Devices, Compositions and Methods for Insect Control

Abstract

The present invention relates to compositions and devices for attracting, trapping and/or monitoring insects, more particularly Carpophilus beetles, such as stone fruit beetle and almond beetle. The described compositions and devices include novel mixtures for attracting Carpophilus beetles, including compositions for selectively attracting and trapping specific species of Carpophilus beetle.

Claims

1-28. (canceled)

29. A composition for attracting Carpophilus beetles, said composition including: ethanol, and a co-attractant mixture including one or more compounds selected from acetaldehyde, ethyl acetate, isobutanol, isopentyl alcohol, and 2-methylbutanol, wherein said composition is a liquid and/or gas mixture; wherein the co-attractant mixture includes one or more compounds selected from: acetaldehyde at a concentration of between approximately 5 l and 170 l/100 ml of corn position, ethyl acetate at a concentration of between approximately 75 l and 125 l/100 ml of composition, isobutanol at a concentration of between approximately 0.1 l and 50 l/100 ml of composition, 2-methyl-butanol at a concentration of between approximately 0.1 l and 40 l/100 ml of composition, and isopentyl alcohol at a concentration of between approximately 0.01 l and 1750 l per 100 ml of composition.

30. A composition according to claim 29, wherein the composition further includes one or more fungal additives.

31. A composition according to claim 30, wherein the one or more fungal additives are produced by yeast of the genus Wickerhamomyces, Pichia or Hanseniaspora.

32. A composition according to claim 31, wherein the one or more fungal additives are from the yeast species Wickerhamomyces rabaulensis, Pichia kluyveri or Hanseniaspora guillermondii.

33. A composition according to claim 30, wherein the one or more fungal additives are selected from isobutyl acetate, isopentyl acetate, 2-phenylethylpropionate, and 2-phenethyl acetate.

34. A composition according to claim 30, wherein the composition includes one or more fungal additives selected from: isobutyl acetate at a concentration of between approximately 0.05 l and 5 l/100 ml of composition, isopentyl acetate at a concentration of between approximately 0.01 l and 150 l/100 ml of composition, or 2-phenethyl acetate at a concentration of between approximately 15 l and 30 l/100 ml of composition.

35. A composition according to claim 30, wherein the fungal additive(s) and co-attractant mixture are present at a ratio of between approximately 1:2 and 1:100 (v/v).

36. A composition according to claim 29, wherein the ethanol is an aqueous ethanol solution.

37. A composition according to claim 36, wherein the aqueous ethanol is between approximately 30 to 85% ethanol.

38. A composition according to claim 29, wherein the concentration of isopentyl alcohol is between approximately 50 l and 1500 l/100 ml of composition.

39. An apparatus for dispensing the composition according to claim 29.

40. An apparatus according to claim 39, wherein the apparatus provides for regulated release of the composition.

41. A device for trapping Carpophilus beetles including a composition according to claim 29.

42. A method of attracting and/or trapping Carpophilus beetles including the step of exposing a Carpophilus beetle infested environment to a composition according to claim 29.

43. A method of monitoring for the presence of at least one Carpophilus beetle including positioning a composition according to claim 29, within an environment that requires monitoring for the presence of Carpophilus beetles.

44. A composition consisting of isopentyl alcohol and aqueous ethanol and optionally one or more fungal additives.

45. A composition according to claim 29 wherein the fungal additives are selected from isopentyl acetate and/or isobutyl acetate.

46. A composition according to claim 29 consisting of isopentyl alcohol and aqueous ethanol.

47. A composition according to claim 29 wherein the aqueous ethanol is between approximately 30 to 65% ethanol.

48. A composition according to claim 29 wherein the isopentyl alcohol is present in a concentration of between 400 and 1500 L/100 mL of composition.

Description

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

[0053] FIG. 1. A sachet dispenser, comprising a heat-sealed low density polyethylene (LDPE) sachet (10) with composition infused wick (20).

[0054] FIG. 2. Release rates using different sachets/components combinations to reproduce the odour of the commercial stone fruit carpophilus co-attractant lure. Ethanol exhibited limited permeation through the LDPE layers and was therefore tested in vials with perforated lids (holes of different diameters).

[0055] FIG. 3. SPME-GC-MS chromatograms of the commercial carpophilus co-attractant in solution (B) and co-attractant in sachets (A). Peak numbering corresponds to the following lure components: 1. acetaldehyde, 2. ethyl acetate, 3. ethanol, 4. isobutyl alcohol, 5. Isopentyl alcohol and 6. 2-methylbutanol.

[0056] FIG. 4. Results of field trials comparing stone fruit beetle catches using the carpophilus lure, CL (without aggregation pheromone) formulated in solution or LDPE sachets. Grey bars represent the mean cumulated catch of stonefruit beetles (C. davidsoni, C. hemipterus and C. humeralis) per trap over the 2 week period of the trial and error bars show the standard error. Lettering above bars show statistical differences between treatments.

[0057] FIG. 5. Field trapping of C. davidsoni using yeasts Pichia kluyveri (Pk), Hanseniaspora guilliermondii (Hg) broth, or sterile Sabouraud dextrose broth control (C). N=20. Letters above bars indicate statistical differences between treatments.

