived simultaneously and interact to enhance mate finding (Christensen TA et al, 2002).

SUBSTITUTE SHEET (RULE 26) Codling moths are attracted by the odor of fruits they feed on. As an example, plant volatiles from apples (Coracini M et al, 2004) such as (£, E)-ct-farnesene (Sutherland ORW et a!, 1974) or from ripening pears such as ethyl (E,Z)-2,4-decadienoate (pear ester) are able to attract both female and male codling moths. Patent application US 2007/0004686: Attractant for apple fruit moth and other insect pests of apple, describes the use of volatile plant compounds of rowan and apple for controlling codling moth.

Some kairomones were investigated for their potentiating effect to codlemone in attracting codling moth males. Pear ester used as adjuvant improved the efficacy of low rates of codlemone in mating disruption (Knight AL et al, 2005). Apple volatiles linalool, (E)-p-famesene, or (Z)-3-hexen-1-ol were as efficient as dodecanol in attracting codling moth males to codlemone (Yang Z et al, 2004). Dispensers releasing a combination of pear ester and codlemone attracted more codling moth males than dispensers releasing codlemone alone under high population densities (Stelinski LL et al, 2006).

D-limonene (1-methyl-4-(1-methylethenyl)-cyclohexane) is a monocyclic terpene (C10H16, CAS No. 5989-27-5) with a lemon-like odor. D-limonene (the R enantiomer) is a major constituent in several citrus oils. Available commercially, it is widely used as a flavor and fragrance additive in perfumes, soaps, foods, chewing gum and beverages. D-limonene is listed in the U.S. Code of Federal Regulation as safe for a flavoring agent. D-Limonene was first registered as an insecticide in 1958. It is effective as a contact spray only.

U.S. Pat. No. 6,440,406 describes a method to attract adult scarabs using compositions of volatiles containing numerous compounds including limonene. However, no use of a pheromone nor additive effect of limonene with a pheromone are reported.

Codlemone used as mating disruptant exerts maximal effects within a relatively narrow dose range. Excess or unsufficient amounts of codlemone may fail to attract codling moth males efficiently. Moreover, mating disruption protocols that rely on decreased codlemone amounts are suited, mainly for economical reasons.

SUBSTITUTE SHEET (RULE 26) Thus, there remain significant unmet needs for an efficient method of codling moth mating disruption using effective pheromone compositions comprising reduced codlemone amounts

These and other objects as will be apparent from the foregoing have been achieved by the present invention.

Summary of the Invention

The present invention provides a composition comprising the combination of the sex pheromone, E8,E10-dodecadien-l-ol (codlemone) and limonene for mating disruption of Codling moth.

The invention further provides a method for mating disruption of codling moth comprising releasing in the crop or orchard an effective amount of a composition comprising the sex pheromone, E8,E10-dodecadien-l-ol (codlemone) and limonene wherein the amount of codlemone is at 5 to 20% by weight relative to the amount of limonene. Preferably, the amount of codlemone is at 10% by weight relative to the amount of limonene.

Also disclosed is a method comprising the steps of releasing separately or concomitantly in the crop or orchard an effective amount of the sex pheromone (E,E)- 8,10-dodecadienol (codlemone) and limonene.

The invention further provides a dispenser for releasing in the crop or orchard an effective amount of a composition. Also disclosed is a dispenser for releasing separately or concomitantly in a crop or orchard an effective amount of the sex pheromone (E,E)-8,10-dodecadienol (codlemone) and limonene.

Other objects and advantages of the invention will become apparent to those skilled in the art from a review of the ensuing detailed description, which proceeds with reference to the following illustrative drawings, and the attendant claims.

SUBSTITUTE SHEET (RULE 26) Brief description of the figures

Figure 1 : Dose-dependent response of male codling moths to codlemone.

The proportion of male codling moths contacting the source at different release rates of codlemone.

Responses of male codling moth to codlemone at different release rates in order to establish a suboptimal attractive dose which could be amenable to improvement by adding host plant volatile products.

Figure 2: Effect of host plant volatile compounds on the attractiveness of codlemone Measurement of the proportion of male codling moth showing activation, wing fanning, take off, upwind flight, and contact with a source consisting of 10 pg/min codlemone admixed with plant volatiles.

Wind tunnel responses of male codling moth to calling females, to codlemone alone and to two-component blends of codlemone (released at 10 pg/min) and plant volatiles (each released at 100 pg/min).

Asterisks indicate significant differences between the responses of males to test solutions containing codlemone alone compared to codlemone admixed with a host plant volatile and to calling females by the Generalized Linear Model at 0.05 type I error rate (n=60 for each treatment).

Figure 3: Time-based comparisons of responses to treatments.

