AU1504101A

AU1504101A - Diamondback moth pheromone composition

Info

Publication number: AU1504101A
Application number: AU15041/01A
Authority: AU
Inventors: Richard Alan Vickers
Original assignee: Commonwealth Scientific and Industrial Research Organization CSIRO
Current assignee: Commonwealth Scientific and Industrial Research Organization CSIRO
Priority date: 1999-11-22
Filing date: 2000-11-21
Publication date: 2001-06-04
Also published as: AUPQ417299A0; WO2001037664A1

Description

WO 01/37664 PCT/AUOO/01417 DIAMONDBACK MOTH PHEROMONE COMPOSITION Field of the Invention: The present invention relates to a composition for use as an attractant 5 for diamondback moths. The composition may be used as the attractant in traps and may also be used to disrupt mating of diamondback moths. The present invention further relates to methods of trapping and disrupting mating of diamondback moths using the composition. 10 Background of the Invention: The composition of the sex pheromone of Diamondback moth, Plutella xylostella, was reported in 1977 by Tamaki and co-workers who proposed a binary mixture containing equal amounts of (Z)-11-hexadecenyl acetate and (Z)-11-hexadecenyl aldehyde. This proved attractive to insects in the field 15 and a number of publications followed, especially field studies, using this mixture. Koshihara & Yamada (1980) showed however that the attractancy of the baits could be increased considerably if small amounts of other compounds were added to the binary mixture. Similar synergistic effects were observed by other workers (e.g. Chisholm et a]. 1982). 20 The binary pheromone mixture has proved quite effective as an attractant in traps. Any possible enhancement in trap catches obtained by adding a synergist has therefore not been an issue in the context of monitoring. The binary mixture has, however, also been used to try to disrupt mating of the insect and in this case the exact composition of the 25 pheromone may be critical. It was successful in reducing damage to crops but the amounts required to achieve an effective level of control were very high. This made the application of mating disruption techniques to control this insect uneconomic under most circumstances. The synergistic effect of a range of other compounds and the large 30 amounts of the binary mixture required for mating disruption could be judged to indicate, as Koshihara & Yamada (1980) themselves speculated, that the binary mixture is incomplete. Alternatively one of its components could have been incorrectly identified. While it may have been expected that if other components are present they would have been found by now, 35 there is no published evidence for any recent re-investigation of the sex pheromone of this insect.

WO 01/37664 PCT/AUOO/01417 2 The big difference between 1977 and now lies in the instrumentation available for pheromone blend analysis. Gas chromatography (GC) has been revolutionised by the appearance of capillary columns which have largely replaced the older packed columns providing much higher resolution and 5 sensitivity. Minor components which may have been missed by Tamaki et al. (1977) would most likely be easily detectable with modern instruments. Based on the information presented above, it was thought to be worthwhile carrying out a new chemical analysis of the contents of the sex pheromone gland of P. xylostella females using gas chromatography. As a 10 result of this analysis, the present inventors have found a component in addition to the two components of the binary mixture proposed by Tamaki et al. (1977). This additional component corresponds to one of the compounds on the list found by Koshihara & Yamada (1980) to increase the numbers of insects caught in traps when added to the binary mixture. In 15 further work presented hereinafter, the present inventors have identified relative amounts of the three components which provide compositions demonstrating improved attraction in traps as compared to previous binary mixtures. 20 Summary of the Invention: The present invention provides a synthetic pheromone composition for use as an attractant for diamondback moths comprising (Z)-11-hexadecenyl acetate, (Z)-11 hexadecenal and (Z)-11-hexadecenol in a weight/weight ratio of 10:2-25:1-5. 25 Detailed Description of the Invention: In a first aspect, the present invention provides a synthetic pheromone composition comprising (Z)-11-hexadecenyl acetate, (Z)-11-hexadecenal and (Z)-11-hexadecenol in a weight/weight ratio of 10:2-25:1-10. 30 Preferably, the weight/weight ratio is 10:5-15:2-5, more preferably, 10:10:4. The composition according to the present invention may be used as an attractant for diamondback moths in traps. The amount of the composition that may be used to bait such traps may be 50 to 2000 tg/trap, preferably 100 35 to 1,000[pg/trap. The compositions may include a suitable solvent such as cyclohexane.