[0058] FIG. 6. GC-EAD responses of female () and male () C. davidsoni adults to headspace volatiles (black, FID chromatogram) of the fruit yeast Pichia kluyveri. Dashed lines indicate consistent physiological responses elicited in beetles antennae and their corresponding chromatographic peaks annotated as follows: 1) ethyl acetate, 2) Isobutyl alcohol, 3) Propyl acetate, 4) Isopentyl alcohol, 5) 2-methyl-1-butanol, 6) Isobutyl acetate, 7) Butyl acetate, 8) Isopentyl acetate, 9) 2-furanmethanol acetate, 10) -caprolactone, 11) Phenylmethyl acetate, 12) Ethyl octanoate, 13) 2-phenylethyl acetate and 14) 2-phenylethylpropionate. IS=Internal standard, IS1=n-octane, IS2=Nonyl acetate.

[0059] FIG. 7. SPME-GC-MS chromatograms of the headspace of the yeast Pichia kluyveri in potato dextrose broth. The odour profile is less complex with less peaks than that of the same yeast grown on peach agar medium. The production of acetaldehyde and ethanol covered by the solvent peak in GC-EAD recordings is made visible by the use of SPME for volatiles sampling.

[0060] FIG. 8. C. davidsoni catches using prototype co-attractant solutions in a stone fruit orchard (Shepparton, VIC). CL corresponds to the control (original Carpophilus Lure co-attractant), CL+IA (co-attractant+Isopentyl Acetate), CL+PA (co-attractant+2-Phenethyl acetate) and CL+IA+PA (co-attractant+Isopentyl Acetate+2-Phenethyl acetate). Letters above bars indicate statistically significant differences. Number of replicates per treatment N=6.

[0061] FIG. 9. Boxplots representing the results of lab-conducted cage dual-choice assays on Carpophilus truncatus responses to odours. Between 50 and 100 beetles were simultaneously exposed to a solution of co-attractant and a solution of co-attractant (control) from which one ingredient at a time was suppressed (treatments). The attraction index was calculated as follows: (number of beetles in test solutionnumber of beetles in control)/(total number of beetles). Hence, positive indices indicate preferences for the test solution whilst negative indices indicate preference for the control (original carpophilus co-attractant). Line in the boxplots correspond to the median, top and bottom of the boxplots represent the 75th and 25th percentiles respectively while error bars depict the 5th and 95th percentiles. White dots are outliers. n correspond to the replication of each experiments. p values result from paired t-tests and stars indicate the level of statistical significance.

[0062] FIG. 10. GC-EAD responses of C. truncatus to a W. rabaulensis synthetic odour blend. 1-Acetaldehyde. 2-Ethanol. 3-Isobutanol. 4-Isopentyl alcohol. 5-2-Methyl butanol. 6-Isobutyl acetate. 7-Isopentyl acetate.

[0063] FIG. 11. Results of field testing results assessing new co-attractants (with Isopentyl alcohol). The original co-attractant treatment (CL) was used as control for comparison with the prototypes. Grey bars show the mean weekly catch of C. truncatus per trap over, white bars represent other species (mostly C. davidsoni, C. hemipterus and C. humeralis). Each treatment was replicated 12 times (6 reps at two field sites). Error bars represent the standard error. Upper and lower case letterings above bars depict respectively the statistical differences between C. truncatus and other carpophilus species catches using different test solutions.

[0064] FIG. 12. Results of field tests assessing new co-attractant solutions (with Isopentyl alcohol). The best performing lure in previous trial (Isopentyl alcohol*2Example 8) was used as control for comparison with other prototypes. Black bars show the mean cumulated catch of C. truncatus per trap over duration of the phase whereas grey bars represent that of other species (mostly composed of C. davidsoni, C. hemipterus and C. humeralis). Treatments were replicated 12 times in total (6 reps in two field sites). Error bars represent the standard error. Upper case lettering above bars depicts statistical differences between C. truncatus catches using different test lures and lower case, those of the other carpophilus species.

[0065] FIG. 13. Results of field assessment testing different concentrations of ethanol used in the simplified co-attractant (Ref; See table 6) Grey bars show the mean weekly catch of C. truncatus per trap over the duration of the trial and the white bars that of other carpophilus species. Treatments were replicated 10 times in total (5 reps in two field sites). The error bars represent the standard error. Lower case lettering above bars are statistical differences between C. truncatus catches using different test lures and the upper case ones, those of the other carpophilus species.

[0066] FIG. 14. Results of field assessment of microbial-derived odour blends. CL corresponds to the original carpophilus co-attractant chosen (control). Black bars show the mean cumulated catch of C. truncatus per trap over the duration of the trial and the grey ones that of other carpophilus species. Treatments were replicated 12 times in total (6 reps in two field sites). The error bars represent the standard error. Upper case lettering above bars are statistical differences between C. truncatus catches using different test lures and the lower case one, those of the other carpophilus species.

[0067] FIG. 15. Result of field assessment of microbial-derived odour blends. CL corresponds to the original carpophilus food attractant chosen as control in this experiment. Black bars show the mean cumulated catch of C. truncatus per trap over duration of the phase whereas grey bars represent that of other species (mostly composed of C. davidsoni, C. hemipterus and C. humeralis). Treatments were replicated 10 times in total (5 reps in two field sites). The error bars represent the standard error. Upper case lettering above bars depicts statistical differences between C. truncatus catches using different test lures and the lower case one, those of the other carpophilus species.