Time-based reactions of male codling moth to different test solutions under the behavioural steps of activation, upwind flight and source contact.

Cumulative proportions of activated male codling moth by test solutions containing codlemone alone (black line) and with single host plant volatiles added plotted against the time elapsed until activation was observed (n=60 for each treatment).

Host plant volatiles that reduced the time until activation of males are above the black line and those that increased the time until activation are below of the black line.

Compounds significantly influencing the responses of males by the Cox proportional hazards model at a type I error rate of 0.05 are marked with an asterisk.

SUBSTITUTE SHEET (RULE 26) Figure 4: Time-based comparisons of responses to treatments.

Take-off and upwind flight behavioural of codling moth males to codlemone admixed to piant voiatiies.

Cumulative proportions of male codling moth showing upwind flight to test solutions containing codlemone alone (black line) and with single host plant voiatiies added plotted against the time elapsed until upwind flight was observed (n=60 for each treatment).

Host plant voiatiies that reduced the time until upwind flight of males are above the black line and those that increased the time until upwind flight are below of the black line.

Compounds significantly influencing the responses of males by the Cox proportional hazards model at a type I error rate of 0.05 are marked with an asterisk.

Figure 5: Time-based comparisons of responses to treatments.

Measurement of time elapsed until activation and upwind flight and of the proportion of codling moth males reaching the source of codlemone admixed with the plant voiatiies.

Cumulative proportions of male codling moth showing source contact to test solutions of codlemone alone (black line) and with single host plant voiatiies added plotted against the time elapsed until source contact was observed (n=60 for each treatment).

Host plant voiatiies that reduced the time until source contact of males are above the black line and those that increased the time until source contact are below of the black line.

Compounds significantly influencing the response of males by the Cox proportional hazards model at a type I error rate of 0.05 are marked with an asterisk.

SUBSTITUTE SHEET (RULE 26) Detailed description of the invention

One object of the present invention is to respond to the need for improved mating disruption methods to the pest insect in particular Codling moth with the particular aim to identify new compounds capable of increasing the attracting effect of the existing combinations used in such method. Another aspect of the invention showed the superior attracting effect of the new mating disruptant.

The inventors particularly investigated the potentiating effect of plant volatile compounds on codlemone (E,E-8, 10-dodecadien-1-ol), the main sex pheromone released by female codling moths to attract conspecific males for mating. The attractive effect of such compositions was tested on male codling moths in a large series of experiments using suboptimal doses of codlemone.

The present invention provides an effective composition that may be used in the mating disruption of the codling moth, a member of the lepidopteran family of insects. This insect pest feeds on various fruits such as apples, pears, apricots and walnuts worldwide.

The codling moth, as defined here, is the common name given to the worldwide pest insect species of fruit crops, Cydia pomonella (Lepidoptera, family Tortricidae), also herein referred to as C. pomonella.

Without being limited to codling moth, it is envisioned that the composition of the invention may be used to control Lepidoptera including Pammene rhediella (Lepidoptera, family Tortricidae).

The composition of the invention has no particular limitation as regards to the kind or the number of pest insects to be controlled insofar as it can be used for mating disruption methods. In addition, there is no limitation on crops, orchards or neighboring fields to which the mating disruptant composition can be applied.

SUBSTITUTE SHEET (RULE 26) As used herein, the following definitions are supplied in order to facilitate the understanding of the present invention.

"A" or "an" means "at least one" or "one or more."

The term "comprise" is generally used in the sense of include, that is to say permitting the presence of one or more features or components.

In a first embodiment, the present invention provides an effective composition comprising codlemone and limonene for mating disruption of the codling moth. Preferably the composition of the invention comprises the combination of the sex pheromone (E,E)-8,10-dodecadienol (codlemone) and limonene.

The term "codlemone" as used herein means the sex pheromone compound (£,£ 8,10-dodecadien-1-ol (CAS No. 33956-49-9) as well as derivatives thereof including isomeric mixtures (see Guerin et al. 1983) where the (E,E) isomer is the major component.

Without being limited to codlemone, a variety of lepidopteran pheromones are suitable for use with the composition of the present invention. Overviews of the pheromones for many insects, including many Lepidoptera, which may be used herein have been described, and include, for example, Mayer and McLaughlin (Handbook of Insect Pheromones and Sex Attractants, CRC Press, Boca Raton, Florida, 1991) and Tamaki [Sex Pheromones, In Comprehensive Insect Physiology Biochemistry and Pharmacology, Vol. 9 Behavior, Kerkut and Gilbert (Ed.), Pergamon Press, New-York, pp. 145-179.