WO 01/37664 PCT/AUOO/01417 3 Since the composition of the invention more closely resembles the natural pheromone composition than previous binary mixtures, it is anticipated that the present composition will also offer advantages in the application of disrupting mating of diamondback moths. In particular, it is 5 believed that lesser, more economic, amounts of the composition will be sufficient. Where the composition is used for disrupting mating, it may be advantageous to use the composition in conjunction with a suitable insecticide (e.g. fibronil), particularly when pest populations are high. That 10 is, insecticide application may be used to help reduce pest populations to a level where mating disruption would be more effective. As with the use as a trap bait, the composition when used as a mating disruptant may include a suitable solvent such as cyclohexane. Dispensing of the composition for the disruption of mating may be achieved using dispensers known to the art. 15 The composition may be dispensed from such dispensers at a rate of about 5-100 mg/ha/hour. In a second aspect, the present invention provides a method of trapping diamondback moths comprising the step of providing an infested area with one or more insect traps containing an amount of a composition 20 according to the first aspect. In a third aspect, the present invention provides a method of controlling the proliferation of diamondback moths, comprising the step of dispensing within an infested area an amount of a composition according to the first aspect, said amount being suitable to disrupt diamondback moth 25 mating. The composition and methods according to the invention thereby offer potential for reducing the damage caused to cruciferous crops (e.g. cabbage, cauliflower, brussel sprouts, broccoli, etc.) by diamondback moth larvae. Throughout this specification the word "comprise", or variations such 30 as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. The invention is hereinafter described with reference to the following 35 non-limiting examples and accompanying figures.