[0068] FIG. 16. Results of a field trial in an almond orchard demonstrating that a simple co-attractant blend of isopentyl alcohol and ethanol (Ref; see table 9) is significantly more attractive than co-attractant blends with ethyl acetate and other yeast odours. Changes in the concentrations of ethyl acetate and yeast esters did not significantly influence C. truncatus attraction (grey bars). The lower catches of non-target species in all treatments involving the Ref solutions indicate that a strong synergism between acetaldehyde (absent in Ref) and ethyl acetate mediate the attraction of other non-target carpophilus (white bars). Lower case and capital letters above bars indicate statistically different weekly catches among treatments for C. truncatus and other non-target species, respectively. Error bars represent the standard errors. Each treatment was replicated 12 times split between two blocks and tested in the field for 7 weeks in Carina (30 January-11 March).

[0069] FIG. 17. Results of a field trial in an almond orchard showing that the new co-attractant blend of isopentyl alcohol and ethanol (Ref; see table 10) catches between 8 and 10 times more C. truncatus (grey bars) than the commercial solution (CL). In addition, the new attractant exhibits a greater specificity reflected in significantly lower catches of non-target Carpophilus species (white bars) than with the old co-attractant. Lower case and capital letters above bars indicate statistically different weekly catches among treatments for C. truncatus and other non-target species, respectively. Error bars represent the standard errors. Each treatment was replicated 12 times split between two blocks and tested in the field for 4 weeks in Carina (25 March-15 April).

[0070] FIG. 18. Results of a field trial in an almond orchard testing the use of different concentrations of isopentyl alcohol in the simplified co-attractant (Ref; see table 11). Grey bars represent C. truncatus weekly capture and white bars that of other carpophilus species. Error bars correspond to the standard error. Lower case and capital letters above bars indicate statistically different weekly catches among treatments for C. truncatus and other non-target species, respectively. The simplified co-attractant tends to catch more beetle when its isoamyl alcohol concentration is doubled (Ref2) while significantly lower catches are obtained when the concentration of isopentyl alcohol was tripled (Ref3). Each treatment was replicated 12 times split between two blocks and tested in the field for 4 weeks in Carina (25 March-15 April).

DETAILED DESCRIPTION OF THE EMBODIMENTS

Example 1Development of the New Lure Dispensers

[0071] The original co-attractant was developed based on fermenting peach juice, and comprised six constituents: acetaldehyde, ethyl acetate, ethanol, isobutyl alcohol, isopentyl alcohol and 2-methylbutanol prepared in aqueous solution according to the method described by Bartelt and Hossain 2006.

[0072] The release rates of co-attractant components formulated in low density polyethylene (LDPE) sachets were investigated (FIG. 1). All compounds except ethanol were found to permeate through the plastic membranes of the dispensers at comparable rates to those of the original co-attractant, but lasting for a significantly prolonged period of time. For instance, the release of highly volatile compounds such as ethyl acetate and acetaldehyde are extended from 1 day in the current solution to 1 week when applied in sachets (FIG. 2).

[0073] The release rate and theoretical longevity of the lure constituents in sachets were tested in gravimetric experiments. Data were used to determine the optimal sachet thickness, size (surface area) and estimated longevity of individual components (FIG. 2).

[0074] A fixed volume (1 ml) of each lure constituent was individually loaded in 50 cm 2 of different thicknesses (50, 100, 150 and 200 m). Sachets were weighed daily for the first 5 days and later at greater time intervals. Release rates of the compounds through different sachet thicknesses were inferred using sachet weight loss over time (FIG. 2). Only the linear part of the weight loss curve was considered in the calculations of release rates. The maximum estimated longevity was deduced from the calculated release rate and corrected based on adjusted sachet size and volume of chemical loaded.

[0075] Of all compounds, only ethanol could not be formulated in sachets. Lab experiments showed that the maximum release rate achievable with thin sachets was in the 25 mg/day range; about a hundred times less than ethanol emission rates in the commercial lure (2.5 g/day). In addition, the results of pilot testing under laboratory conditions suggest that equal emission rates and longevity could be achieved using ethanol gel instead of ethanol in solution.

TABLE-US-00001 TABLE 1 Combinations of different sachets used to recreate the odour of the original carpophilus co-attractant solution. Release rate Load Est. long. Release rate (commercial solution) Compound Thickness (m) Size (cm) (ml) (days) (mg/day) (mg/day) acetaldehyde 200 5 2.5 2 7 50 43 ethyl acetate 200 5 5 2 15 136 110 Isobutyl alcohol 50 2.5 2.5 1 200+ 4.9 2.9 Isopentyl alcohol 50 2.5 2.5 1 300 3.4 3.3 2-methyl-butanol 100 5 5 1 200+ 4.3 0.6 Notes: In grey: release rates of compounds in commercial solution Compositions loaded on a wick and sealed within the sachet. Ethanol cannot be formulated in sachet and is instead applied as a 45% solution (200 ml) in 11 cm diameter plastic container.

[0076] The similarity between the solution and sachet version of the co-attractant was confirmed by Solid-phase microextraction (SPME) sampling and GC-MS-FID analysis. (FIG. 3).

Example 2Field Studies of Sachet Lures

[0077] Lures in sachets were tested in an almond orchard in a trial that initially aimed to compare the attraction of the nut beetle C. truncatus to the commercial (stone fruit beetle) co-attractant, formulated in solution and sachets. However, as populations of stone fruit beetles (both C. davidsoni and C. hemipterus) were also present, this enabled data on sachet effectiveness to be collected for these species.

