Patent Application
20070122376
The present invention relates to new pheromones and method of preventing infestation of the swede midge, Contarinia nasturtii in Brassica vegetables, in particular Brussels sprouts, cauliflower and broccoli. In particular the present invention relates to sexual pheromones of C. nasturtii, and their use for the monitoring of the presence of C. nasturtii, as well as controlling said insect, method for its use, as well as compounds having such an activity.
The object of the present invention is to obtain a possibility to reduce infestation of Brassica vegetable plants by C. nasturtii. The object of the present invention is to obtain a possibility to determine the quantitative presence as well as the emergence time of C. nasturtii for monitoring purpose, and to obtain an efficient control of C. nasturtii by forecasting optimal timing of plant protection measures based on results of the above mentioned monitoring.
A further object of the invention is to obtain an environmentally acceptable control of C. nasturtii by applying the sexual pheromones of C. nasturtii to interrupt mating and thereby reproduction of the pest in the treated cultures.
Cecidomyiidae, gall midges, is a family encompassing about 5000 species (Gagné 1989) of which several are important noxious insects within the agricultural area all over the world. One of these gall midges is C. nasturtii, which infests Brassica vegetables, in particular Brussels sprouts, cauliflower and broccoli. The female C. nasturtii lays its eggs in the tips (buds) of cruciferous host plants, where the larvae feed and develop. Feeding damage near the growing tips induces distortion and gall-like symptoms. When the larvae have become fully developed they will go down into the soil and form cocoons, in which they will pass into the chrysalis stage and then either emerge to start the next generation cycle, or begin a diapause, which can be one or two winters long. In springtime, the larvae will leave the winter cocoon and create a summer cocoon in which it will develop to chrysalis and then to an adult midge. The emergence is determined by temperature and moisture. Mating is thought to occur shortly after emergence. Both sexes are known to leave the emergence field (due to crop rotation usually not a cruciferous crop) and once mated females will look-for a field with a Brassica crop to lay their eggs.
One reason for high damage levels is the lack of a good monitoring method for determining the time of emergence The (monitoring) method used today means sifting and floatation of soil samples, which are then analyzed under microscope to determine in which development stage the chrysalises are. The analyses are carried out by someone skilled in the art and are very time consuming. Soil samples of a few fields are, however, only analyzed. As the emergence is dependent on the local climate (moisture and temperature) the accuracy of (the monitoring) becomes low with regard to other fields. Furthermore, the adults appear very suddenly and for a short time period, and as the control actions must be carried out prior to egg laying, the method is very uncertain. It requires a very efficient communication system as well, in order to reach all farmers with the results of the prognostication (monitoring) and recommendation concerning the time for controlling action. Currently, the only available monitoring method is based on the exposition of yellow pan traps filled with water and a detergent. The traps do not catch species-specifically, but rather all phytopagous insects are attracted and, therefore, the analysis of the trap content is laborious and the tiny swede midges can be detected and identified only by well trained personnel. Furthermore, the trapping efficiency of yellow traps is low for the swede midge and, with rather low populations, the crop is frequently damaged by C. nasturtii even when no midges are detected in traps. Temperature-based forecasting methods have been developed but failed to give accurate, field-specific prognosis.
In pheromone based monitoring methods a limited number of traps baited with a pheromone related to the intended insect, in particular a sexual pheromone, in synthetic form, are used. A pheromone trap is the most sensitive monitoring system known today. Using such, even very low population densities can be detected. Since pheromones are highly specific with respect to the attracted insect species, traps baited with such pheromones catch predominantly the targeted species, which greatly facilitates the analysis of the traps content and the detection of the respective species.
Noxious insects, as well as all other insects, can vary a lot in number from year to year and their occurrence during the season may vary too. The difficulty is thus to know if and when they need to be controlled. The trap catches will, however, provide an answer to three essential questions, viz: 1) if the insect is present in the field; 2) when the insect is present in the field; and 3) approximately how many there are. The answer to the first question is essential to prevent or eliminate unnecessary control treatments as a matter of precaution, using insecticides. Having the answer to question no. 2), an optional control treatment can be made at the right time, i.e. when the insect is there.
Pheromone baited traps do not provide an exact measure of the population density but the catch result denotes if there is a low, an average, or a high risk infestation. Pheromone based monitoring systems are used today for about 200 different noxious insects, in particular moths. With such monitoring traps the use of insecticides can be lowered with 50 to 75%. Unnecessary control treatment can thus be avoided, which not only reduces the farmer's costs but also limits the adverse effects on the environment Should the trap catches indicate that control treatments are necessary, one will be able to carry out the control at the most sensitive development stage of the insect.
