Background of the Invention: The composition of the sex pheromone of Diamondback moth, Plutella xvlostella, was reported in 1977 by Tamaki and co-workers who proposed a binary mixture containing equal amounts of (Z)-ll-hexadecenyl acetate and (Z)-11-hexadecenyl aldehyde. This proved attractive to insects in the field 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 al. 1982).

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 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 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, there is no published evidence for any recent re-investigation of the sex pheromone of this insect.

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 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. zlostella females using gas chromatography. As a 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 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.

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)-ll-hexadecenol in a weight/weight ratio of 10: 2-25: 1-5.

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.

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 2000g/trap, preferably 100 to 1, 000yg/trap. The compositions may include a suitable solvent such as cyclohexane.

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 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 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.

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 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 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 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 non-limiting examples and accompanying figures.

Brief description of the accompanving figures: Figure 1 provides a gas chromatograph trace (Trace A00445) of an extract of P. ylostella pheromone glands. Trace A00444 is from an extract of other body tissues.

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.

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 xj,-Iostella females using gas chromatography. The alternative approach of investigating the sensory sensilla in the males using 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 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 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 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 carried out with varying numbers of glands, 10 to 30, pooled together to increase the amount of pheromone collected. Following extraction with

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, 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 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 (Z)-1l-hexadecenol.

Example 2: Formulation of synthetic pheromone compositions and field trials The identification in Example 1 of the third component (C) (ie. (Z)-11- 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 bioassav in which the 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.

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 Tanglefoott-coated liner, 90mm wide and 170mm long that was fixed to the base with a paper clip.

Baits consisted of 100 n. g of pheromone dissolved in 10 al of cyclohexane

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)-ll-hexadecenol, So as not to 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 showed that the formulations including (Z)-l1-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 each of the blends below arranged as a Latin square. Composition Description Average no. of insectscaught 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

Compositions C and D are more attractive than either A or B with a greater than 99.9% probability.

Trial 2.

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. Composition Description Average no. of insectscaught 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: Z11-160Ac Z11-1GALD Z11-160H E 10 0 0 F10 10 0 G 10 0 2 H 10 10 2 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 separation. Traps were rotated one position in a clockwise direction every ca 2 weeks. Compositions I-M were each loaded with a total of 1000jug of

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. al.. 1995, from a local Diamondback moth 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 times each week over the period of the trial (58 days).

Table 3. Ratio of components, Trial 3. Z11-160Ac Z11-16ALD Z11-160H I 10 10 0 J 15 5 0 K 10 10 2 L 15 5 2 M 10 10 4 N1280

Table 4. Initial distribution of traps, Trial 3. I1 J1 K1 L1 M1 N1 01 02 12 J2 K2 L2 M2 N2 N3 03 I3 J3 K3 L3 M3 M4 N4 04 14 J4 K4 L4 L5 M5 N5 05 I5 J5 K5

O-trap containing virgin females (non-svnthetic control) 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

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 Compositions I-NI. 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 Fg for Compositions I-M vs 100 ßg for Composition N) may have contributed to the difference in bait efficacy in the long term. 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. CATCH IN 5 TRAPS Day I J K L M N O 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

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 including (Z)-ll-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.

References Chisholm M. D., Steck, W. F., Underhill, E. W. & Palaniswamy, P. (1982) Field trapping of diamondback moth Plutella xlZlostella using an improved four- component sex attractant blend, J. Chent. Ecol. 9,113-118.

Koshihara, T. & Yamada, H. (1980). Attractant activity of the female sex pheromone of diamondback moth, Plutella xszlostella, (L.), and analogue. Jap.

J. Appl. Ento. Zool. 24,6-12.

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). Appl. Ent. Zool, 12, 108-210.

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 Joumal of Pest Managment 41,201-204.

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 be considered in all respects as illustrative and not restrictive.