[0078] Lures were tested in black funnel traps (2317 cm, Bioglobal, Queensland, Australia), held in a metal ring fixed to a metal fence picket at 1.5 m height. A piece of insecticide (32 cm, dichlorovos) impregnated plastic strip was placed inside the traps to kill captured beetles. 200 ml of commercial lure or 45% ethanol (used with sachets) solutions was placed in open plastic containers (11.0 cm diameter) covered with mosquito netting. Sachets were suspended inside the traps by means of paperclips. Traps baited with different lures were arranged in a randomized block complete with a minimum of 30 m spacing between traps. Traps were serviced, trapped beetles collected, and lures replaced, weekly.

[0079] Over a two-week period, the sachet lures caught significantly more beetles than the same lure formulated in solution. C. hemipterus made up the vast majority of the beetles caught, with few C. davidsoni present in traps. C. hemipterus, prefers decaying fruits and is therefore considered more a nuisance than a pest. By contrast, C. davidsoni attack ripening fruits and cause significant crop damage. Both species are known to be strongly attracted to the carpophilus lure. The predominance of C. hemipterus in this specific trial may reflect an existing bias in the resident beetle population in the almond orchard.

Example 3Field Studies of Sachet Lures

[0080] The useability and effectiveness of the co-attractant formulated in sachets was field tested in December 2019 at two almond orchards located near Mildura, Victoria. A freshly prepared co-attractant solution was used as a control (i.e. no dispensers), and ethanol was dispensed in different ways across treatments, i.e. in plastic vials, in solution or both. Traps were arranged in a 64 randomized complete block design replicated at both sites (n=12). Beetles were collected and the lures replaced after one week.

[0081] Stone fruit beetles are often found in almond orchards in spring. The trial aimed to test lure effectiveness with the nut beetle C. truncatus, with the results revealing an impact of the sachets on the stone fruit pests, C. davidsoni, C. hemipterus and C. humeralis catches. [0082] Treatments comprising co-attractant components in sachets caught significantly more beetles than the co-attractant in solution. [0083] Greatest catches were obtained when sachets were applied with ethanol in solution as opposed to in vials or vials and solution simultaneously; reaching up to 8 times more the number of beetles caught with the co-attractant solution (FIG. 4).

Example 4Identification and Testing of 2-Phenylethylacetate as a New Co-Attractant Component for Carpophilus Attacking Stone Fruit

[0084] Our laboratory studies investigating the influence of yeasts on carpophilus oviposition and larval development suggest that fruit microbiome plays a crucial role in beetle survival: C. davidsoni pupal mass, survival to adulthood was found to be greater on fruit media inoculated with Pichia kluyveri and their development time shorter compared to when the same medium was inoculated with Hanseniaspora guillermondii; another yeast isolated from the guts of wild caught beetles (Baig et al. 2020).

Attraction of Stone Fruit Attacking Carpophilus to Yeast Odours in Field Studies

[0085] Beetle attraction to yeast volatiles was tested in a stone fruit orchard in Bunbartha (Northern Victoria). Volatiles were applied in the form of high-density yeast cultures of the two gut-associated yeasts P. kluyveri and H. guillermondii prepared in a potato dextrose broth, while a sterile potato broth was used as procedural control. Twenty replicates of each treatments placed in McPhail traps with a cube of insecticide were hung in trees in randomized transects with a 7 m minimum distance between traps. Beetles were collected and yeast broth was replaced weekly.

[0086] In line with lab performance studies, traps baited with P. kluyveri attracted significantly more beetles than those baited with H. guillermondii and sterile broth (FIG. 5).

Example 5Identification and Comparison of Yeast Odour Actives

[0087] P. kluyveri and H. guillermondii volatiles were collected and analysed by GC-MS. Statistical analysis indicated that 70% of the dissimilarity between the two yeast odours was linked to two chemicals: Isopentyl acetate and 2-phenylethyl acetate.

[0088] C. davidsoni sensitivity to the different compounds present in the odour of the attractive yeast P. kluyveri was tested using gas-chromatography coupled with electroantennography (GC-EAD). In total, 14 compounds elicited consistent responses with C. davidsoni (FIG. 6). Of these compounds, four are already present in the existing carpophilus lure (ethyl acetate, isobutyl alcohol, Isopentyl alcohol and 2-methyl-1-butanol) while responses to the two others (ethanol, acetaldehyde) also present in the headspace of P. kluyveri; could not be observed (covered by the solvent peak). However, beetles GC-EAD responses to these compounds were observed in another study (FIG. 7). Most of the other responses were elicited by esters (mostly ethyl esters, cyclic esters and acetates); including Isopentyl acetate and 2-phenyl ethyl acetate, and their amplitudes appeared widely dose-dependent. Volatiles collections and analysis conducted in parallel with electrophysiological studies (GC-EAD, FIG. 6) revealed a strong sensitivity of beetle antennae to two dominant esters in the yeast odour profile; namely Isopentyl acetate and 2-phenethyl acetate.

Example 6Field Testing of New Synthetic Blends to Attract Carpophilus Attacking Stone Fruits

[0089] Synthetic blends consisting of the combination of the existing carpophilus lure with two new potential yeast attractants; namely Isopentyl acetate and 2-phenylethyl acetate; were formulated based on their respective ratio to ethyl acetate (Table 2) and tested in the field.

