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Enhancement of refuges for Helicoverpa armigera (Lepidoptera: Noctuidae) used in the resistance management plan for cotton (Gossypium hirsutum L.) containing Bollgard II® traits
Agriculture, Ecosystems and Environment 135 (2010) 328–335
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Enhancement of refuges for Helicoverpa armigera (Lepidoptera: Noctuidae) used in the resistance management plan for cotton (Gossypium hirsutum L.) containing Bollgard II1 traits Stewart J. Addison * Monsanto Australia, PO Box 92, Harlaxton, Queensland 4350, Australia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 4 February 2009 Received in revised form 22 October 2009 Accepted 22 October 2009 Available online 27 November 2009
Helicoverpa armigera (Hu¨bner) egg densities were sampled across interfaces between cotton containing Bollgard II1 traits and pigeonpea or unsprayed non-transgenic cotton refuges to determine the effectiveness of the refuge crops for Bollgard II1 resistance management plans (RMPs) against H. armigera. Additionally, a commercially available moth-attraction technology (Magnet1) was included at interfaces with both refuge crops to determine whether the use of such an attractant technology enhances the effectiveness of the refuges. Where the refuge crop was unsprayed non-transgenic cotton, there was no consistent change in the egg-density profile across the interface, suggesting that the Helicoverpa moths were not detecting and responding to the transition from Bollgard II1 to nontransgenic cotton. However wherever Magnet was applied, there was a consistent increase in egg density on or near the Magnet-treated cotton rows. This suggests that moth-attraction technology has potential to increase the efficiency of non-transgenic cotton refuges by increasing oviposition there so that the refuge produces more individuals unselected for Bt trait resistance. However, it is not yet known how best to deploy insect-attraction technology in non-transgenic cotton refuges. Pigeonpea was a very attractive refuge crop, with Helicoverpa egg densities an order of a magnitude higher than the adjacent Bollgard II1 crop and with no spillover of this increased oviposition into cotton. Application of moth attractant to the edge of the pigeonpea refuge did not alter the egg-density profile across the crop interface, suggesting that the combination of moth-attraction technology with pigeonpea refuges would not increase the effectiveness of the refuge. These studies identify avenues for improving the effectiveness of refuge crops for the management of Bt resistance in H. armigera. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Bacillus thuringiensis Magnet Transgenic crops Pigeonpea Oviposition Bt resistance Insect-attraction technology
1. Introduction The use of spatial and temporal refuges have long been advocated to delay insect adaptation to transgenic plants as part of resistance management plans (RMPs) (Roush, 1994; Tabashnik, 1994) and currently are a mandated part of the RMP for cotton (Gossypium hirsutum) containing the Bollgard II1 traits against Helicoverpa armigera (Lepidoptera: Noctuidae) in Australia (OGTR, 2002, 2006). The refuges are aimed to produce susceptible individuals that will mate with moths from Bollgard II1, including any individuals carrying alleles for resistance to the additional proteins produced from genes incorporated into the cotton genome from Bacillus thuringiensis (Bt) (which comprises the Bollgard II1 trait), thus reducing the probability of homozygousresistant individuals occurring in field populations.