Chemical insecticides to noxious insects will, with regard to the impact on the environment and their quite often broad control spectrum (i.e. they target many different insects), be replaced by biological control methods. Such methods utilize basic knowledge of the ecology and biochemistry of the specific insects. The precision, i.e., the desired species specificity can thus become very high. The methods, which are the commercially most successful ones, are based on the sexual pheromones or other pheromones of insects. The use of pheromones in insect pest management is based on the use of naturally occurring compounds at naturally occurring amounts and concentrations; which means maximum consideration of the environment.
When controlling noxious insects with sexual pheromones one imitates and utilizes the natural situation in which a female attracts a male by emitting the sexual pheromone, which will be spread as a mist in the wind direction. A male who comes into the pheromone plume responds by flying against the wind towards the female. For control purposes, a large number of synthetic pheromone sources are applied in an area that will thus be flooded with pheromone. In this scent environment the female's own signal will be drowned and the male will have no possibility to find her. The technique is called mating disruption and is a commonly spread control method.
It has been shown that females use a sexual pheromone in order to attract the males in approximately 10 gall midge species (for a review, see Harris and Foster, 1999; Hillbur, 2001). However, chemical identifications of pheromones have so far only been made in the pea midge, C. pisi (Hillbur et al. 1999, 2000, 2001), the orange wheat blossom midge, Sitodiplosis mosellana (Gries et al. 2000), and the Douglas-fir cone gall midge, C. oregonensis (Gries et al. 2002).
FIGS. 2 shows the Mass spectra of two EAD peaks;
a shows mating disruption in C. Nasturii: damage in Brussels sprouts low plant half with distorted sprout;
b shows mating disruption in C. Nasturii: damage in Brussels sprouts high plant Half with cork damaged sprouts.
It has now been found possible to control C. nasturtii by prognostication (monitoring), or mating disruption, with a mixture of the pheromones (2S, 9S)-diacetoxyundecane, (2S,10S)-diacetoxyundecane and 2S-acetoxyundecane.
Insect Rearing
Larvae of C. nasturtii (Kieffer) were collected in August 2000 from heavily attacked Brussels sprouts of a field near Kerzers (Canton Freiburg, Switzerland). Infested growing tips were placed on a peat-soil mixture where larvae pupated when they had completed their development. The pots containing the pupae were stored in cages, and from the emerging adults a continuous laboratory culture was established. Similarly, larvae from a field near Ins (Canton Bern, Switzerland) were added to the culture in 2001.
C. nasturtii larvae were reared on cauliflower, Brassica oleracea convar. botrytis, cv. Candid Charm. Food plants were grown in pairs in pots (diameter 13 cm) in a substrate consisting of 85% peat and clay (Floraton no. 5, Floragard, Oldenburg, Germany) in a greenhouse at 15-30° C. with additional light supplied during winter months. Plants were kept free of aphids and white flies by spraying them, if necessary, once with pymetrozine (0.025% a.i.) not later than 3 weeks before they were used as food plants. When plants had 8 to 10 true leaves and a tip diameter of 5-10 mm, they were transferred to climate rooms with 22° C., R.H.75%, and L/D=16/8. In there, they were placed into cages (50×50×50 cm) made of perspex with the two side walls consisting of a fine-meshed screen. The cages contained 100-200 midges, males and females that were allowed to oviposit on the plants for 24 hrs. Then the plants were removed, replaced by the next batch, and incubated at the above mentioned conditions. Every day, the tips of the plants, where larvae became visible 5-6 days after oviposition, were misted with tap water.
If larvae were used to supply the culture, aboveground plant parts were cut off on day 17 after oviposition before the pots with substrate containing the pupae were placed in the oviposition cages where midges were allowed to hatch. If midges were supposed to be used for EAG-work or for the collection of pheromone gland extracts, aboveground plant parts were cut off on day 15. Males for wind tunnel experiments were prevented from getting a chance to mate by placing glass cylinders over pots. In the morning after the night when the first midges had hatched, these cylinders, containing usually only males, were set aside before females started to emerge.