[0090] Field trials were conducted in a peach orchard in Invergordon (Victoria). Treatments included the original carpophilus lure (CL: control), the original lure combined with Isopentyl acetate (CL+IA) or with 2-phenylethylacetate (CL+2PA) or both (CL+IA+2PA). Six replicates of each odour blend were placed in black funnel traps held on pickets at 1.5 m height, arranged in randomized transects. Beetles were collected and synthetic blends replaced weekly over a 5-week period.

[0091] These compounds were tested either separately or together in the existing Carpophilus co-attractant and compared with the original co-attractant in the field. The trial was conducted over a five week period during which the beetles were collected and the lure solutions renewed weekly. The lure compositions investigated are described in Table 2.

TABLE-US-00002 TABLE 2 Composition of the synthetic lures used as treatments in stone fruit orchard trial. Treatments Lure composition (vol/100 mL) (CL) Acetaldehyde: 65.4 L Carpophilus Lure Ethanol : 44.3 ml Ethyl acetate: 104.4 L Isobutanol: 33.8 L Isopentyl alcohol: 74.1 L *2-methyl-butanol: 24.4 L Water: 55.4 mL (CL + IA) Acetaldehyde: 65.4 L Carpophilus Lure + Ethanol : 44.3 mL Isopentyl Acetate Ethyl acetate: 104.4 L Isobutanol: 33.8 L Isopentyl alcohol: 74.1 L 2-methyl-butanol: 24.4 L *Isopentyl acetate: 142 L Water: 55.4 mL (CL + 2PA) Acetaldehyde: 65.4 L Carpophilus Lure + Ethanol : 44.3 mL 2-Phenylethyl Acetate Ethyl acetate: 104.4 L Isobutanol: 33.8 L Isopentyl alcohol: 74.1 L 2-methyl-butanol: 24.4 L *2-Phenylethyl acetate: 24 L Water: 55.4 mL (CL + IA & 2PA) Acetaldehyde: 65.4 L Carpophilus lure + Ethanol : 44.3 mL Isopentyl Acetate + Ethyl acetate: 104.4 L 2-Phenylethyl Acetate Isobutanol: 33.8 L Isopentyl alcohol: 74.1 L 2-methyl-butanol: 24.4 L *Isopentyl acetate: 124 L *2-Phenylethyl acetate: 24 L Water: 55.4 mL

[0092] The co-attractant to which the yeast compound 2-phenethyl acetate was added was observed to result in the capture of significantly more beetles than the original co-attractant alone (FIG. 8). Further, the addition of isopentyl acetate (alone or in combination with 2-phenethyl acetate) to the co-attractant was observed to reduce lure attractiveness (FIG. 8).

[0093] Studies determining optimal sachet size and thickness, lure concentration, release rates and lure longevity were performed as described in Table 3. Results showed that 2-phenethyl acetate is suitable for dispensing in sachets and longevity can be extended to over 70 days.

TABLE-US-00003 TABLE 3 Sachet description, lure formulation, release rates and lure longevity (based on GC-MS analysis) for all components used in field studies of Example 6. Thickness Size Load Est. long. Release rate Compound (m) (cm) (ml) (days) (mg/day) acetaldehyde 200 5 2.5 2 40 50 ethyl acetate 200 5 5 2 15 136 Isobutyl alcohol 50 2.5 2.5 1 200+ 4.9 Isopentyl alcohol 50 2.5 2.5 1 300 3.4 2-methyl-butanol 100 5 5 1 200+ 4.3 2-phenylethylacetate 50 5 5 2 70+ 28 Ethanol applied as a 45% solution (200 ml) in 11-cm diameter plastic container.

Example 7Co-Attractant Compositions Targeting Carpophilus truncatus

[0094] The commercially available carpophilus co-attractant exhibits some degree of attraction to C truncatus under laboratory and field conditions. The effect of volatiles within the existing co-attractant was therefore investigated in laboratory conducted bioassays (FIG. 9). Key attractants and repellents were identified leading to a simplified version of the existing co-attractant. Further lab testing showed that lure attraction could be augmented by varying the concentrations of one of the key attractants.

TABLE-US-00004 TABLE 4 Compositions of the test and control solutions used in cage bioassays Test solutions Control Experiment Composition Vol* Composition Vol* w/o Acetaldehyde 163.5 l acetaldehyde Ethyl acetate 261 L Ethyl acetate 261 L vs Isobutyl alcohol 84.5 l Isobutyl 84.5 l CL Isopentyl alcohol 185.8 l alcohol 185.8 l 2-methyl-butanol 61 l Isopentyl 61 l In ethanol 45% alcohol 45% w/o ethanol Acetaldehyde 163.5 l 2-methyl- vs Ethyl acetate 261 L butanol CL Isobutyl alcohol 84.5 l In ethanol Isopentyl alcohol 185.8 l 2-methyl-butanol 61 l In water w/o Isobutyl Acetaldehyde 163.5 l alcohol Ethyl acetate 261 L vs CL Isopentyl alcohol 185.8 l 2-methyl-butanol 61 l In ethanol 45% w/o Isopentyl Acetaldehyde 163.5 l alcohol Ethyl acetate 261 L vs Isobutyl alcohol 84.5 l CL 2-methyl-butanol 61 l In ethanol 45% w/o 2-methyl- Acetaldehyde 163.5 l butanol Ethyl acetate 261 L vs Isobutyl alcohol 84.5 l CL Isopentyl alcohol 185.8 l In ethanol 45%