* Tel.: +61 7 4634 8300; fax: +61 7 4634 8500. E-mail address: [email protected]. 0167-8809/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.agee.2009.10.015
The Australian Bollgard II1 RMP has five key components which contribute to the establishment and maintenance of both spatial and temporal refuges for H. armigera in the cotton-growing regions of eastern Australia (Monsanto, 2004). These components are (1) use of refuge crops (either non-cotton crops or nonBollgard II1 cotton) to produce Bt-susceptible moths on-farm, (2) a defined planting window for Bollgard II1 to reduce the number of H. armigera generations potentially exposed each season, (3) end-of-season pupae destruction by cultivation (southern areas) and trap crops (central Queensland) to remove any Bt-selected individuals from the population between cotton seasons, (4) control of cotton volunteers and stub cotton to remove the risk of creating mixtures of Bollgard II1 and conventional cotton that may allow survival of H. armigera larvae heterozygous for Bt resistance genes, and (5) spray limitations, including no use of Bt sprays in refuges. Of these RMP components, the maintenance of effective refuges is the most costly for growers, requiring significant amounts of land close to Bollgard II1, scarce irrigation water, and on-going
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management input during the season to ensure that refuge plants are healthy and remain attractive to H. armigera moths. In contrast, production systems elsewhere in the world have lower refuge requirements because cropping systems involve Bollgard II1 in small areas within diverse multi-cropping systems involving crops attractive to Helicoverpa (such as in India), or natural refuge areas that produce large numbers of unselected moths (most of southern and south-eastern cotton areas in the USA (US EPA, 2007)). Therefore, any techniques that enhance the performance of refuges are of particular interest to the Australian cotton industry. In considering the role of refuges in management of Bt resistance for Bollgard II1, little use has been made of what is known of H. armigera moth host-selection behavior (Parsons, 1940; Hedin, 1976; Hopper, 1981; Farrow and Daly, 1987; Drake, 1990; Fitt, 1991; Fitt and Farrow, unpublished data cited in Fitt, 1991) and pre- and post-alighting responses (Jayaraj, 1982; Jallow and Zalucki, 1995, 1996; Jallow, 1998), or exploiting existing semiochemical technology (Ag Biotech Australia, 2004) that can influence H. armigera moth behavior. This paper evaluates approaches to enhancing refuge effectiveness for Bollgard II1 in Australia that exploit this knowledge and suggests options that may lead to improvements in refuge effectiveness. Taken together, these previous studies have two implications for refuge-enhancement studies. Firstly, H. armigera moths strongly prefer pigeonpea for oviposition over cotton (Jayaraj, 1982). At a pigeonpea/cotton interface, moths are highly likely to respond by turning back into the pigeonpea, as Fitt (1991) reports for a maize/cotton interface. Secondly, while there are differences between cotton cultivars in ovipositional preference under choice situations, this is mostly related to leaf trichome density, and cultivars with similar leaf hairiness characteristics are likely to have a similar ranking of oviposition preference (Jallow, 1998). This means that, at an interface between two cotton cultivars (such as occurs at the boundary of Bollgard II1 and unsprayed refuge cotton), H. armigera moths are unlikely to lay differential numbers of eggs on either side of the boundary if the cultivars have similar pubescence characteristics. These considerations lead to some specific predictions about what may occur at interfaces between pigeonpea and unsprayed cotton refuges and Bollgard II1, with and without the addition of a moth feeding-attractant. Firstly, at the interface between Bollgard II1 and non-transgenic refuge cottons, there should be no change in egg density where the insect-attractant was not applied because moths are unlikely to detect the cotton refuge/ cotton crop interface. However, there may be a significant change in egg numbers where the attractant was applied, resulting from attraction of moths to the treated rows. Secondly, at the boundary of Bollgard II1 and a pigeonpea refuge, there should be a marked change in H. armigera egg density. Thirdly, because of the strong moth turning-response recorded by Drake and Fitt (1990) and Fitt and Farrow (unpublished data, Fitt, 1991) when moths left an attractive crop, any increase in egg densities in Bollgard II1 adjacent to pigeonpea should be restricted to rows close to the crop interface. Finally, at a Bollgard II1/pigeonpea interface there may not be any additional response in egg density where moth attractant was applied (compared to where it was not applied) because of the strong positive stimuli coming from the pigeonpea. These specific predictions were tested by recording Helicoverpa egg densities across a series of crop interfaces between Bollgard II1 and either pigeonpea or non-transgenic cotton, with and without a moth attractant (Magnet1). The primary objective of the study was to evaluate the effectiveness of the alternate refuge crops, with the secondary aim being to assess the likely impact of H. armigera moth attractants on refuge effectiveness, using Magnet as an example of this type of product.
