Brigitte Frérot, Michel Guillon,1 Paul Bernard,2 Luc Madrennes, Benoit de Schepper,1 Fréderic Mathieu and Armelle Coeur D'Acier
Unité de Phytopharmacie et des Médiateurs chimiques, INRA, 78026 Versailles Cedex, France
Key words - sex pheromone, mating disruption, head space, release rate, Sesamia nonagrioides, Noctuidae, Lepidoptera
Introduction
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The technique of reducing populations by disrupting pheromonal communication with synthetic sex pheromones offers the potential of controlling insect pests without incurring the problems associated with insecticide use. Moreover the use of mating disruption techniques provides an alternative means of reducing insect populations poorly controlled by insecticides. The corn stalk borer Sesamia nonagrioides (Lef.) which is a major pest on maize crops in the South of France and all around the Mediterranean area is insufficiently controlled by insecticides due to the fact that larvae develop as stalk borers and are therefore inaccessible to insecticide sprays. The difficulties to control populations by usual insecticide sprays make this insect a good candidate to develop population control by mating disruption in industrial crops.
In Europe mating disruption concerns mainly orchard and vineyard pests (Minks & Cardé 1995). The reason why no trials have been undertaken against industrial crop pests could be the lack of reliable sprayable dispensers which allow conventional spray equipment or helicopter to be used (Frérot 1990). Moreover in European farming methods, especially on large scale, manual application dispensers might be considered as unthinkable. On the other hand, dispenser longevity appears to have also inhibited the use of sprayable microencapsulated formulations (Charmillot & Vickers 1991). New technologies for formulating sprayable pheromones have renewed interest for the use of mating disruption techniques for industrial scale crops as cereals. Sprayable formulations of synthetic pheromone seemed more compatible with large scale agriculture. The method will create a multitude of artificial point sources of pheromones. Recently, Suckling & Angerilli (1996) demonstrated that mating disruption is achieved better if it is caused by an uniform cloud of sex pheromone, though Sanders (1982) showed in wind tunnel conditions that a few strong sources disrupted males more effectively than many diffuse sources. However, this has not been proved in field experiments and the fact remains that early experiments with microencapsulated sex pheromones were efficient (Hall & Marrs 1989). Sprayable dispensers or liquid pheromone formulations will renew interest for diffuse sources.
Successful use of this method requires the release of large amounts of pheromone over the target area during all the flight period of adult insects. Although the mechanism of disruption is still the subject of conjecture and might be multi mechanism (Cardé & Minks 1995), the quality of the released blend is also essential to success. Little information (Knight 1995; Svensson et al. 1995; Bradley et al. 1995; Witzgall et al. 1996) was available on the release rate of multicomponent pheromones released during mating disruption trials. Recent advances in field electroantennogram technique (Karg et al. 1990) allow the estimation of air permeation by synthetic pheromone, but it remains difficult to conclude about ambient pheromone concentration and moreover the blend released cannot be evaluated by this technique (Karg et al. 1994). Information about the quality and the concentration of the blend released during the trial is needed to understand success or failure.
Thus, our study was undertaken to determine the relative efficacy of potential communication disruptants of Sesamia nonagrioides. The control of release characteristics of both liquid and solid sprayable formulations, i.e.: quantity and quality of the blend released in the field during trials, was set up. Comparision of efficiency of the two sprayable formulations and first results on mating disruption against S. nonagrioides are presented in this paper.
Materials and methods
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Field test lay out
The experimental area was situated in main region of maize production, Tarn (France). The field tests, located near La Gardiolle consisted of four blocks of maize field sharing the same characteristics of cultures: i.e. Sowing late April-begining of May, cv Cecilia, density: 49 500 to 76 400 plants/ha. The field did not receive insecticide treatment during the first generation of S. nonagrioides.
Disruption trials were conducted only on the second generation, on two plots immediatly adjacent - Plot I (14.5 ha); Plot II (12 ha). Plot I received a sprayable solid formulation of synthetic pheromone whereas Plot II was spread with liquid formulation of synthtetic pheromone. Treatments, whatever the formulation, were applied by helicopter at the same dose of pheromone:100 g/ha. A second spray was made at the dose of 50 g/ha for Plot II, 15 days after the first spray.