The term "limonene" as used herein means the monocyclic monoterpene 1-methyl-4- (l-methylethenyl)-cyclohexane, in its D form (D-limonene equivalent to (+)-limonene or the R enantiomer (R)-(+)-limonene; CAS No. 5989-27-5) or its L form (L-limonene equivalent to (-)-limonene or the S enantiomer (S)-(-)-limonene; CAS 5989-54-8), or the racemic mixture termed dipentene (CAS No. 138-86-3) and derivatives thereof.

SUBSTITUTE SHEET (RULE 26) Surprisingly, t e Applicant has found that the composition of the present invention comprising the combination of codlemone and limonene shows superior attracting activity towards codling moth maies than combinations using other known plant volatile compounds. More significant was the demonstration by the Applicant that the combination of codlemone and limonene showed attracting activity of codling moth males comparable to that of calling females, an effect that was achieved with only some of the other plant volatile compounds tested by the Applicant (see Figure 2).

The term "attracting activity" as used herein means an effect similar to the effect that a female of a pest insect has for attracting conspecific males.

As shown in the Examples, the composition comprising codlemone and limonene has a synergistic effect compared to a composition comprising codlemone alone, and this effect, superior to all other compositions using codlemone tested by the Applicant, is not inferior to the effect of a codling moth female releasing codlemone.

US Pat. 6,440,406 showed no significant effect of limonene used alone on adult scarabs and poor selectivity in attracting male or female scarabs.

Another aspect of the invention describes the superior activity of the composition including limonene even though the composition contains decreased amounts of codlemone compared to the higher doses required to attract males using codlemone alone. This implies a major economic advantage over existing codlemone formulations considering the moderate cost of limonene compared to codlemone.

Suitable formulations of the composition may be prepared from these volatile compounds in isolated or impure form. However, as a practical matter, it is expected that substantially pure volatile compounds will be formulated with an inert carrier. The practitioner skilled in the art will also recognize that these volatile compounds may be formulated in single or separate compositions, as well as in liquid or solid form. Liquid carriers for use herein include but are not limited to water or organic solvents, such as polyols, esters, methylene chloride, alcohol (such as C1-C4 alcohol) or vegetable oil, although vegetable oils and alcohols are preferred. Suitable vegetable oils include olive oil, sesame oil, peanut oil, canola oil, cottonseed oil, corn oil, soybean oil, mineral oil, as well as methylated forms of these oils, or mixtures thereof. Aromatic and linear hydrocarbon solvents may also be included. The active ingredient mixture

SUBSTITUTE SHEET (RULE 26) may also be incorporated in a solid substrate, such as clays, diatomaceous earth, silica, polyvinyl chloride, polystyrene, polyurethanes, or other synthetic polymers, ureaformaldehyde condensates, and starches. Other useful solid support matrices include expanded vermiculite and paraffinic, bees wax or rubber.

Applicants have surprisingly found that the weight ratio of the compounds in the composition that is an effective mating disruptant for codling moth is about 10:1 (limonene.codlemone).

Preferably the amount of codlemone is at 5 to 20% by weight relative to the amount of limonene and even more preferably the amount of codlemone is at 10% by weight relative to the amount of limonene.

The composition of the present invention is used in an amount effective to induce the measured male responses.

"An effective amount" in the case of an attractant response, may be defined as the quantity of the composition that efficiently attracts codling moth males to the source of the combination in numbers significantly higher than males are attracted to a source of codlemone used alone. In the case where the desired response is disruption of mating by confusing or inhibiting the male moth, an effective amount is defined as that quantity of the composition which disorientates males at a rate significantly higher than disruption achieved by a source of codlemone used alone.

The compounds may be dispersed together from the same source or separately from adjacent sources, in which case the released mixture shall be adjusted to the described ratio of codlemone and limonene.

The content of the codlemone in the mating disruptant of the present invention can be the same as the codlemone content of conventional mating disruptants comprising, as the major component, the most efficient codlemone content to disorient males. It is because the mating disruptant of the present invention has nothing different from the conventional mating disruptant except that it contains or is used in conjunction with a specific amount of limonene which is not used in current compositions containing codlemone.

SUBSTITUTE SHEET (RULE 26) It is contemplated that the composition of the present invention represents a minimal composition and thai it can be used as a combination with other compositions containing codlemone insofar as such combinations can be used for the mating disruption method.

Field use of the pheromone composition of the invention shall involve dispenser or disseminator (controlled release substrate) known in the art. The precise amounts of each of the codlemone and limonene components to be released for efficient mating disruption shall be closely adjusted to prevailing weather and field or orchard conditions and continuously monitored formulations.

A dispenser refers to any kind of device known by the skilled in the art that can for example be constituted from any one of a range of natural or synthetic polymers or other substrate forming or not forming an impermeable envelope around the compositions and that may or may not include a slow-release membrane and that allows controlled release into the air of the compositions.