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2. Materials and methods 2.1. Unsprayed non-transgenic cotton refuge – 2005 This section of the study used two large Bollgard II1 fields (areas 0.36 and 0.55 km2) with adjoining areas of unsprayed, irrigated non-transgenic cotton at Cecil Plains, Queensland (278350 S, 1518150 E). In Field 1 (Bollgard II1 cultivar Sicot 289B), rows were oriented 548 east of north (approximately north-east to south-west) with the cotton refuge of cultivar Sicot 80 located on the north-east side of the field, 50 m across a minor public road with no roadside vegetation between the field and the refuge. Six millilitres of Magnet insect-attractant were applied to the upper surface of a 1-m section of crop canopy on the end of each of 60 Bollgard II1 rows (60 m), i.e. 360 mL on 60 m of row, on the northeast boundary of the field weekly from 21 February 2005 (Julian Day 52) for 2 weeks using a fabricated sprayer with a ‘dribble’ nozzle. The Magnet and no-Magnet sample sites were approximately 350 m apart within the field, with two sampling transects in each area 40 m apart. Sampling points were at the row ends (0 m) and then 3, 10, 50 and 200 m into the Bollgard II1 crop along the rows. All Helicoverpa eggs were counted on a 1-m section of crop row at each sampling point prior to each Magnet application and again 3 days after spraying (DAS). In Field 2 (Bollgard II1 cultivar Sicot 80B), crop rows were oriented 368 west of north (approximately north-west to south-east and at right angles to rows in Field 1), with eight rows of unsprayed non-transgenic Sicot 80 refuge in the south-west side of the field, with the remainder of the field being Sicot 80B cotton. Threehundred and sixty milliliters of Magnet was applied to a 60 m section of the sixth non-transgenic row in from the south-western field edge (Row 0), as for Field 1 at this site. Eggs were sampled in Row 0 and 3, 10, 50 and 200 m (rows) from the treated row; thus Row 3 was the first Bollgard II1 row. The Magnet and no-Magnet sample areas in this field were approximately 300 m apart. Sampling sites in Fields 1 and 2 were 550 m apart at their closest point. Sampling transects in the two fields were oriented at 1808 to each other, and directly towards and away from the expected predominant wind direction (north-east). During the 2004/2005 growing season, approximately 75% of Heliothine eggs on the Darling Downs were H. armigera by February 2005 (D. Murray, personal communication). Fifteen-minute interval meteorological data for the experimental period were obtained from weather stations operated at Horrane (278320 17.7400 S, 1518140 4.5100 E), Norwin (278330 42.9000 S, 1518200 31.4400 E), and Tyunga (278400 12.9600 S, 1518170 38.7400 E) by the Darling Downs Cotton Grower’s Association. These weather stations triangulate the experimental site at distances of between 7.0 and 8.7 km over near-flat ground. Wind direction and speed data were plotted as polar scatter plots for the Helicoverpa flight period (17.45 (sunset) to midnight; Drake and Fitt, 1990) for the days between Magnet application on Julian Days 52 and 59 and the subsequent egg counts three days later. Direction was plotted as the deviation angle from north, and the speed as the distance from the centre of the graph. 2.2. Unsprayed non-transgenic cotton refuge – 2006 In February 2006, the effect of Magnet application on Helicoverpa egg distribution was assessed in three Bollgard II1 fields at the Cecil Plains site. Three Magnet applications were made to 100 m of crop row in each field at weekly intervals from 2 February 2006 (Julian Day 33), as previously. In each field, four sampling transects 20 m apart were established in each treated area and egg counts made 4, 16 and 400 m (rows) from the Magnet-treated row. Helicoverpa egg counts commenced 3 days after the first Magnet application and from then on were made
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prior to each Magnet application and again 3 days later. All Helicoverpa eggs were counted on a 1-m section of crop row at each sampling site. In 2006, Fields 1 and 2 were planted to the same Bollgard II1 cultivar throughout (Sicot 289BR) while the third field contained two cultivars (DP570B and Sicot 289B) with the 4 and 16 m samples being DP570B and the 400 m sample Sicot289B. These two cultivars were phenologically and morphologically extremely similar, including leaf pubescence characteristics. During the 2005/2006 growing season, approximately 70% of Heliothine eggs on the Darling Downs were H. armigera by January 2006 (D. Murray, personal communication). 