Two untreated control plots (UCP) were for the first (9 ha) located closed to experimental plots and for the second (2.5 ha) located 3 km away. Insecticide control plot (ICP - 25 ha) located 5 km away from the experimental plots received a single application by helicopter of Lambdacyhalothrin at 15 g active ingredient/ha (0.3 l/ha of Karate).
According to Mazomenos' sex pheromone identification of S. nonagrioides (1989) the following blend was used for the trials Z11-16Ac 69%, Z11-16OH 8%, Z11-16Al 8%, 12-Ac 15%. For plot I, the sex pheromone was first dissolved in a specific extender (Elf-Atochem) and then included in technical polymer microgranules (Elf-Atochem) of 80 µm diam. Miscellaneous adjuvants and inert clay were mixed and pulverisation was applied at the dose of 25 kg/ha for 100 g of pheromone.
Concerning plot II, synthetic sex pheromone was dissolved in the specific extender (QSP 1L) and was applied by helicopter at low volume (40 l/ha).
Traps, built specifically for sexual trapping of S. nonagrioides by Plant Protection Institute were used. They were baited either with virgin females or with synthetic lures. Three virgin females kept in wire mesh containers and renewed twice a week were placed in traps for Plot II, UCP and ICP. Each experimental plot and control plot contained a trap baited with a cap filled with 1000 µg of synthetic pheromone. Pheromone caps were renewed each fortnight and traps were checked three times per week.
Control of infestation
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In the first generation, before mating disruption experiments, 1500 stalks (300 stalks in 4 areas chosen following a diagonal plus 300 stalks selected at ramdom) per experimental plot were controlled and specific damage was noted on the plants.
In second generation, 500 plants per experimental and control plots were dissected in order to assess species of insect and larvae instars. The 500 stems were chosen in four spots following a diagonal plus 100 stems at random in each plot.
Control of release rate
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Evolution of quality and quantity of pheromone which was released during field trials was followed up during eight weeks. Sachets containing 5 g of the microgranule formulation used for treatment on plot I were hung in the field and sampled each week for analyses. Two replications per treatment per week were made. Each sachet represented 1/50 ha, i.e.: 200 m2.
Analyses were made by head space collection of air borne volatils on resin (Frérot et al., 1992). Samples were placed in a cylindrical glass container (5 cm x 2.5 d) connected on one side with a trap filled with 5 g of SupelpackTM 2 (16/50 mesh ; purified résin Amberlite XAD 2, SUPELCO) and on the other side, downwind with an air purifier filled with 40 g of Amberlite XAD 16 (SUPELCO). The air flow (100 ml/min.) was generated for 24 h by a vacuum pump and flown through the device from the air purifier cartridge to the effluvia trap cartidge. Desorption of the resin contained in the trap cartridge was achieved by washing the resin in a minimal volume of CH2Cl2 (0.5 ml). After a few minutes of maceration and shaking, the solution was filtered over glass wool and then stored at -27°C before analyses. Effluvia sampling was obtained under the following conditions: 25 ± 2°C and 80 ± 5% R.H.and quantities of each sample introduced were equivalent to 92 mg of pure pheromone.
Quantity of the remaining pheromone was evaluated by complete extraction of solid samples in ethanol. The complete sachet was placed in 80 ml of ethanol (organic solvents are not compatible with the technical polymer). The resulting solution was shaken using a vortex for 10 min, then filtered over glass wool and analysed using the following GC conditions.
Analytical procedure
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Gas chromatographic analyses (GC) were carried out with a 5890 Hewlett Packard gas chromatograph fitted with a split-splitless injector (240°C) and a flame-ionization detector (260°C). The fused silica capillary column used was a CPSil8 CB (25 m x 0.32 ID, Chrompack) with temperature programming from 90°C to 140°C at 20°C/mn and from 140°C to 260°C at 5°C/mn. Helium was used as the carrier gas at 10 psi pressure. Two µl were injected.