It is also contemplated that the composition of the invention may be used as a detector or monitoring agent by utilizing the composition in a lure (or attractant) designed for insect trapping especially Lepidoptera. Preferably the insect is codling moth. Traps may be baited with the novel composition of the invention and the catch tabulated to determine size and location of an infestation. Economic use of appropriate pest management systems can be determined in this manner. Furthermore, it is also contemplated that the composition of the invention may be used for mass trapping for the purpose of removing codling moth from a particular zone or geographic region.

In an particular embodiment of the invention, the composition of the invention may be used as vehicle to attract and concentrate the insects to areas treated with chemicals such as insecticides or chemosterilizants, so that only limited but still effective amounts of the insecticide or chemosterilizant are used, that is, an amount that is lethal for an exposed insect or at least sub lethal but sufficient to incapacitate the insect in regard to mating activity.

SUBSTITUTE SHEET (RULE 26) Another object of the present invention is to provide a method for mating disruption of the codiing moth, which comprises the steps of releasing in the crop or orchard an effective amount of the composition according to the invention. Preferably the codling moth is Cydia pomonella. The aim of the present method is to control the population of codling moth.

Moreover, the invention provides a method of mating disruption using this mating disruptant that shows by all parameters measured superior efficacy over other currently used combinations comprising codlemone together with other plant volatile compounds.

In particular it is provided a method for mating disruption of the codling moth, which comprises the steps of releasing separately or concomitantly in the crop or orchard an effective amount of the sex pheromone (E,E)-8,10-dodecadienol (codlemone) and limonene.

The invention also contemplates a dispenser for releasing in the crop or orchard an effective amount of the composition of the invention.

Alternatively it is also provided a dispenser for releasing separately or concomitantly in the crop or orchard an effective amount of the sex pheromone (E,E)-8, 10- dodecadienol (codlemone) and at least one host plant volatile compound.

Preferably said at least one host plant volatile compound is limonene.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications without departing from the spirit or essential characteristics thereof. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of

SUBSTITUTE SHEET (RULE 26) the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

Various references are cited throughout this Specification, each of which is incorporated herein by reference in its entirety.

The foregoing description will be more fully understood with reference to the following Examples. Such Examples, are, however, exemplary of methods of practicing the present invention and are not intended to limit the scope of the invention.

Examples:

Compounds

Codlemone:

(E,E)-8,10-dodecadien-1-ol (codlemone, >99.8%) from Siegfried Ltd. (Zofingen, Switzerland).

Eleven plant volatile compounds were tested in combination with codlemone:

hexan-1-ol (hereafter hexanol, >99% purity),

(Z)-3-hexen-1-ol (>98%),

(E)-2-hexen-1-al (>99%),

ethyl (E,Z)-2,4-decadienoate (pear ester, >97%),

(Z)-3-hexenyl acetate (>98%),

R(+)-limonene (limonene, >98%),

(±)-linalool (linalool, >97%),

β-caryophyllene (caryophyllene, >98.5%),

methyl salicylate (>99%) supplied by Sigma-Aldrich Chemie (Buchs, Switzerland), (E)- -farnesene (beta-farnesene, >90%) from Bedoukian Research, Inc. (Danbury, CT, USA),

4,8-dimethyl-1 ,3(E),7-nonatriene (dimethyl-nonatriene, -94%) from Givaudan (Dubendorf, Switzerland),

Ethanol was used as solvent (analysis grade, >99.8%, Merck AG, Dietikon, Switzerland).

SUBSTITUTE SHEET (RULE 26) Wind tunnel

The experiments were conducted in a wind tunnel. A wind tunnel of non-reflecting glass was equipped with centrifugal ventilators at both ends that operated simultaneously to move air across its length at 30cm/s through active charcoal filters at the up- and down-wind ends. Overhead illumination was provided by 36W fluorescent tubes (Philips type TLD36W/83o) at 36 kHz. Two groups of 6 tubes 120 cm long (total illuminated length 190 cm) hung 12 cm above the top of the wind tunnel. The light intensity was regulated with a potentiometer to produce ca. 10 lux along the wind tunnel floor. Light was dispersed using a Perspex Prisma® crystal- clear plastic sheet under the fluorescent tubes and by placing crepe paper on the roof of the wind tunnel. The wind tunnel was housed in a walk-in climate chamber (Schaller Uto AG, Bern, Switzerland) that allowed the air stream to be maintained at 18 ± 0.5°C and 85 ± 2% R.H. during experiments. The wind tunnel is designed so that the combinations are released in a sustained manner. These conditions were adequate for males to fly and make contact with calling females in the wind tunnel.