2.3. Unsprayed pigeonpea refuge – 2005 This site was located at Dalby Agricultural College, Australia (278090 S, 1518170 E) in a Bollgard II1 (cultivar Sicot 71B) cotton field with an immediately adjacent unsprayed and irrigated pigeonpearefuge field. Weekly from 18 February 2005 (Julian Day 49) for 3 weeks, 360 mL of Magnet insect-attractant was applied over 60 m of the outside row of the pigeonpea refuge adjacent to the Bollgard II1 crop (Row 0) as for the 2005 cotton-refuge experiments. An untreated 60 m section of the crop interface approximately 250 m away was designated as a control area. In each area, two transects 60 m apart were established with sampling points in the treated row (Row 0) and then Row 3 (i.e. 2 rows into the Bollgard II1), and Rows 10, 50 and 200. All Helicoverpa spp. eggs were counted on a 1m section of crop row at each sampling site prior to each Magnet application and again 3 days later. 2.4. Data structure and statistical analysis Magnet is a patented mixture of floral volatiles, terpenoids and green leaf volatiles that attracts moths of H. armigera but the attractive effect of the plant volatiles disappears within 4–6 days of application (Ag Biotech Australia, 2004). In these experiments, Magnet was applied at weekly intervals for several weeks, with egg counts done just prior to Magnet application and then again 3 days later. The egg counts made on the day of Magnet application reflect the distribution of ovipositing Helicoverpa moths over the previous few days while the 3-days post-treatment egg count quantifies moth responses to the Magnet application in a repeated-measures data structure. Data sets were analyzed utilizing this data structure in a restricted maximum likelihood (REML) analysis with the pretreatment counts as a covariate and the random model that minimized deviance using GenStat Version 9 (Payne et al., 2006). The test statistic was the Wald statistic divided by its degrees of freedom, with the statistic being distributed approximately as the chi-square distribution (Payne et al., 2006). This gives tests of
significance for the fixed effects that are equivalent to the F-tests in ANOVA but with an optimal random model fitted to the covariance patterns in the repeated-measures structure. In 2006, there were no pre-treatment counts for the first Magnet treatment but there were for the second and third applications. Additionally, in 2006 there was only one treatment, i.e. Magnet, in each field, so all fields were included in a single REML analysis that considered Fields, Distance from Magnet and Julian Date. Two alternative data structures were investigated for this year’s data. Firstly, the three sets of 3 days after spraying data were analyzed without pre-treatment count as a covariate. Secondly, this analysis was repeated for the second and third application dates with their respective pre-treatment counts as covariates, as was done for the 2005 data. The 2005 pigeonpea-refuge data and the 2006 data were transformed by the H(x + 0.5) and log10 (x + 1) transformations, respectively, because residuals were not normally distributed. Where data were transformed and there were pre-treatment covariate data, the pre-treatment data were transformed onto the same scale as the post-treatment data for analysis. Because of the scale on which Magnet affects moth behavior, the Magnet and no-Magnet areas were located some hundreds of metres apart in each field. Consequently, the treatment main effects (i.e. Magnet vs. no-Magnet) were inevitably confounded with ‘position in the field’ effects and so were not used to assess the impact of the treatments. Instead, treatment effects were sought in the Treatment Distance interaction term. That is, different egg-density profiles where Magnet was, and was not, applied were used as evidence that Magnet was affecting moth distribution and oviposition. Additionally, given the random variations in moth distribution in the absence of any attractive treatments, the Distance response in no-Magnet areas was expected to be variable over time and space. In contrast, if Magnet is having an effect then the Distance response in Magnet areas should be consistent over time and space. Additionally, differential variation of moth numbers over the field could lead to a Treatment Time interaction, but this is another component of the confounding of treatment and field position discussed previously. Finally, if the unsprayed refuge of non-transgenic cotton or pigeonpea was having an effect on the egg distribution in the Bollgard II1 field, then a Distance main effect was expected regardless of whether Magnet was applied or not. 3. Results 3.1. Unsprayed non-transgenic cotton refuge – Field 1, 2005 At this site, there were differences between the Magnettreated and untreated areas and a significant Julian Day TreatTreatment interaction (Table 1) that reflect differences in moth
Table 1 Wald tests for the REML analysis of Helicoverpa egg count data from Bollgard II1 adjacent to unsprayed non-transgenic cotton and pigeonpea, 2005. Fixed term in REML analysis
Pre-treatment egg count Julian Day Treatment Distance Julian Day Treatment Julian Day Distance Treatment Distance Julian Day Treatment Distance *
Cotton refuge (Field 1)
Cotton refuge (Field 2)
Pigeon pea refuge
d.f.
Wald statistic/d.f.
d.f.
Wald statistic/d.f.
d.f.
Wald statistic/d.f.