WO 01/37664 PCT/AUOO/01417 4 Brief description of the accompanying figures: Figure 1 provides a gas chromatograph trace (Trace A00445) of an extract of P. xylostella pheromone glands. Trace A00444 is from an extract of other body tissues. 5 Figure 2 provides graphical results for trap catches using Compositions I and M described hereinafter. Figure 3 provides graphical results for trap catches using Compositions J and L described hereinafter. 10 Example 1: Analysis of an extract of Plutella xylostella pheromone glands As mentioned above, it was considered that it would be worth carrying out a new chemical analysis of the contents of the sex pheromone gland of Plutella xylostella females using gas chromatography. The alternative approach of investigating the sensory sensilla in the males using 15 electrophysiological techniques was rejected. Single cell studies rather than the simpler electroantennogram would be required for the necessary degree of compound selectivity to be achieved and the small size of the insect and the fragility of its antenna makes such an undertaking very difficult. Koshihara and Yamada (1977) used an indirect method of collection in 20 which a number of female insects were allowed to release the pheromone inside a glass flask. The pheromone deposited on the walls was then collected using a solvent. In the present instance, it was decided instead to excise pheromone glands from the females and extract the pheromone from the glands. Each method has advantages and disadvantages. The present 25 choice was based on the fact that there was available methods for purifying the pheromone which removes most of the troublesome impurities. In addition, a method to discriminate between pheromone components and impurities using computer manipulated graphics to facilitate the comparison of GC traces was developed. Direct extraction from the glands also 30 minimizes the oxidation and other decomposition of pheromone products which can occur by the indirect method. This was important because one of the components reported by Koshihara and Yamada (1977) is an aldehyde which is a relatively unstable compound. After establishing a culture of the insect, a series of collections were 35 carried out with varying numbers of glands, 10 to 30, pooled together to increase the amount of pheromone collected. Following extraction with WO 01/37664 PCT/AUOO/01417 5 hexane and removal of high boiling point components using an evaporative technique they were injected into a gas chromatograph containing a Polyethylene Glycol Column (SGE BP20). Extracts produced a consistent pattern of peaks, Trace A00445 on Figure 1. Their number was, however, 5 rather high suggesting that not all of them were components of the pheromone. The pheromone glands in diamondback moth are very small and it is difficult to remove them without also including other body tissues. However, by comparing the GC traces with ones obtained from insect material from further up the abdomen, Trace A00444 on Figure 1, the peaks 10 arising from other body tissues were eliminated. The traces indicated the presence of the two components identified by Tamaki et aL., marked as A and B on Figure 1. A third component, C, was also found which corresponds to one on the list found by Koshihara & Yamada (1980) to increase the numbers of insects caught in traps when it was added to the binary mixture, that is 15 (Z)-11-hexadecenol. Example 2: Formulation of synthetic pheromone compositions and field trials The identification in Example 1 of the third component (C) (ie. (Z)-11 20 hexadecenol), does not in itself prove that it is a behaviourally active component of the P. xylostella pheromone. That is, it is possible that the third component was a precursor of one of the other components so that its presence might be coincidental. The best way of determining whether it actually contributes directly to the attraction is by a bioassay in which the 25 old binary blend is compared with the new formulation containing the third component. Trapping of insects in an infested field using sticky traps baited with different pheromone compositions was considered the quickest and most effective method to resolve the matter. 30 Trial 1. A four by four grid of traps, with 20m separation between them. was set up containing four different pheromone formulations in a Latin square distribution. The traps were constructed from waxed cardboard, measuring 85mm from base to apex and housed a replacable Tanglefoot*-coated liner, 35 90mm wide and 170mm long that was fixed to the base with a paper clip. Baits consisted of 100 tg of pheromone dissolved in 10 [tl of cyclohexane WO 01/37664 PCT/AUOO/01417 6 applied to the lumen of a 20mm length of surgical rubber tubing (internal diameter: 4.5mm, outer diameter: 6.5mm) placed centrally on the liner. Each trap contained the binary blend at two different ratios and two different formulations of a blend including (Z)-11-hexadecenol. So as not to 5 overwhelm the traps and to enhance any positive effect all the baits were made up with a concentration one tenth that normally used in commercial baits used for monitoring purposes. This was set up in a field of brussel sprouts in South Australia, The experiment was replicated three times using fresh baits. The numbers of insects caught at the different traps clearly 10 showed that the formulations including (Z)-11-hexadecenol was much more attractive to the insects than the old binary blend (Table 1). Table 1. Average numbers of insects caught over three weeks in 4 x 4 grid of traps, for 15 each of the blends below arranged as a Latin square. Composition* Description Average no. of insects caught A. Binary Mixture formulation 1 6.6 ± 3.7 B. Binary Mixture formulation 2 14.5 ± 9.4 C. New Blend formulation 1 138.1 ± 32.6 D. New Blend formulation 2 93.7 ± 35.3 * Composition (ratios) as follows: (Z-11- (Z-11- (Z-11 Composition hexadecenyl hexadecenal hexadecenol acetate (Z11 - 16 ALD) (Z11 - 16 OH) (Z11 - 16 OAc) A 10 10 0 B 10 3 0 C 10 10 2 D 10 3 2 WO 01/37664 PCT/AUOO/01417 7 Compositions C and D are more attractive than either A or B with a greater than 99.9% probability. Trial 2. 5 In a second trial conducted in the same manner as Trial 1. "New Blend' formulation 2 (H) was more attractive than binary compositions of acetate/aldehyde and acetate/alcohol (see Table 2). Table 2. 10 Composition Description Average no. of insects caught E. Acetate alone 10.3 ± 7.1 F. Binary Mixture formulation 1 33.3 ± 13.5 G. Acetate/Alcohol 10:2 mixture 12.5 ± 11.3 H. New Blend formulation 1 123.3 ± 61.0 * Composition (ratios) as follows: Z11 - 16 OAc Z11 - 16 ALD Z11 - 16 OH E 10 0 0 F 10 10 0 G 10 0 2 H 10 10 2 15 Trial 3. A third trial was conducted at Lembang, Indonesia. There were five replicates of each of seven treatments, details of which are given in Table 3. Water traps were initially set out as shown in Table 4, with at least 20 m 20 separation. Traps were rotated one position in a clockwise direction every ca 2 weeks. Compositions I-M were each loaded with a total of 1000sg of WO 01/37664 PCT/AUOO/01417 8 pheromone onto red surgical rubber tubing. Composition N consisted of 100 pg of pheromone loaded on to grey rubber septa. The component proportions of these baits reflected the composition of virgin female pheromone glands as determined by Zilahibalogh et. a]., 1995. from a local Diamondback moth 5 population. The compositions were not replaced during the trial. Virgin females derived from a laboratory culture were placed individually in small cylinders with mesh-covered ends and were suspended from the underside of the lids covering the water traps. The protocol called for replacement of females when they died. Catches were recorded 2 to 3 10 times each week over the period of the trial (58 days). Table 3. Ratio of components, Trial 3. Z11-16OAc Z11-16ALD Z11-160H I 10 10 0 J 15 5 0 K 10 10 2 L 15 5 2 M 10 10 4 N 12 8 0 15 Table 4. Initial distribution of traps, Trial 3. I1 Ji K1 Li M1 N1 01 02 12 J2 K2 L2 M2 N2 N3 03 13 J3 K3 L3 M3 M4 N4 04 14 J4 K4 L4 L5 M5 N5 05 15 J5 K5 0 - trap containing virgin females (non-synthetic control) 20 The greatest catch was achieved with Composition M. with which a total 1107 of moths was caught over the period of the trial (equivalent to 3.8 moths/trap/day) (Table 5). This composition comprised OAc:ALD:OH in a 1:1:0.4 ratio. In contrast, compositions with the same ratio of OAc:ALD but without OH (Composition I) resulted in a catch of 606 moths, or 2.1 WO 01/37664 PCT/AUOO/01417 9 moths/trap/day (Figure 2). When OH was added to a 2.5:7.5 ratio of ALD:OAc, the total catch increased from 287 (Composition J) to 896 (Composition L) (Figure 3). Composition N performed poorly in comparison with all of the 5 Compositions I-M. Although none of the latter had an ALD:OAc ratio corresponding exactly with that of N (6:4), Composition I was similar (ALD:OAc = 5:5) but caught far more moths. Whilst differences in pheromone loading (1000 sig for Compositions I-M vs 100 sg for Composition N) may have contributed to the difference in bait efficacy in the long term, 10 there was already a significant difference between the two baits after only eight days, where Compositions N and I averaged 2.2 and 4.6 moths/trap respectively. Table 5. Raw data. Trial 3. 15 CATCH IN 5 TRAPS Day I J K L M N 0 number 1 1 1 16 19 36 0 0 4 12 2 31 24 60 4 66 6 7 4 44 22 51 5 40 8 3 5 41 20 28 2 70 12 10 4 42 32 58 4 50 14 31 4 78 128 74 1 15 16 11 3 34 50 55 4 11 19 23 3 63 77 87 4 7 21 12 5 24 44 33 3 2 23 13 8 29 72 26 0 0 26 47 15 56 56 37 1 1 28 48 14 34 46 23 2 3 30 18 0 21 17 15 2 0 34 37 16 27 49 30 0 3 35 21 24 1 43 17 3 3 37 26 19 17 14 8 6 11 40 13 22 29 10 33 3 5 42 30 20 27 14 37 3 3 49 60 38 78 53 111 6 13 51 36 14 40 27 93 2 13 54 52 10 52 18 112 3 7 56 35 3 33 10 41 3 3 58 60 53 41 51 42 2 2 Sum 606 287 858 896 1107 63 328 WO 01/37664 PCT/AUOO/01417 10 Conclusion The field trials have established that the pheromone contained in the sex pheromone glands of females of P. xylostella is significantly different from the binary blend currently used for monitoring. Compositions 5 including (Z)-11-hexadecenol out performs the old binary compositions by a factor of 4-10 as measured by the number of insects which are attracted into a trap.