Insect preparation

For insect preparation, pupae of C. pomonella originating from Andermatt Biocontrol AG (Grossdietwil, Switzerland) were removed from the corrugated cardboard strips in which they had pupated and the sexes were separated. Adult males and females emerged into plastic cages (30cm 30cm ^χ 30cm, mesh size: 24, type BUGDORM-I, Megaview Science Education Services Co., Ltd., Taichung, Taiwan) in a climate chamber (Rosemund AG, Liestal, Switzerland) at 25°C and 65% R.H. in the photophase (18 hrs.) and 18°C and 85% R.H. in the scotophase (6 hrs.).

Release of compounds

For the release of test stimuli, stock solutions of synthetic chemicals were made up in ethanol and were dispensed from an ultrasound evaporator at the head of the wind tunnel. The ultrasound evaporator consisted of a syringe pump (type CMA 400, CMA Microdialysis AB, Solna, Sweden) that pumped the test solution at 10 μΙ/min from a 5 ml gas-tight syringe (Hamilton type 81527, Milian SA, Meyrin, Switzerland) into PTFE micro tubing (1.5 m long, 1.02 mm o.d., 0.56 mm i.d., Hamilton type 90674)

SUBSTITUTE SHEET (RULE 26) connected by a PTFE micro tubing connector (3 cm long, 1.57 mm o.d., 0.97 mm i.d., Hamilton type 20919) to a borosilicate glass capillary (50 mm long, 1 mm o.d., type GC 00-10, Ciark Electromedical instruments, Pangbourne, England) with a drawn out tip (10-20 mm tip length, 30-40 μιη i.d. tip opening). A frequency generator (Wavetek FG-5000A, Willtek Communications GmbH, Ismaning, Germany) producing a square-wave signal (ca. 80 kHz, 40V amplitude) was connected to a piezo-ceramic disc (25 mm diameter, Philips PXE5 25/2.0) that hung from the glass capillary. This caused the latter to oscillate and produce an aerosol of the test solution that broadened to ca. 10 mm at 50 mm from the source. The oscillating glass capillary was installed in the middle of the upwind end of the wind tunnel at 30 cm from the floor. A metal grid cylinder (15 mm long, 30 mm o.d.) placed over and along the axis of the capillary protected the latter against damage by moths attempting to contact the chemical source. Oscillations of the capillary tip dispersed the solution as micro- droplets that evaporated within a few cm downwind of the release point to create an invisible plume at 10-15 cm from the source. The ratio of codlemone and host plant volatiles was 1 to 10 in our experiments. When the responses of males to calling females were tested, a single calling female C. pomonella was presented in a glass tube (25mm o.d., 21 mm i.d., 125 mm long) closed at either end with curtain netting (1.5 mm mesh). The tube was placed on a platform 30 cm high at the upwind end of the wind tunnel with its long axis parallel to the wind direction.

Experimental settings

Experiments were performed with 2-4 day-old males within 0.5 to 3 hour of the onset of the scotophase, when females called. Males were transferred from the rearing cabinet to the environmental cabinet housing the wind tunnel (both at the same environmental conditions) twenty minutes before the experiments. A single male was presented in a glass tube (125 mm long, 25 mm o.d., 21 mm i.d.) on a stand (30 cm high, i.e. at the same height as the oscillating capillary) at the downwind end of the wind tunnel (the upwind end of the glass tube was 35 cm from the downwind end of the wind tunnel) in each test. Sixty males were tested per treatment with the exception of tests with different release rate of codlemone when on average 24 males were tested for each dose. Having put the glass tube with the male on the stand, first the downwind cap and then the upwind plastic cap on the glass tube was removed and the door of the wind tunnel was closed. Real-time recording started

SUBSTITUTE SHEET (RULE 26) immediately by starting the OBSERVER software package (version 5.0, Nodus Information Technology, Wageningen, The Netherlands) that ran on a portable PC. The same person put the glass tube on the stand, removed the caps, closed the door and started the software. Two minutes were allowed for the male to respond to each test treatment during which the following behaviours were recorded by eye and the time at which each behaviour criterion began was recorded by pressing the appropriate key of the software protocol: moth activation (movement in the release tube), wing fanning, take off, upwind flight (upwind flight of at least 15 cm towards the odour source), passing the midline of the flight zone in the wind tunnel (158.5 cm in total from the upwind end of the release tube to the protection grid over the glass capillary tip), close-in (approaching to within 15 cm of the metal grid) and contact (contact to the metal grid by the moth).