Gas chromatographic-mass spectrometric (GC-MS) analyses were used to confirm identities of compounds. GCMS was carried out with a Nermag R10-10C quadrupole mass spectrometer coupled to a Girdel 32 gas chromatograph equipped with a split-splitless injector and a CPSil8 CB fused silica capillary column (25 m x 0.32 ID, Chrompack) with temperature programming from 90°C (2 min) to 240°C at 8°C/mn. Electron impact (EI) mass spectra were obtained at 70 eV, the instrument scanning from 40 to 350 amu. Calibration curves were obtained by GC for 12Ac and for Z11-16Ac and following equations were obtained for 12Ac Surf=(8.13732 x Quantity)-21.15515. The same equation was applied to Z11-16Al, Z11-16OH and Z11-16Ac Surf=(8.66254 x Quantity)-3.85434.
Results
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Release rate of pheromone
Figure 1a shows results obtained in laboratory conditions by collecting compounds released by liquid and solid sprayable formulations compared with pure pheromone. It appeared that both solid and liquid formulations behaved similarly. The release rate of the pure pheromone was higher than that of the sprayable pheromone indicating that the specific extender and the technical polymer slowed down the release rate of the pheromone. However, Figure 1b shows that the blend released was different from the blend introduced. Release rate of pheromone components depended mainly on the molecular weight and the polarity of the molecule. 12Ac was the main compound released even if present only at the ratio of 15%. The main component of the pheromone blend Z11-16Ac was released at the same rate as Z11-16Al (2% to 5%) and the most polar compound the Z11-16OH represented only 0.1% to 0.5% of the released blend.
Laboratory results showed a constant release rate which lasted 30 days, but solid formulation kept in natural conditions and analysed after 30 days showed no detectable compounds, demonstrating that there was a disparity between natural and laboratory conditions. Thus, analyses of pheromone quantities remaining in sachet were made for the solid formulation and it was decided to apply a second spray.
Results of release rate from the solid formulation showed that the release rate during the first week in the field was very high, i.e.: 309 mg/ha/hr. After one week 50% of the load had been released but in the following weeks the release rate decreased considerably and reached 17.5 mg/ha/hr the third week, a level under that which the mating disruption is known to be achieved. Regular emission was not obtained under natural conditions and was probably dependant on the ratio between synthetic pheromone, extender and technical polymer.
Figure 1 Results of effluvia collections: a) release rate of solid and liquid sprayable formulations; b) quality of the blend released
Field trapping and damage level before and after mating disruption experiment
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Fourteen days before mating disruption treatment, damage rates per experimental plots Plot I and II, were assessed at 2.6 %, whereas untreated plots UCP were respectively 1.3 % and 4.1 % and insecticide treated field was 6.3 %. Field trapping with either virgin females or synthetic lures before mating disruption experiment evidenced
Figure 2 Results of male trapping in plot I, treated with solid sprayable formulation and in the untreated control plot (UCP)
a high level of males caught in UCP (116 per trap) compared with ICP (27), Plot I (47) and Plot II (46), both almost equivalent (Figures 2 to 4). After mating disruption treatments, traps continued to catch males in both control plots (UCP, ICP). On plot I a reduction of catches was achieved during the first 12 days, then catches resumed showing that mating disruption was not achieved. These results corroborated the release rate results, demonstrating that after three weeks the amount of pheromone was too low to allow mating disruption. On plot II reduction of catches was achieved throughout the experiment. Comparison with control plots confirmed that the second half dose spray was necessary.
Figure 3 Quantity of pheromone released in the field from solid sprayable formulation of pheromone. Results were obtained by complete extraction. During 8 weeks 64.1 g/ha were released, 35.9 g/ha remained in solid formulation
Figure 4 Results of male trapping in mating disruption plot treated with liquid formulation (Plot 2) and in control plot (ICP)
Last control of damage (25/09) showed (Table 1) that Plot II was significantly different from other plots with the lowest level of damage attesting that pheromonal communication was disrupted.