WO 01/37664 PCT/AUOO/01417 11 References Chisholm M.D., Steck, W.F., Underhill, E.W. & Palaniswamy, P. (1982) Field trapping of diamondback moth Plutella xylostella using an improved four 5 component sex attractant blend. J. Chem. Ecol. 9, 113-118. Koshihara, T. & Yamada, H. (1980). Attractant activity of the female sex pheromone of diamondback moth, Plutella xylostella, (L.), and analogue. Jap. J. Apple. Ento. Zoo]. 24, 6-12. 10 Tamaki, Y., Kawasaki, K., Yamada, H., Koshihara, T., Osaki, N., Ando, T., Yoshida, S. & Kakinohana, H. (1977). Z-11-Hexadecenal and Z-11 hexadecenyl acetate: Sex pheromone components of the diamondback moth (Lepidoptera: Plutellidae). Apple. Ent. Zoo], 12, 108-210. 15 Zilahibalogh G.M.G., Angerilli N.P.D., Borden J.H., Meray M., Tulung M. and Sembel D. (1995). Regional differences in pheromone responses of diamondback moth in Indonesia. International Journal of Pest Managment 41, 201-204.

WO 01/37664 PCT/AUOO/01417 12 It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to 5 be considered in all respects as illustrative and not restrictive,

Claims

1. A synthetic pheromone composition comprising (Z)-11-hexadecenyl acetate, (Z)-11-hexadecenal and (Z)-11-hexadecenol and, optionally, a 5 suitable solvent.

2. A synthetic pheromone composition according to claim 1 having a (Z)

11-hexadecenyl acetate:(Z)-11-hexadecenal:(Z)-11-hexadecenol component weight/weight ratio of 10:2-25:1-10. 10 3. A synthetic pheromone composition according to claim 1 having a (Z) 11-hexadecenyl acetate: (Z)-11-hexadecenal:(Z)-11-hexadecenol component weight/weight ratio of 10:10:4. 15 4. A method of trapping diamondback moths comprising the step of providing an infested area with one or more insect traps containing an amount of a composition of any one of the preceding claims. 5. A method according to claim 4, wherein said amount is about 50-2000 20 pig/trap. 6. The method according to claim 4, wherein said amount is about 100 1000 [tg/trap. 25 7. A synthetic pheromone composition according to any one of claims 1 to 3, further comprising a suitable insecticide. 8. A method of controlling the proliferation of diamondback moths comprising the step of dispensing within an infested area an amount of a 30 composition according to any one of claims 1 to 3 or 7 which is sufficient to disrupt diamondback moth mating. 9. A method according to claim 8, wherein the composition is dispensed at a rate of about 10-100mg/ha/hour.