1 1 1 4 1 4 4 4
8.08** 19.58*** 4.16* 0.80NS 11.77*** 0.82NS 3.76** 1.21NS
1 1 1 4 1 4 4 4
113.76*** 79.72*** 3.76NS 1.69NS 13.63*** 1.39NS 5.05*** 1.53NS
1 2 1 4 2 8 4 8
115.37*** 31.42*** 0.02NS 24.54*** 7.64*** 4.52*** 1.78NS 0.50NS
Wald statistic/degrees of freedom test significant at P < 0.05. Wald statistic/degrees of freedom test significant at P < 0.01. Wald statistic/degrees of freedom test significant at P < 0.001. NS Not significant (P > 0.05). **
***
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in egg density in Rows 0–3 (average of 9.8 and 7.6 eggs m1 on Days 55 and 62, respectively) compared to the rest of the field (average of 2.8 and 2.3 eggs m1 across the rest of the field on the same two sampling dates) (Fig. 1A and B). The overall egg density in Row 0 was significantly higher than in Rows 10, 50 and 200 (all P < 0.05) while the non-Magnet area showed the opposite pattern, with Row 0 with lower egg densities than Rows 10, 50 and 200 (all P < 0.05).
activity in different parts of the field over the duration of the experiment. Specifically, the part of the field that did not receive Magnet in Row 0 on Julian Day 52 had 2.5 times the number of eggs 3 days later compared to the area where the Magnet was applied in Row 0; however, 3 days after the Day 59 treatment the egg densities were approximately the same. These differences are not interpreted as indicating actual Treatment effects, but are rather artifacts of the experimental layout imposed by Magnet’s mode of action. There were large differences between sampling dates, with oviposition pressure falling markedly between Julian Day 55 and Julian Day 62. As indicated previously, from the refuge-enhancement perspective the Distance main effect term and the interaction term involving Treatment and Distance are the critical lines of the REML table (Table 1). The absence of a Distance main effect across the cotton refuge/Bollgard II1 interface indicates that the Helicoverpa moths are not detecting and responding to the crop boundary, in contrast to the very clear response at the pigeonpea/Bollgard II1 interface reported later in this paper. This suggests that there is not any difference in attractiveness/apparency of the two types of cotton in this experiment. In the area where no-Magnet was applied, the pattern of egg densities across the field was markedly different on the two 3-DAS sampling dates (Fig. 1). On Day 55, egg counts rose sharply from the refuge edge (Row 0 – 4.8 eggs m1) to be 13–20 eggs m1 for the rest of the field (Fig. 1A). On Day 62, egg counts were much lower (mean = 4.2 eggs m1), and rose gradually from Row 0 (2.6 eggs m1) to reach 7.1 eggs m1 at Row 200 (Fig. 1B). In contrast, the area where the Magnet was applied in Row 0 showed identical patterns on the two sampling dates with sharp increases
In this field, there were no statistical differences between the Magnet-treated and untreated areas (P = 0.053) but there was a significant Julian Day Treatment interaction (Fig. 2), again reflecting differences in moth activity in different parts of the field over the duration of the experiment. As for Field 1, there was no Distance main effect, again suggesting that the moths did not respond to the cotton refuge/cotton crop interface in the way they responded to the pigeonpea/cotton crop boundary. Again there was a large difference between the two 3-DAS sampling dates, with oviposition pressure in Field 2 falling between Julian Day 55 and Julian Day 62 (Fig. 2). For the Treatment and Treatment Distance effects, the pattern of significant differences for this field is the same as recorded in the other field with an unsprayed non-transgenic cotton refuge, and the opposite of that recorded where pigeonpea was the refuge crop (Table 1). That is, there were higher egg densities close to the site of the Magnet application than further into the field (Fig. 2), with egg density in sampling Row 3 (i.e. the first Bollgard II1 row) significantly higher than further in the field (P < 0.05).
Fig. 1. Helicoverpa egg density (SEM) on Bollgard II1 adjacent to an unsprayed nontransgenic cotton refuge in Field 1 with and without Magnet application in Row 0, 2005, 3 days after Magnet application on (A) Julian Day 52, and (B) Julian Day 59.
Fig. 2. Helicoverpa egg density (SEM) on Bollgard II1 adjacent to an unsprayed nontransgenic cotton refuge in Field 2 with and without Magnet application in Row 0, 2005, 3 days after Magnet application on (A) Julian Day 52, and (B) Julian Day 59.