Extracts For EAG And Chemical Identification
The pots with infested soil were put in glass cages (28×28×33 cm) and kept at L/D 18/6, 25° C., and RH 70% for the adults to emerge. After emergence, males and females were transferred to separate cages. Females were kept under the conditions mentioned and males were kept at L/D 18/6, 15° C., and RH 75%. Gland extracts were prepared by excising the ovipositors of females exhibiting calling behavior, i.e. extending their ovipositor (see Harris and Foster (1999) for a detailed description). The ovipositors were collected in a vial kept in liquid nitrogen and then extracted for 1-2 min redistilled hexane (LabScan). Extracts were kept in sealed glass capillaries at −18° C. until use.
Extracts For Preliminary Wind Tunnel Assays
Calling females were collected from the laboratory culture and submersed in batches in hexane (p.a. grade) for 15 min at room temperature. The supernatant was then decanted and concentrated to the highest concentration tested by blowing off the hexane with N2.
Electrophysiological Recordings
Male antennal response to ovipositor extracts was analyzed by gas chromatography 25 with electroantennographic detection (GC-EAD). The GC used was a Hewlett Packard 6890 GC with flame ionization detection and an Innowax column (30m×0.25 mm ID, Hewlett Packard, USA). The column temperature was programmed from 80° C. (2 min hold) to 230° C. at 10° C./min. The antennal recordings were made using a Plexiglass holder with two wells connected to an amplifier by gold wire electrodes (JoAC, Lund, Sweden; Zhang et al., 1997; Hillbur, 2001). Male insects were mounted in the holder as described by Hillbur et al. (2000). The antennal preparations were continuously exposed to a charcoal filtered and humidified air stream (0.3 m/s) through a glass tube (8 mm diam.). Antennal signals were amplified (JoAC) before they were recorded and analyzed with ElectroAntennoGraphy software (Syntech, Hilversum, The Netherlands).
Analyses
Analyses were carried out by coupled gas chromatography mass spectrometry (GC-MS). A Hewlett-Packard gas chromatograph 5890 (Palo Alto, Calif., USA) was linked to a double focusing sector-field mass spectrometer VG 70/250 E (Vacuum Generators, Manchester, UK) operated at 70 eV. Alternatively, a quadrupole instrument (MD 800/GC 8060, Fisons, Ismaning, Germany) was employed. Using helium as carrier gas, separations were achieved with a 60 m×0.25 mm-ID, 0.25 μm film DB5-MS fused silica column (J & W Scientific, Folsom, Calif., USA) under the following conditions: 2 min 50° C., then programmed to 280° C. at a rate of 5° C./min. In addition, a 50 m×0.25 μm-ID, 0.27 mm film FFAP (Macherey & Nagel, Düren, Germany) fused silica column was operated under the following conditions: 3 min 50° C., then programmed to 220° C. at a rate of 3° C./min. Separation of stereo isomers was carried out with a 25 m×0.25 mm-ID fused silica column coated with a 1:1 mixture of heptakis-(6-0-tert.-butyldimethylsilyl-2,3-di-0-methyl)-cyclodextrin and OV1701 using hydrogen as carrier gas. NMR-spectra were recorded with a Bruker AMX-400 (Karlsruhe, Germany); chemical shifts (S) are given in parts per million relative to tetramethylsilane; coupling constants I are given in Hertz.
Syntheses
Fine chemicals and solvents were purchased from Aldrich or Merck and were of highest purity available. Purifications of synthetic products were carried out by flash chromatography on silica gel (silica 32-63, 60 Å, ICN-Biomedicals, Eschwege, Germany) at 1.3 bar using mixtures of ethyl acetate and hexane.
(S)-Dec-9-ene-2-ol 8
Under argon atmosphere a Grignard reagent was prepared from 10.0 g (56.6 mmol) 7-bromohept-1-ene and 2.06 g (84.8 mmol) freshly ground magnesium cuttings in 50 ml abs. tetrahydrofuran at 60° C. After cooling to room temperature, the solution was added dropwise to 3.3 g (4 ml, 56.7 mmol) (S)-methyloxiran and 1.1 g (5.7 mmol) copper-I-iodide dissolved in 40 ml tetrahydrofuran at −78° C. Subsequently, the solution was warmed to room temperature and stirred for an additional hour. After addition of 300 ml saturated aqueous ammonium chloride solution, the mixture was extracted with 200 ml ethyl acetate. The aqueous layer was separated and extracted three times with 200 ml of a 1:1 mixture of ethyl acetate and hexane. The combined organic extracts were dried over magnesium sulphate and concentrated in vacuo. Purification by flash 35 chromatography (10% ethyl acetate in hexane) furnished 6.2 g (39 mmol, 70%) of (S)-2-dec-9-ene-2-ol.