[0095] Fifty beetles were simultaneously exposed to a solution of co-attractant (control) and a solution of co-attractant from which one additive at a time was suppressed (treatments, see table 4). The attraction index was calculated as follows: (number of beetles in test solutionnumber of beetles in control)/(total number of beetles). Hence, positive indices indicate preferences for the test solution whilst negative indices indicate preference for the control (original carpophilus co-attractant) (FIG. 9)

[0096] Cage experiments demonstrated that the commercial co-attractant was significantly less attractive in the absence of ethanol and Isopentyl alcohol which appear to be the main attractants. In addition the co-attractant was significantly more attractive in the absence of 2-methylbutanol thus demonstrating its repellent effect. Acetaldehyde did tend to act as an attractant though this could not be clearly demonstrated in subsequent experiments whilst ethyl acetate and isobutyl alcohol seem to not influence beetle's orientation. Hence, the co-attractant chosen for testing in the field consisted of a reduced version of the commercial attractant comprising only Isopentyl alcohol in 45% ethanol.

[0097] Further cage bioassay experiments showed that increased concentrations of Isopentyl alcohol contribute to an increase in lure efficacy. Therefore, different concentrations of this compounds were tested in the field.

Example 8Design of Yeast-Derived Co-Attractant Mixture

[0098] As co-attractants are derived from microbial odours, beetle attraction towards yeast odours isolated from the insect's gut was tested (Farrukh Baig, PhD thesis, 2020). The results demonstrated that C. truncatus is strongly attracted to the odour of the yeast Wickerhamomyces rabaulensis. Chemical analysis of the yeast headspace revealed strong similarities in composition with the commercial co-attractant. However, components occurred at ratios differing from those of the commercial solutions, and in addition other candidate attractants were identified by electrophysiological techniques (GC-EAD) (FIG. 10). Synthetic blends of the yeast odours were developed and field tested.

[0099] The headspace odour of W. rabaulensis was observed to comprise almost all the compounds from the co-attractant except ethyl acetate. In addition, Isopentyl acetate and isobutyl acetate accounted for a significant part of the odour profile and elicited strong antennal responses from beetles.

[0100] A synthetic blend of the observed composition was used for field testing at low and high (30) concentrations (See Example 10). The effect of the two esters was tested in the yeast blend formulation and also in combination with the commercial co-attractant in two separate trials (See Examples 9 and 10).

Example 9Field Trials of Modified Co-Attractant Mixture, for Attracting Carpophilus truncatus (Almond Beetle)

[0101] In the first field trial, the reduced version of the commercial attractant was evaluated both in solution and in its sachet version, with each treatment being replicated 12 times (6 reps at two field sites). The commercial formulation of the co-attractant (CL) was used as a control (Table 5).

TABLE-US-00005 TABLE 5 Composition of the test co-attractants developed using laboratory bioassays and tested in the field. Treatments Lure composition Liquid base CL (control) Acetaldehyde: 65.4 L /100 ml 250 ml of a solution of Ethyl acetate: 104.4 L / 100 ml ethanol:water (45:55) 2-methylpropan-1-ol: 33.8 l /100 ml Isopentyl alcohol: 74.3 l / 100 ml 2-methyl-butanol: 24.4 l /100 ml [Isopentyl- Isopentyl alcohol: 150 l / 100 ml OH] 2 (solution) [Isopentyl- Isopentyl alcohol: 2 sachets (50 m, 2.5 2.5 OH] 2 (sachet) cm, 1 ml load)

[0102] It was observed that traps baited with Isopentyl alcohol prepared in 45% ethanol caught significantly more C. truncatus beetles compared to the commercial co-attractant. In addition, the number of non-target species individuals was significantly lower than with the original solution (FIG. 11).

[0103] In a subsequent trial, the reduced co-attractant was assessed using a higher concentration of Isopentyl alcohol. The best performing solution in the previous trial was, this time used as control (Table 6). Treatments were replicated 12 times in total (6 reps in two field sites).

TABLE-US-00006 TABLE 6 Composition of variants of the reduced co-attractant formulated in solution and sachet Treatments Lure composition Liquid base [Isopentyl-OH]*2 (solution) - Isopentyl alcohol: 150 l / 100 ml 250 ml of a solution of control ethanol:water (45:55) [Isopentyl-OH]*6 (solution) Isopentyl alcohol: 450 l / 100 ml [Isopentyl-OH]*6 (sachet) Isopentyl alcohol: 1 sachet (50 m, 5 5 cm, 2 ml load)

[0104] There was no significant difference observed between the reduced co-attractant and the reduced co-attractant containing a higher concentration of Isopentyl alcohol. However, there appears to be a clear trend for greater catches with the more concentrated lure (FIG. 12).

[0105] In another trial conducted in almond orchards the simplified co-attractant was tested at different ethanol concentrations and confirmed 45-50%, corresponding to that of the original co-attractant, as optimal (see Table 7 and FIG. 13).

TABLE-US-00007 TABLE 7 Lure composition using different concentrations of ethanol in the simplified co-attractant Treatment Lure composition Liquid base 45% EtOH Isopentyl alcohol: 150 l / 100 ml 250 ml ethanol:water (45:55) 65% EtOH Isopentyl alcohol: 150 l / 100 ml 250 ml ethanol:water (65:35) 85% EtOH Isopentyl alcohol: 150 l / 100 ml 250 ml ethanol:water (85:15)

Example 10Fungal Additive Composition Trials for Attracting Carpophilus truncatus (Almond Beetle)

[0106] Synthetic blends of the attractive yeast W. raubulensis were assessed at two different concentrations: a concentration that corresponds to natural volatiles emissions of cultured yeasts (Wr low) and a more concentrated blend adjusted to match the ethanol concentration of the co-attractant whilst preserving the ratios of other components of the blend (Wr high). In a last treatment, the two newly identified yeast esters (Isopentyl acetate and isobutyl acetate) were added to the original co-attractant (Table 8). Each treatment was replicated 12 times in total (6 reps in two field sites).