Statistical analysis of results

For the statistical analysis of the results, the proportion of males showing each behavioural element per treatment was calculated (number of males responding under each behavioural category over the total number of males tested). These treatment proportions were then compared using the Generalized Linear Models (GLM) with binomial distribution and logit link function. Kaplan-Meier curves were used for time-based representation of responses of males to different test solutions. These response-time relationships were compared using the Cox proportional hazards model. All statistical analyses were performed using the statistical software R with a type I error rate of 0.05.

In contrast to several previous studies where the responses of moths to test stimuli were recorded in wind tunnels, we used the Generalized Linear Model (GLM) with binomial distribution and logit link function to analyse the behavioural responses of male C. pomonella. The behavioural steps generally quantified in wind tunnel experiments are activation, wing fanning, take-off, upwind flight and odour source contact. Such responses of males show a binomial distribution (for instance, activated or not). In contrast to ANOVA, where data are assumed to be measured at intervals or ratio scales (Anderberg MR, 1973), GLM can handle such binomial data sets and can be used to compare the proportion of moths showing a specified behavioural step between treatments.

SUBSTITUTE SHEET (RULE 26) Example 1 :

Increasing the attractiveness of codlemone to codling moth males can be regarded as a key to increasing the efficiency of mating disruption of codling moth. In addition to the enhancing effects of secondary pheromone components (Knight AL et al, 2005; Witzgall P et al, 2001 , Bartell RJ et al, 1988), we now know that host plant compounds can also enhance the attractiveness of male C. pomonella to codlemone (Yang Z et al, 2004; Knight AL et al, 2005). However, results published so far have not led to a clear indication as to the use of plant volatiles for mating disruption. According to Yang Z et al, (2004), admixing plant volatiles such as either hexenyl butanoate, methyl salicylate or (Z)-3-hexenyl benzoate to an underdosed level of codlemone resulted only in the enhancement of the early behavioural steps of activation and wing fanning, but not the later steps from upwind flight to source contact. In contrast, admixing the female sex gland component dodecan-1-ol to an underdosed-level of codlemone did not influence activation and wing fanning, but this secondary sex pheromone component strongly influenced the later behavioural steps from upwind flight to source contact in two separate studies (Arn H et al, 1985; Witzgall P et al, 2001 ). Witzgall et al. also found that admixing codlemone ester, (E,E)-8,10-dodecadienyl acetate, to an underdosed level of codlemone only influenced the late behavioural steps. By contrast Yang Z et al. (2004) did find that some compounds like linalool, β-farnesene and (Z)-3 hexen-1-ol influenced the whole behavioural repertoire of male C. pomonella when admixed with codlemone.

In an effort to more closely characterize the effect of plant volatiles on codling moth attraction, we first examined the responses of male C. pomonella to codlemone at different release rates under our experimental conditions in order to establish a suboptimal attractive dose which could be amenable to improvement by adding host plant volatile products. Results in Figure 1 show that codlemone released by 100 pg/min was the optimal dose with 70% of the codling moth males contacting the source. Release rates between 1 and 100 pg/min were suboptimal and attracted less moths, whereas release rates higher than 100 pg/min reduced the number of males that succeeded in contacting the source. At the release rate of 10 pg/min codlemone, a high proportion of male C. pomonella showed activation (97.7%), wing fanning

SUBSTITUTE SHEET (RULE 26) (91.6%) and take off (85.0%), but a significantly lower proportion (33.3%, GLM, Estimate -2.443, Standard error 0.454, z value -5.381 , p value < 0.001 ) showed upwind flight, passed the midline, approached and contacted the source. At release rates below 10 p/min and above 1000 pg/min, the males contacted the source in less than 15 per cent of cases. Noteworthy, the 70% proportion of source contact measured at codlemone released at 100 pg/min was very close to that observed with calling females (63%, Figure 2).

Example 2:

Various host plant volatiles (released at 100 pg/min) were tested for their potentiating effects to a suboptimal release rate of 10 pg/min codlemone on behaviour of codling moths. The proportion of males showing activation (95- 00%) and wing fanning (90- 98.3%) responses to codlemone admixed with any of the plant volatiles was no different to the response to codlemone alone or to calling females. Except for methyl salycilate, none of the host plant volatiles added to codlemone affected the proportion of males taking off compared to codlemone alone or to calling females (Fig. 2). Methyl salycilate added to codlemone resulted in a reduction in the proportion of males taking off to 68.3% (Fig. 2). Adding β-farnesene, pear ester, linalool and limonene to codlemone significantly increased the proportion of male C. pomonella undertaking upwind flight, passing the midline, closing-in and making source contact compared to the proportion responding to codlemone alone (also summarized in Table 1 ). The levels of responses under these four behavioural criteria to these treatments were no different to that recorded for calling females (also summarized in Table 2). The other host plant volatiles had no effect on the proportion of male C. pomonella undertaking upwind flight, passing the midline, closing-in and making source contact.