(S)-2-Benzyloxydec-9-ene
To a suspension of 3.2 g (133 mmol) sodium hydride (freed from paraffin) in 105 ml ice cold abs. tetrahydrofuran was added 6.2 g (39.5 mmol) (S)-dec-9-ene-2-ol under argon. After stirring for 2 h, 470 mg (1.3 mmol) tetra-n-butyl ammonium iodide were added, followed by dropwise addition of 13.5 g (9.4 ml, 78.9 mmol) benzyl bromide, while stirring was continued for 12 h. Subsequently, 50 ml saturated aqueous ammonium chloride solution were added at 0° C. followed by 50 ml hexane. The aqueous layer was separated and extracted four times with 100 ml of a 1:4 mixture of ethyl acetate/hexane. The combined organic layers were dried over magnesium sulphate and concentrated in vacuo. Flash chromatography (5% ethyl acetate in hexane furnished 9.7 g (39.4 mmol, 100%) (S)-2-benzyloxydec-9-ene.
(2R,9S)-9-Benzyloxydecane-1,2-diol
A vigorously stirred solution of 6.0 g (24.3 mmol) (S)-2-benzyloxydec-9-ene in 300 ml of a 1:1 mixture of water and tert-butanol was cooled to −10° C. After addition of 29.2 g AD-Mix (i.e. 1.2 g per mmol alkene) the mixture was stirred at 4° C. for 40 h. Subsequently, 30.0 g (158 mmol) sodium disulfite were added, and the mixture was stirred at 20° C. for another hour. The brownish suspension was extracted three times with 250 ml ethyl acetate. The organic layers were combined, dried over magnesium sulphate, and concentrated in vacuo. Flash chromatography (ethyl acetate and hexane 1:1) furnished 4.2 g (15 mmol, 62%) (2R,9S)-9-benzyloxydecan-1,2-diol as a colorless oil.
(2R,9S)-9-Benzyloxy-1-(p-tolylsulfonyloxy)-decane-2-ol
To an ice cold solution of 4.6 g (16.4 mmol) (2R,9S)-9-benzyloxydecane-1,2-diol in 50 ml abs. pyridine was slowly added a solution of 2.9 g (15 mmol) p-toluene sulfonyl chloride in 20 ml abs. pyridine. Stirring was continued for 5 h at 0° C. and 12 h at 4° C. Subsequently, 150 ml ice water (brought to pH 5 with 0.1% hydrochloric acid) were added and 60 ml diethyl ether. Subsequently, the aqueous layer was extracted four times with 120 ml diethyl ether. The combined organic layers were washed three times with 100 ml of an aqueous, saturated solution of copper-II-sulphate and two times with 100 ml brine. The organic layer was dried over magnesium sulphate and concentrated in vacuo. Flash chromatography (10% ethyl acetate in hexane) furnished 4.6 g (10.5 mmol, 64%) (2R,9S)-9-benzyloxy-1 (p-tolylsulfonyloxy) decane-2-ol.
(2S,9S)-2-Benzyloxyundecane-9-ol
To 11.8 ml of a commercially available solution of 20% methyl magnesium chloride (31.4 mmol) in tetrahydrofuran were added 20 ml abs. tetrahydrofuran. The solution was cooled to −78° C., and 595 mg (3.14 mmol) copper-I-iodide were added. After stirring for 15 min, a solution of 4.55 g (10.5 mmol) (2R,9S)-9-benzyloxy-1(p-tolylsulfonyloxy)-decane-2-ol in 10 ml abs. tetrahydrofuran was added over a period of 2 h. The solution was warmed to −30° C. and stirred for another hour. Subsequently, 150 ml saturated aqueous ammonium chloride and 50 ml diethyl ether were added at 0° C. The aqueous layer was extracted three times with 150 ml ethyl acetate. The combined organic layers were dried over magnesium sulphate and concentrated in vacuo. Flash chromatography (16% ethyl acetate in hexane) furnished 2.6 g (9.4 mmol, 90%) (2S,9S)-2-benzyloxyundecane-9-ol
(2S,9S)-Undecane-2,9-diol
To a solution of 1.95 g (7 mmol) (2S,9S)-2-benzyloxyundecane-9-ol in 30 ml ethanol was added 20 mg Pd/C-catalyst. Hydrogenation was carried out over a period of 12 h at a pressure of 40 atm. After completion of the reaction, the catalyst was filtered off over silica, and the solution was concentrated in vacuo. Flash chromatography (ethyl acetate:hexane 1:1) yielded 1.2 g (6.4 mmol, 92%) (2S,9S)-undecane-2,9-diol.