3.2. Unsprayed non-transgenic cotton refuge – Field 2, 2005
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Fig. 4. Helicoverpa egg density (SEM) on Bollgard II1 3 days after Magnet applications, 2006. For each field, means with the same letter are not significantly different (P < 0.05). Fig. 3. Pooled wind direction and speed data around the 2005 non-transgenic cotton refuge experimental site (Cecil Plains district, Queensland) during the Helicoverpa oviposition period (sunset–midnight) for the six post-treatment nights. The direction the wind is blowing from is plotted as the deviation angle from north and the speed as the distance from the centre of the graph.
3.3. Wind direction data, Cecil Plains – 2005 The wind direction during the Helicoverpa oviposition period was consistent for both experimental periods at the three meteorological station sites that triangulated the experimental site, and so was pooled (Fig. 3). Over these six evenings, the wind blew from the north-east to east for 82% of the moth flight period (average angle = 698 east of north) at speeds that averaged 13 km h1 and ranged up to 28 km h1. 3.4. Unsprayed non-transgenic cotton refuge – 2006 The treatment effects for this experiment were examined at the interaction level rather than the main-effect level because there were significant interactions in the REML analysis (Table 2). There was a Julian Day Field interaction, reflecting differing changes in egg counts over the three dates in the three fields, i.e. the natural variation that existed in Helicoverpa egg pressure. The Julian Day Distance interaction was not significant, indicating that the changes in egg density with distance from the Magnet application were the same over the three sampling dates. This consistency suggests that the changes with respect to distance were not due to some random fluctuation in egg density, but rather reflect a consistent change with respect to the treatment of interest, i.e. distance from the Magnet application. The important interaction was Field Distance (Fig. 4). Examination of the two-way table,
Table 2 Wald tests for the REML analysis of Helicoverpa egg count data from Bollgard II1 adjacent to unsprayed non-transgenic cotton, 2006. Fixed term in REML analysis
d.f.
Wald statistic/d.f.
Julian Day Field Distance Julian Day Field Julian Day Distance Field Distance Julian Day Field Distance
2 2 2 4 4 4 8
147.12*** 282.54*** 12.39*** 2.95* 1.96NS 7.36*** 1.92NS
* ***
Wald statistic/degrees of freedom test significant at P < 0.05. Wald statistic/degrees of freedom test significant at P < 0.001. Not significant (P > 0.05).
NS
and t-tests between distances for each Field, indicate that distances of 4 and 16 m were not different in Field 1 (P > 0.05) but were significantly different in both of the other two fields (P < 0.05). Additionally, the relationships between the 4 and 16 m distances and the 400 m distance were different in the different fields, with the 400 m distance having variously more, equal or fewer eggs than both the 4 m and 16 m distances. This indicates that the scale of Magnet effects on Helicoverpa egg distribution is relatively small-scale when Magnet application is restricted to a single crop row. The analysis of data from the second and third Magnet applications with pre-treatment counts as a covariate showed the same relationships at the Field Distance level as the analysis with three dates but without pre-treatment counts as a covariate (data not shown). That is, Distances 4 and 16 were not different in Field 1 (P > 0.05) but were significantly different in both of the other two fields (P < 0.05). 3.5. Unsprayed pigeonpea refuge – 2005 At this site, there was a significant Julian Day main effect and significant Julian Day Treatment interaction (Table 1). These results indicate that (a) the Helicoverpa ovipositional pressure varied through time (with oviposition pressure falling markedly during the experimental period (Fig. 5)), and (b) this change in oviposition pressure through time was different in different sections of the field. Note also that the pattern of significant differences is the opposite of that recorded where the refuge crop was non-transgenic cotton (Table 1). That is, when the refuge crop was unsprayed non-transgenic cotton there was no significant Distance main effect but a significant Treatment Distance interaction, but for the pigeonpea refuge the opposite pattern occurred. There was a highly significant Distance main effect and Julian Day Distance interaction, but no Treatment Distance interaction. Fig. 5 provides an understanding of these effects. There was the same Distance profile for both Treatments on each of the three sampling dates with a significant increase in Row 0 compared to all other Distances (P < 0.05). However, the increase in Row 0 was numerically largest when the oviposition pressure was highest (Julian Day 52) (Fig. 5A). The spike of oviposition at the margin of the pigeonpea refuge resulted in the highly significant Distance main effect while the change in response in Row 0 as egg pressure declined during the experiment led to the significant Julian Day Distance interaction.