Example 3:

The time-based reactions of male C. pomonella to the plant volatile compounds were analysed under the behavioural steps of activation, upwind flight and source contact. Codlemone released at 10 pg/min activated 81 % of the males within the first 10 seconds of experiments and 87% within the first 20 seconds (Fig. 3). Three host plant compounds, hexanol, limonene and methyl salycilate, influenced the reaction time of male C. pomonella to codlemone (summarized in Table 3). Admixing hexanol or

SUBSTITUTE SHEET (RULE 26) limonene to codlemone decreased the reaction time of male C. pomonella: in the first 10 seconds of experiments the proportion of activated males increased to 95% and 92%, respectively. Admixing meihyi saiyciiate to codiemone increased reaction time: only 53% of males were activated in the first 10 seconds of experiments, and only 65% in the first 20 seconds (Fig. 3).

Example 4:

Effect of plant volatiles on the upwind flight behavioural step of codling moth males was determined. Limonene, pear ester and beta-farnesene admixed to codlemone shortened significantly the time till the upwind flight response of male C. pomonella to codlemone was induced, and the other host plant volatiles tested had no effect on this behavioural step (Fig. 4, also summarized in Table 3). Whereas 33% of C. pomonella males showed upwind flight to codlemone within the 120 second experimental period, following admixture of limonene, pear ester or β-farnesene to codlemone this proportion was reached within 26, 23 and 25 seconds, respectively.

Example 5:

When codlemone was released at 10 pg/min, the first source contact happened only after 20 seconds of exposure. After 60 seconds the proportion reaching the source increased to 23% and to 33% for the whole experimental period (120 seconds). Admixing limonene, pear ester, β-farnesene or linalool to codlemone significantly decreased the time required by male C. pomonella to contact the odour source: the 33% source contact level observed to codlemone within 120 seconds was reached within 37 seconds when limonene was admixed to codlemone, and within 41 seconds when pear ester was added to the pheromone (Fig. 5, also summarized in Table 3). Similar source contact levels were observed for linalool and β-farnesene added to codlemone within 68 and 62 seconds, respectively.

We show here that by adding limonene to codlemone the response time of male C. pomonella was shortened and the proportion of males attracted to the pheromone source increased. We conclude that combining limonene with synthetic codlemone should enhance the efficiency of mating disruption control method through ensuring engagement of males with dispensers to the detriment of females.

SUBSTITUTE SHEET (RULE 26) The results of Examples 1-5 and their statistical analysis are summarized in Tables 1-3.

Table 1 shows a summary of results (estimate, standard error, z value and p value) from the Generalized Linear Model analysts of the behavioural responses of C. pomonella to host plant volatiles (released at 100 pg/min) admixed to codlemone (released at 10 pg/min). Only compounds influencing behaviour of C. pomonella are listed.

SUBSTITUTE SHEET (RULE 26) Table 1 : Limonene showed a statistically significant strong positive effect on the four criteria measured: upwind flight, passing midline, close-in and contact to the

Table 2 shows a summary of results (estimate, standard error, z value and p value) from the Generalized Linear Model analysis of the behavioural responses of male C. pomonella to host plant volatiles (released at 100 pg/min) admixed to codlemone (released at 10 pg/min) compared to calling females. Only plant compounds causing effects the same as calling females are listed.

Table 2: Limonene showed a statistically significant strong positive effect on the four criteria measured, upwind flight, passing midline, close-in and contact to the codlemone source.

SUBSTITUTE SHEET (RULE 26) Table 3 shows the summary of results (estimate, standard error, z value and p value) from the Cox proportional hazards analysis of the time-dependent behavioural responses of C. pomonella to host plant volatiles (released at 100 pg/min) admixed to codiemone (released at 10 pg/min). Only compounds influencing the reaction time of C. pomonella are listed.

Table 3: Limonene showed a statistically significant strong decreasing effect on the reaction time of the three criteria measured, activation, upwind flight and contact to the codiemone source.

The GLM revealed a highly significant (PO.001 ) correlation in time elapsed until activation and upwind flight with the probability of an individual reaching the

SUBSTITUTE SHEET (RULE 26) treatment source, i.e. treatments that induced males to accomplish these behaviours quicker were also more likely to successfully attract these males to the source.

SUBSTITUTE SHEET (RULE 26) References

Anderberg MR. 1973. Cluster analysis for applications. Academic Press, pp. 359.

Arn H, Guerin PM, Buser HR, Rauscher S and Mani E. 1985. Sex pheromone blend of the codling moth, Cydia pomonella: evidence for a behavioural role of dodecan-1- ol. Experientia 41 : 1482-1484.