Table 1 Percentages of damage obtained after dissection of 500 plants on each plot. Different letters mean a significant difference (p<= 0.05)
| % of damage on stalk | % of damage on corn cob | |
| Plot I. solid sprayable formulation | 6.8 a | 0.4 a |
| Plot II. liquid sprayable formulation | 1.8 b | 0 b |
| ICP. insecticide control plot | 13.2 a | 0.6 a |
| UCP. untreated control plot 1 | 9.4 a | 1.4 a |
| UCP. untreated control plot 2 | 31.5 c | 2.5 c |
Observations of instar larvae (Table 2) showed that in all the cases there were no young larvae L1 and L2. Plot II was the only one to have no L3 and no L5 evidencing that matings had not occured late and early during the adult flight period. In Plot II the number of L4 was the lowest, even if not significant. The most abundant stages in all plots considered were L3 followed by L4. Considering that 30 to 35 days are necessary at an average temperature of about 20°C, which were the average field conditions, for eggs to reach the third and fourth instar larvae (Hilal 1978), mating had occured around August 27th for L3 and around August 22nd for L4. This information added to results of field trapping allowed us to conclude that Plot II was fairly well protected until August 22nd. To reach fifth instar about 40 days were required under the current climatic conditions. Consequently L5 would have been oviposited around August 15th, period during which mating disruption was not achieved in plot I.
Table 2 Mean numbers of each instar larvae per dissected plant
| L1 | L2 | L3 | L4 | L5 | L6 | |
| Plot I. solid sprayable formulation | 0 | 0 | 0.05 ab | 0.03 a | 0.01 ab | 0 a |
| Plot II. liquid sprayable formulation | 0 | 0 | 0 a | 0.01 a | 0 ab | 0 a |
| ICP. insecticide control plot | 0 | 0 | 0.14 b | 0.06 a | 0.1 ab | 0 a |
| UCP. untreated control plot 1 | 0 | 0 | 0.09 ab | 0.04 a | 0.03 ab | 0 a |
| UCP. untreated control plot 2 | 0 | 0 | 0. 28 c | 0.16 b | 0.06 b | 0.2 b |
Conclusions
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Disrupting pheromonal communication with synthetic sex pheromone, formulated for application by helicopter was obtained against S. nonagrioides leading us to consider the method as promising against cereal pests (Table 3).The final assessment of damage showed a significant reduction of larvae population in plot II whereas plot I treated with solid formulation was not different from control plots. This negative result in plot II was directly correlated with the insufficient quantity of pheromone released after three weeks. These controls of the infestation appeared to be very important in order to reach a conclusion on the efficacy of the trials and validity of field trapping. Athough field trappings gave information about insects flying in the field, they were poorly correlated with the level of damage.
Table 3 Final results from field assessment data. Percentage of efficacy were calculated as follows: (% of damage in the control plot - % of damage in the considered plot)*100/ %of damage in the control plot. * mean difference significant p <= 0.5
| UCP I | UCP II | ICP | |
| Plot I | 27.6% | 79% | 48.5% |
| Plot II | 80.9%* | 94.4%* | 86.4%* |
The method used to control the release rate in terms of quantity and quality requires a minimum of specific equipment, but is necessary for the understanding of mechanisms and limitation of the mating disruption. Results obtained on the quality of the blend released indicate that special attention must be paid when the target insect produces a multicomponent sex pheromone. Release rate of compounds is directly related to molecular weight and polarity more than to quantity introduced. These physical properties could limit the development of such a method to monitor pest populations. In the case of S. nonagrioides, reinvestigation of the pheromone produced by the females (Frérot et al. 1996) did not evidence 12Ac and Z11-16Al either on the gland surface or in gland extracts. Further investigation will be undertaken to verify the effect of each component on chemical communication between males and females. In addition, the solid formulation will be modified and improved in order to calibrate and keep the release rate more regular.
Acknowledgements
The authors are pleased to thank Sara Cowley for reviewing the English.
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