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Fitt, 1991). The short-range movement takes place close to the ground (1–4 m) (Drake and Fitt, 1990) and is the class of movement of most interest to refuge enhancement. Within the short-range movement category, not much is known about the behavior of Helicoverpa moths with respect to field interfaces between more and less attractive crops. However, this is an important issue for refuge enhancement. It has been often suggested (e.g. Parsons, 1940; Hedin, 1976; Hopper, 1981) that volatiles emanating from flowering cotton and maize may act as long-range attractants or arrestants for Helicoverpa females, thus allowing moths to concentrate within areas of flowering hosts. Drake and Fitt (1990) and Fitt and Farrow (unpublished data, cited in Fitt, 1991) observed moth behavior at cotton–maize crop boundaries as well as at edges of cotton fields bounded by fallow land. They indicated that up to 60% of H. armigera moths flying out of the maize turned back rather than fly over adjacent cotton crops. This turning behavior occurred at both downwind and upwind sides of the crop although moths tended to travel further from the maize edge before turning on the downwind side than on the upwind side. The moths were clearly able to perceive the patch boundaries and to respond very quickly. Similar turning behavior was observed to a lesser extent at the boundary of cotton and fallow ground. Additionally, once in the attractive crop they tend not to leave but rather remain there, presumably to feed and lay eggs. From a refuge-enhancement perspective, this behavior underpins the use of refuge crops such as pigeonpea, maize and sorghum as an option in Bollgard II1 RMPs (Monsanto, 2004). 4.1. Unsprayed non-transgenic cotton refuges, 2005
Fig. 5. Helicoverpa egg density (SEM) on Bollgard II1 adjacent to an unsprayed pigeonpea refuge with and without Magnet application in Row 0, 2005, 3 days after Magnet application on Julian Days 49, 56 and 63.
Importantly, there was no Treatment Distance interaction. This indicates that the change in egg density with respect to the pigeonpea was the same whether Magnet was applied in Row 0 or not. From this, it can be concluded that where the adjacent refuge crop was pigeonpea, the addition of Magnet at the edge of the refuge field did not change the response of the Helicoverpa moths to the refuge crop, as measured by the post-treatment egg density. 4. Discussion Studies of H. armigera moth movement indicate that this species undertakes both short-range movement (up to few kilometres) and long-range movement (hundreds of kilometres) at various times of the year (Farrow and Daly, 1987; Drake, 1990;
There were no consistent changes in H. armigera egg density across the Bollgard II1/cotton interfaces in the absence of Magnet. This result is consistent with the predictions based on the studies of moth ovipositional behaviour by Jallow (1998). However, where Magnet was applied at the Bollgard II1/cotton interface, there was a consistent increase in egg density at or close to the interface. Because Magnet insect-attraction technology (Ag Biotech Australia, 2004) contains plant volatiles that are responsible for the attractiveness of various plants to Helicoverpa moths seeking nectar prior to oviposition, moth reactions to such insectattractant applications would be expected to mimic moth responses to an attractive plant, and where the insect-attractant was applied to non-transgenic cotton close to a Bollgard II1/nontransgenic cotton boundary, it would be expected that H. armigera moths would respond to this application by spending more time in, and laying more eggs on and near, the treated rows. This is what occurred (Figs. 1 and 2). The wind during the oviposition periods of these trials was of a direction and strength (Fig. 3) that would have carried moths to the west–south–west, i.e. into the sampled Bollgard II1 crop in Field 1 and away from the sampled crop in Field 2. At any crop interface that elicited the turning-response observed by Drake and Fitt (1990) and Fitt and Farrow (unpublished data, cited in Fitt, 1991), this may result in a shift in the position of any peak of oviposition downwind. In the 2005 trials, a more marked increase in egg density close to the Magnet-treated strip in Field 1 was recorded than in Field 2 (Figs. 1 and 2) and this is consistent with the displacement of the Magnet-induced egg peak away from the sampled area in Field 2 and into the adjacent cotton refuge. Sampling on both sides of the narrow Magnet-treated strip would have resolved this and should be incorporated into future trial designs. In addition, this result suggests that when laying out nontransgenic cotton refuges on which narrow strips of insectattractant technology are to be used, the refuge areas should be positioned so they are downwind of Bollgard II1 during the