Bartell RJ, Bellas TE and Whittle CP. 1988. Evidence for biological activity of two further alcohols in the sex pheromone of female Cydia pomonella L. (Lepidoptera: Tortricidae). J Austral Entomol Soc 27:11-12.

Birch AN, Begg GS and Squire GR. 201 1. How agro-ecological research helps to address food security issues under new IPM and pesticide reduction policies for global crop production systems. J Exp Bot. Jun;62(10):3251 -61. Epub 201 1 Jun 8.

Christensen TA and Hildebrand JG. 2002. Pheromonal and host-odor processing in the insect antennal lobe: how different ? Curr Opin Neurobiol. Aug;12(4):393-9.

Coracini M, Bengtsson M, Liblikas I and Witzgall P. 2004. Attraction of codling moth males to apple volatiles. Entomol Experim et Applic 1 10:1-10.

Dudareva N, Negre F, Nagegowda DA and Orlova I. 2006. Plant Volatiles: Recent Advances and Future Perspectives. Critical Reviews in Plant Sciences, 25; 5, 417- 440.

Guerin PM, Arn H, Blaser C and Lettere M. 1983. Z, E-8, 10-Dodecadiene-1-ol, attractant for male Pammene rhediella. Entomologia Experimentalis et Applicata 33, 346.

Knight AL and Light DM. 2005. Developing action thresholds for codling moth (Lepidoptera: Tortricidae) with pear ester- and codlemone-baited traps in apple orchards treated with sex pheromone mating disruption. The Canadian Entomologist 137:(6) 739-747.

Matsumoto S. 2010. Molecular mechanisms underlying sex pheromone production in moths. Biosci Biotechnol Biochem. 2010;74(2):223-31. Epub 2010 Feb 7. Review.

Roelofs WL, Comeau A, Hill A and Milicevic G. 1971. Sex attractant of the codling moth: characterization with electroantennogram technique. Science 174:297-299.

Stara J, Nadova K and Kocourek F. 2006. Insecticide resistance in codling moth (Cydia pomonella). J Fruit Ornam Plant Res 14:99-106.

Stelinski LL, Gut LJ and Miller JR. 2006. High-performance mating disruption of codling moth, Cydia pomonella. Workshop on arthropod pest problems in pome fruit production, Lleida (Spain).

Sutherland ORW, Wearing CH and Hutchins RFN. 1977. Production of a-farnesene, an attractant and oviposition stimulant for codling moth, by developing fruit of ten varieties of apple. J Chem Ecol 3:625-631.

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Witzgall P, Stelinski L, Gut L and Thomson D. 2008. Codling moth management and chemical ecology. Annu Rev Entomol. 53:503-22. Review.

SUBSTITUTE SHEET (RULE 26) Witzgall P, Bengtsson M, Rauscher S, Liblikas I, Backman AC, Coracini M, Anderson P and Lofqvist J. 2001. Identification of further sex pheromone synergists in the codling moth, Cydia pomonella. Entomol Experim etApplic 101 :131 -141.

Witzgall P, Kirsch P and Cork A. 2010. Sex pheromones and their impact on pest management. J Chem Ecol. Jan;36(1 ):80-100. Review.

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SUBSTITUTE SHEET (RULE 26)

Claims

Claims

1. A composition comprising the combination of the sex pheromone (E,E)-8, 10- dodecadienol (codlemone) and limonene.

2. The composition according to claim 1 , wherein the amount of codlemone is at 5 to 20% by weight relative to the amount of limonene.

3. The composition according to claim 1 , wherein the amount of codlemone is at 10% by weight relative to the amount of limonene.

4. The composition according to any of claims 1 to 3, for mating disruption of the codling moth.

5. A method for mating disruption of the codling moth, which comprises the steps of releasing in a crop or orchard an effective amount of the composition according to any of claims 1 to 3.

6. A method for mating disruption of the codling moth, which comprises the steps of releasing separately or concomitantly in the crop or orchard an effective amount of the sex pheromone (E,E)-8,10-dodecadienol (codlemone) and limonene.

7. A dispenser for releasing in the crop or orchard an effective amount of the composition according to any of claims 1 to 3.

8. A dispenser for releasing separately or concomitantly in a crop or orchard an effective amount of the sex pheromone (E,E)-8,10-dodecadienol (codlemone) and at least one host plant volatile compound.

9. The dispenser of claim 8, wherein said at least one host plant volatile compound is limonene.

SUBSTITUTE SHEET (RULE 26)

10. Use of the composition according to any of claims 1 to 3, as a lure designed for insect trapping.

11. The use of claim 10, wherein the insect is codling moth.

12. Use of the composition according to any of claims 1 to 3, for mass trapping of codling moth.

SUBSTITUTE SHEET (RULE 26)