TABLE-US-00008 TABLE 8 Test treatments involving volatiles synthetic blends of the yeast W. rabaulensis. Treatments Lure composition Liquid base CL Acetaldehyde: 65.4 l /100 ml 250 ml of a (Control) Ethyl acetate: 104.4 L / 100 ml solution of 2-methylpropan-1-ol: 33.8 l /100 ml ethanol:water Isopentyl alcohol: 74.1 l / 100 ml (45:55) 2-methyl-butanol: 24.4 l /100 ml Wr (Low) Acetaldehyde: 5.4 l /100 ml 250 ml of a 2-methylpropan-1-ol: 0.83 l /100 ml solution of Isopentyl alcohol: 3.53 l /100 ml ethanol:water 2-methyl-butanol: 0.15 l /100 ml (1.5%) Isopentyl acetate: 0.015 l /100 ml Isobutyl acetate: 0.1 L / 100 ml Wr (High) Acetaldehyde: 162 l /100 ml 250 ml of a 2-methylpropan-1-ol: 24.8 l /100 ml solution of Isopentyl alcohol: 105.8 l /100 ml ethanol:water 2-methyl-butanol: 4.53 l /100 ml (45:55) Isopentyl acetate: 0.45 l /100 ml Isobutyl acetate: 3 L / 100 ml CL + esters Acetaldehyde: 65.4 l /100 ml 250 ml of a Ethyl acetate: 104.4 L / 100 ml solution of 2-methylpropan-1-ol: 33.8 l /100 ml ethanol:water Isopentyl alcohol: 74.1 l / 100 ml (45:55) 2-methyl-butanol: 24.4 l /100 ml Isopentyl acetate: 0.45 l /100 ml Isobutyl acetate: 3 L / 100 ml

[0107] The two yeast blends demonstrated a trend for attracting more beetles than the original carpophilus lure (FIG. 14), especially at greater concentrations. However, the lure combining the new esters with the original co-attractant caught significantly more C. truncatus.

[0108] A further field trial aimed to evaluate the influence of the two new esters on C. truncatus attraction in the W. rabaulensis yeast blend. Wr [high] treatment used in previous trial was tested with and without these esters and compared with the commercial co-attractant (CL) used as control (Table 9). Each treatment was replicated 10 times in total (5 reps in two field sites). The error bars represent the standard error.

TABLE-US-00009 TABLE 9 Lure composition of treatments used to test the influence of yeast esters. Treatments Lure composition Liquid Base CL Acetaldehyde: 65.4 l /100 ml 250 ml ethanol: Ethyl acetate: 104.4 L / 100 ml water 2-methylpropan-1-ol: 33.8 l /100 ml (45:55) Isopentyl alcohol: 74.3 l / 100 ml 2-methyl-butanol: 24.4 l /100 ml Wr adj. Acetaldehyde : 137 L / 100 ml 2-methylpropan-1-ol: 21 L / 100 ml Isopentyl alcohol: 90 L / 100 ml 2-methyl-butanol: 4 l /100 ml Isobutyl acetate: 0.4 l/100 ml Isopentyl acetate: 2.5 L / 100 ml Wr adj. w/o esters Acetaldehyde : 137 L / 100 ml 2-methylpropan-1-ol: 21 L / 100 ml Isopentyl alcohol: 90 L / 100 ml 2-methyl-butanol: 4 l /100 ml

[0109] The yeast-derived blend tended was observed to result in the capture of more C. truncatus and less of other beetle species than the original co-attractant (FIG. 15). While esters increased beetle catches, compound ratios of the yeast blend alone appeared to already increase lure efficacy and specificity (FIG. 15).

Example 11Trial Comparing Fungal-Derived Blends with Simplified Co-Attractants (Ref) for Attracting C. truncatus (Almond Beetle)

[0110] The best yeast-derived blend and some variants of differing concentrations of ethyl acetate and esters were compared with the simplified co-attractant (comprising only ethanol and isopentyl alcohol) and simplified co-attractants combined to yeast esters (Table 10).