(2S,9S)-Diacetoxyundecane [2S,9S]
A solution of 1.40 g (7.4 mmol) (2S,9S)-undecane-2,9-diol in 20 ml abs. pyridine was cooled to 0° C. After addition of a small amount of 4-dimethylaminopyridine, 2.3 g (2.1 ml, 22.3 mmol) acetic anhydride were added dropwise, and the mixture was stirred for another two hours at room temperature. Usual workup followed by flash chromatography (7% ethyl acetate in hexane) furnished 1.9 g (6.9. mmol 93%) (25,9S)-diacetoxyundecane
(2S, 10S)-Undecane-2,10-diol
Under an argon atmosphere a Grignard reagent was prepared from 2.5 g (10.9 mmol) 1,5-dibromopentane and 795 mg (32.6 mmol) freshly ground magnesium cuttings in 25 ml abs. tetrahydrofuran at 60° C. After cooling to room temperature, the solution was added dropwise to 1.24 g (1.5 ml, 21.7 mmol) (S)-methyloxiran and 420 mg (2.17 mmol) Cu-I-iodide, dissolved in 10 ml abs. tetrahydrofuran at −78° C. Subsequently, the solution was warmed to room temperature and stirred for another hour. After addition of 250 ml of saturated aqueous ammonium chloride solution, the mixture was extracted with 100 ml ethyl acetate. The aqueous layer was separated and extracted three times with 150 ml of a 1:1 mixture of ethyl acetate and hexane. The combined organic extracts were dried over magnesium sulphate and concentrated in v-acuo. Purification by flash chromatography (20% hexane in ethyl acetate) furnished 1.1 g (5.84 mmol, 54%) (2S, 10S)-undecane-2,10-diol.
(2S,10S)-Diacetoxyundecane [2S,10S]
A solution of 1.1 g (5.8 mmol) (2S, 10S)-undecane-2,10-diol in 15 ml abs. pyridine was cooled to 0° C. After addition of a small amount of 4-dimethylaminopyridine, 1.79 g (1.66 ml 17.5 mmol) acetic anhydride were added dropwise, and the mixture was stirred for another two hours at room temperature. Usual work up followed by flash chromatography (10% ethyl acetate in hexane) furnished 1.38 g (5.1 mmol, 87%) (2S, 10S)-diacetoxyundecane.
(2S)-Undecane-2-ol
A Grignard reagent was prepared from 2.8 g (14,7 mmol) 1-bromooctane and 900 mg (37.0 mmol) freshly ground magnesium cuttings and reacted with 853 mg (1.03 ml, 14.7 mmol) (S)-methyloxiran. The preparation procedure and work up followed the protocol as described for the synthesis of (S)-dec-9-ene-2-oI. After flash chromatography of the crude product, (20% ethyl acetate in hexane), 2.0 g (11.6 mmol, 79%) (2S)-undecane-2-ol were obtained.
(2S)-Acetoxyundecane
Acetylation of 2.0 g (11.6 mmol) (2S)-undecane-2 followed the protocol as described above for the acetylation of the undecane diols. Work up followed by flash chromatography (5% ethyl acetate in hexane) furnished 2.3 g (10.7 mmol, 93 °h). (2S)-acetoxyundecane.
Wind tunnel experiments were performed in the 2nd to 4th hour of the photophase at 22° C. and 73% r.h. in a wind tunnel described in detail by Rauscher et al. (1984). Compounds were diluted in hexane (p.a. grade). For the bioassays, 10 p 1 of the stimulus to be tested was applied onto a piece of fitter paper (Chromatographiepapier “Schleicher & Schuell AG” Auer-Bittmann Soulié AG, Zurich, Switzerland, 69.2 gr/m2, 1 cm2)which was then placed in the air stream at least 20 sec before the first male was tested. For each stimulus, a series of five males were tested individually, requiring a total of 5-10 min for each series. For each series, a new piece of filter paper was loaded with the respective stimulus.
Males were kept individually in glass tubes (10 cm, 15 mm i.d.) closed at one end with a piece of screen (mesh size 1×1 mm) until they were tested. With these tubes they were transferred into the wind tunnel, where the tube was placed on a pole in horizontal position with the opening upwind, at a distance of 130 cm downwind from the odor source. Time recording was then started and the following steps recorded. Activation by wing-fanning, take off, directed flight towards the odor source, flights closer than 30 cm from the source (“close in”), landing on the source or elsewhere. The test was stopped when no activity or take off was registered during 2 mm or when the insect landed elsewhere.
Of the recorded parameters only the number of successful landings on the source were analyzed
The blend with the highest attractiveness in the wind tunnel was tested in the field. Treatments tested were either dispensers consisting of red rubber septa (used as lids for serum bottles, VWR International, S-220 09 Lund, Schweden, weight=1.7g), or dental cotton rolls (Hillbur et al., 2000), or control traps without pheromone dispensers. Cotton rolls were cut and one third was used per dispenser. Both dispenser types were loaded with a blend of 500 ng (2S,9S)-diacetoxyundecane, 1000 ng (2S,10S)-diacetoxyundecane, and 10 ng (2S)-acetoxyundecane, diluted in 20 μl of hexane. Delta traps made of waxed cardboard were provided by PheroNet AB. They had a height of 10 cm and a sticky insert of 15.5×9 cm. Dispensers were positioned 1-2 cm above the insert.
The field assay was performed in a plot with broccoli (B. oleracea convar botrytis var. italica cv. Fiesta) two weeks before harvest. At this stage, plants were 50-70 cm in height, the leaf canopy was closed, and between broccoli plants numerous weeds were growing. Pots containing substrate with 150-250 pupae each of laboratory-reared midges were placed in the field. Two groups of 5 pots each were arranged in the centre of the field, 8 m apart from each other. The pots were replaced by a new batch 7 days later. Groups of traps were placed at 6 locations, all between 3.5 and 5 m from the nearest release point. Each trap group consisted of 6 traps, belonging to the three different treatments, each exposed at two different heights above ground. Traps with the same treatment were fixed on the same pole at 20 or 55 cm above ground, and at 30-40 cm from the next pole of the same group. The sticky inserts were replaced 5 times within the 10 day experimental period and midges were counted with a binocular microscope.
The blend of the pheromone used in the field assays for monitoring was tested for its effect on mating disruption in the field. Two equally designed and planted plots of Brussels sprouts (Brassica oleracea var. gemmifera cv. Helemus) were used to perform two release experiments each (release 1, release 2; see below) with the midges. The dimensions of the plots were 16×16 m each with a distance of 1 m between the rows and about 40 plants per row (640 plants per plot). Before the experiments both plots had no Brassica planted for several years and therefore no natural population of C. nasturtii. The two plots were located about 500 m apart and separated by a row of greenhouses excluding movements from one plot to the other. One plot was treated with pheromone for mating disruption, the other plot was not treated and used as control.
The pheromone applied for mating disruption on the treated plot was distributed with dispensers made of whole dental cotton rolls (diameter 1 cm). Each dispenser was covered by a delta-formed 10 cm wide sheet made of white plastic foil as rain protection (10 cm high; open to the sides) and mounted at a height of 45 cm. Dispensers were spaced in a rectangular 2×2 m grid over the entire plot.
The dose of pheromone applied per square metre on the treated plot was 125 μg (2S,9S)-diacetoxyundecane, 25 μg (2S,10S)-diacetoxyundecane, and 250 μg (2S)-acetoxyundecane. In order to achieve this dose each dispenser was loaded with a blend of 50 μg (2S, 9S)-diacetoxyundecane, 100 μg (2S,10S)-diacetoxyundecane, and 1 μg (2S)-acetoxyundecane, diluted in 100 μl of hexane. Freshly loaded dispensers were installed three days prior to each of the two release experiments.
For each release experiments, 12 pots per plot containing substrate with 150-250 pupae each of laboratory-reared midges were placed in the field. The total expected emergence of adults midges was about 1000 males and 1000 females per plot and release.
In release 1, the pots were placed on 20 Jul. 2004 in a row with a distance of 30 m from the treated and control plot in western direction, each. The plants on the plots were about 50 cm high by that time and had an age of 10 weeks from planting. The design tested whether mating disruption in a target field is effective even when the emergence area is not treated by the, pheromone.