TABLE-US-00010 TABLE 10 Test treatments involving volatiles synthetic blends of the yeast W. rabaulensis, the simplified co-attractant and combinations of the two. Treatments Composition Liquid base Pheromones CL + EA + Acetaldehyde: 65.4 l /100 ml ethanol:water no Esters Ethyl acetate: 104.4 L / 100 ml (45:55) 2-methylpropan-1-ol: 33.8 l /100 ml 250 ml / trap Isopentyl alcohol: 74.3 l / 100 ml 2-methyl-butanol: 24.4 l /100 ml Isobutyl acetate: 3 l / 100 ml Isopentyl acetate: 0.45 L / 100 ml CL + EA Acetaldehyde: 65.4 l /100 ml (Wr) + Ethyl acetate: 20 L / 100 ml Esters 2-methylpropan-1-ol: 33.8 l /100 ml Isopentyl alcohol: 74.3 l / 100 ml 2-methyl-butanol: 24.4 l /100 ml Isobutyl acetate: 3 l / 100 ml Isopentyl acetate: 0.45 L / 100 ml Ref Isopentyl alcohol: 400 L / 100 ml Ref + EA + Isopentyl alcohol: 400 L / 100 ml esters Ethyl acetate: 104.4 L / 100 ml Isobutyl acetate: 3 l / 100 ml Isopentyl acetate: 0.45 L / 100 ml Ref + EA Isopentyl alcohol: 400 L / 100 ml (Wr) + Ethyl acetate: 20 L / 100 ml esters Isobutyl acetate: 3 l / 100 ml Isopentyl acetate: 0.45 L / 100 ml CL + EA + Acetaldehyde: 65.4 l /100 ml Esters*3 Ethyl acetate: 104.4 L / 100 ml 2-methylpropan-1-ol: 33.8 l /100 ml Isopentyl alcohol: 74.3 l / 100 ml 2-methyl-butanol: 24.4 l /100 ml Isobutyl acetate: 9 l / 100 ml Isopentyl acetate: 1.35 L / 100 ml Ref + EA + Isopentyl alcohol: 400 L / 100 ml esters*3 Ethyl acetate: 104.4 L / 100 ml Isobutyl acetate: 9 l / 100 ml Isopentyl acetate: 1.35 L / 100 ml

[0111] The simplified co-attractant (Ref) was observed to result in the capture of three to four-fold more C. truncatus than any other treatment (FIG. 16). The addition of ethyl acetate and the yeast esters to the simplified solution considerably reduced the number of captured beetles suggesting the existence of an antagonistic effect between these compounds and Isopentyl alcohol. In addition, the simplified co-attractant yielded significantly lower captures non-target carpophilus species than the other treatments.

Example 12Trial Comparing the Simplified 2-Component Co-Attractant (Ref) with the Commercially Available Co-Attractant (CL) in the Presence of Synthetic Commercial Pheromones

[0112] The simplified co-attractant (Ref, including isopentyl alcohol and ethanol) and the original commercially available co-attractant solutions were tested in the presence of the commercial carpophilus pheromones (Table 11).

TABLE-US-00011 TABLE 11 Test treatments involving the simplified and original attractants tested in the presence of commercial synthetic pheromones. Treatments Composition Liquid base Pheromones Ref + Phero Isopentyl alcohol: 400 L / 100 ml ethanol:water 1 trispecies CL + Phero Acetaldehyde: 65.4 l /100 ml (45:55) septum Ethyl acetate: 104.4 L / 100 ml 250 ml / trap 2-methylpropan-1-ol: 33.8 l / 100 ml Isopentyl alcohol: 74.3 l / 100 ml 2-methyl-butanol: 24.4 l /100 ml

[0113] The simplified co-attractant was observed to increase by eight to ten times the capture of C. truncatus (FIG. 17) compared to the original solution which attracted significantly more non-target species (predominantly C. hemipterus). Hence, the greater capture and specificity of the simplified co-attractant was proved to be more specific and therefore more suitable for use in attract & kill and monitoring of C. truncatus.

Example 13Optimization of the Concentration of Isopentyl Alcohol in the Simplified Co-Attractant (Ref)

[0114] Different concentrations of isopentyl alcohol prepared in 45% ethanol were tested in a field trial to determine the optimal concentration to use in the simplified co-attractant (Table 12).

TABLE-US-00012 TABLE 12 Test treatments involving the simplified co-attractant (Ref, including isopentyl alcohol and ethanol) with varying concentrations of isopentyl alcohol. Treatment Composition Liquid base Pheromones Ref Isopentyl alcohol: 400 L / 100 ml ethanol: water no Ref 2 Isopentyl alcohol: 800 L / 100 ml (45:55) Ref 3 Isopentyl alcohol: 1.4 mL / 100 ml 250 ml / trap

[0115] Co-attractant solutions in which the concentration of isopentyl alcohol was doubled (Ref2) tended to catch more C. truncatus than the reference solution (Ref) and significantly more than the co-attractant in which the concentration of isopentyl alcohol was tripled (Ref3) (FIG. 18). All solutions yielded negligible captures of non-target carpophilus species. The results indicate that doubling the concentration of isopentyl alcohol may yield significantly greater C. truncatus captures over longer trapping periods.

REFERENCES

[0116] Bartelt R J, Hossain M S (2006) Development of synthetic food-related attractant for Carpophilus davidsoni and its effectiveness in the Stone Fruit orchards in Southern Australia. J Chem Ecol 32:2145-2162. doi: 10.1007/s10886-006-9135-7. [0117] Hossain M S, Bartelt R J, Hossain M A B M, et al (2008) Longevity of pheromone and co-attractant lures used in attract-and-kill stations for control of Carpophilus spp. Entomol Exp Appl 129:148-156. doi: 10.1111/j.1570-7458.2008.00769.x. [0118] Torr S J, Hall D R, Phelps R J, Vale G A (1997) Methods for dispensing odour attractants for tsetse flies (Diptera: Glossinidae). Bull Entomol Res 87:299-311. [0119] Baig F et al. (2020) Yeasts Influence Host Selection and Larval Fitness in Two Frugivorous Carpophilus Beetle Species. J Chem Ecol. 46: 675-687. [0120] Baig F et al. (2020) Chemical ecology of Carpophilus beetles and their yeast symbionts. PhD thesis. Queensland University of Technology. (Published 25 Aug. 2020).