In release 2, the pots were placed on 9 Aug. 2004 randomly distributed within the treated and control plot, each. The plants on the plots were about 65 cm high by that time and had an age of 13 weeks from planting. The design tested whether mating disruption in a target field is effective for midges emerging from within the planted plot.
Furthermore the general effectiveness of mating disruption was controlled with a test for pheromone-trap-shut down. For latter, four pheromone traps each (as used in the field assays for mating disruption with cotton dispenser) were randomly distributed over the treated and control plot. From release 2 on, the sticky inserts of those 8 traps were changed and counted in three- or four-day intervals for about six weeks.
Damage for both release experiments was scored at harvest time by the end of September 2004. Randomly starting within each row, every fifth plant was chosen for damage scoring and taken out, i.e. about 130 plants each were scored for the control and the treated plot. Each plant was scored for early damage from release 1-midges (lower plant half) and later damage from release 2-midges (higher plant half). Damage types scored were the number of distorted sprouts and the number of sprouts with cork damage. Additionally the number of dwarf sprouts at the basis of the plants was counted.
Electrophysiology
GC-EAD analysis of ovipositor extract revealed two peaks that elicited a response in the male antenna.
Chemistry
The mass spectra of the two EAD-active peaks (
Using a synthetic sample to check for retention times and using single ion monitoring mass spectrometry (m/z 43, 87) we found a target compound in the natural extract in extremely small amounts. However, due to its low concentration, we were unable to determine the stereoisomeric composition of the natural product.
Response to female extract: The number of males flying upwind and landing on the odor source increased with increasing doses of female extract on the filter paper as shown in
Response to synthetics and mixtures: The identified pheromone components did not elicit responses directed towards the odour source as shown in
Optimizing the relative concentration of (2S)-acetoxyundecane: In blends with 1.5 ng/dispenser of (2S5,9S)-diacetoxyundecane and 3.0 ng/dispenser of (2S,10S)-diacetoxyundecane the concentration of (2S) was varied in order to find the optimal relative concentration of this compound as shown in
A total of 1500-2500 males were released for this experiment and a total of 1543 males were captured in the exposed traps during 10 days. None of the control traps without pheromone dispensers ever contained a male C. nasturtii as shown in
Traps with cotton rolls as dispensers captured 88.4%±2.35 of all midges captured 15 (mean±s.e., N=5 dates of counting midges), indicating that the pheromone blend released from the cotton rolls was more attractive than that from the rubber septa, which may be due to the release rate from and absorbance to the septa.
The assay was conducted in a field that had little C. nasturtii damage in recent years. Hence, the indigenous population was likely to be low during the experiment. Therefore, a comparison between the estimated number of released midges and the captured males suggests a high recapture rate, probably above 50%, despite the simultaneous presence of females that were probably calling during some time of the assay. This indicates a very high efficiency of the trap within the tested range of 3-5 m.
In this preliminary field assay that was set up in Brussels sprouts in the region of Ins (Kanton Bern, Switzerland) two traps captured male midges for 6 weeks. From the 30 sticky papers of these traps, a total of 100 male midges were identified based on a molecular diagnosis method (Frey et al., submitted) and all turned out to be C. nasturtii. Thus, there is evidence that the tested blend is not only attractive to C. nasturtii males but that this attractiveness is also species-specific to a substantial degree. However, to consolidate this finding, more evidence is needed from all C. nasturtii flights over the whole season. Furthermore, the developed trap must be tested in various regions with C. nasturtii populations throughout the world so that species-specificity of the catches can be assessed for the respective regions.
The application of the pheromone results in a full trap shut-down of pheromone traps in the treated plot (release 2) indicating that the mixture and dose applied effectively interrupts mating of C. nasturtii within the treated plot (see
Both release experiments show a significant reduction of damage caused by the infection by C. nasturtii (
part two of the release experiment for investigating the effect of mating disruption in the Swede midge Contarinia nasturtii with the tested sexual pheromone mixture and dosage. Damage in low plant half is caused in the period of part one of the release from distance (without pheromone traps), damage in high plant half is caused in the period of part two of the release within the plots combined with testing trap shut down of pheromone traps. Reduction in damage is calculated as the relative difference (given in %) between pheromone treated plot and the untreated control plot.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0400302-6 | Feb 2004 | SE | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/SE05/00186 | Feb 2005 | US |
| Child | 11502896 | Aug 2006 | US |