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evening Helicoverpa oviposition period. Existing meteorological data could be used to determine whether wind direction during the period from sunset to midnight is consistent enough during the cotton-growing season to implement this option. Alternatively, applying the strips of insect-attractant technology some distance in from the edge of the refuge would avoid displacement of the oviposition peak by the wind into Bollgard II1 regardless of the wind direction. Such approaches to the placement of the insectattractant technology would increase the probability that moths are more likely to be ‘arrested’ by the insect-attractant within the non-transgenic cotton and remain there to lay eggs, thus avoiding increasing egg lay in Bollgard II1 crops as opposed to on the refuges and so increasing refuge effectiveness. 4.2. Unsprayed non-transgenic cotton refuges, 2006 The Helicoverpa oviposition pressure across Field 1 in 2006 was very high (50–60 eggs m1) while it was only 6–19 eggs m1 in the other two fields (Fig. 4). That the response to the insect-attraction technology was different between the fields with high and more moderate egg-pressure suggests that when Helicoverpa egg pressure is very high, attractant technology may not affect egg distribution but that it may at more moderate oviposition pressures. Magnet is designed to attract moths from relatively short distances at times when a moth is seeking a nectar source (Ag Biotech Australia, 2004), rather than as an oviposition stimulant per se. Perhaps at high moth densities there was interference between females that masks the attraction response to this particular insect-attraction technology. 4.3. Insect-attraction technology to increase effectiveness of refuges One difference that is apparent between the cotton-refuge fields and the pigeonpea-refuge field is that, in the former, the application of the insect-attraction technology at the refuge/crop interface significantly altered the Helicoverpa oviposition response at the crop/refuge interface, but this did not occur when the refuge crop was pigeonpea. It seems likely that the attractiveness of the Magnet volatiles was swamped by the volatiles/stimuli being detected from the pigeonpea, but Magnet was clearly detectable by Helicoverpa moths when the refuge crop was unsprayed nontransgenic cotton. This points to there being potential to enhance the effectiveness of unsprayed non-transgenic cotton refuges, but not pigeonpea refuges, through the use of insect-attraction technology by increasing the attractiveness/‘apparency’ of the refuge compared to Bollgard II1. What is not known at this stage is the optimal application pattern of the attraction technology to achieve this effect. Possible options include spraying of refuge edge rows, or alternating sprayed and unsprayed rows in the refuge so that attraction of Helicoverpa moths is maximized while minimizing cost. 4.4. Pigeonpea refuges The egg-density profiles across these crop interfaces showed up to an order of magnitude higher egg density in pigeonpea than in the adjacent Bollgard II1 crop. Oviposition-choice studies have shown that pigeonpea is much more attractive than cotton for oviposition by H. armigera (Jayaraj, 1982) while cotton was ranked in the mid-range by Firempong and Zalucki (1990) along with crops such as soybean and lucerne. Jallow and Zalucki (1995, 1996) and Jallow (1998) tested a range of host plants and found that most female moths ranked maize, sorghum, and tobacco highest, followed by cotton, with the least preferred plants being cowpea and lucerne. These studies make clear that while cotton is susceptible to H. armigera damage, it is not a preferred plant for
oviposition. The strong preference for pigeonpea over cotton documented here is consistent with these results. Additionally, based on the strong turning behavior observed by Drake and Fitt (1990) and Fitt and Farrow (unpublished data, Fitt, 1991) when moths leave a highly attractive crop, a narrow zone of effect of the pigeonpea into the Bollgard II1 may have been expected. In these trials, the higher egg density in the Bollgard II1 was restricted to the row closest to the pigeonpea. This data indicates that once flying Helicoverpa moths have been attracted to/‘arrested by’ (sensu stricto Fitt, 1991) the pigeonpea, they do not then move into adjacent Bollgard II1 and lay eggs. This suggests that pigeonpea can be a source of large numbers of unselected individuals without inadvertently increasing the numbers of individuals exposed to the Bollgard II1 genes in the adjacent cotton. The relative strengths of the attractive stimuli coming from the pigeonpea and the moth attractant would appear to be the key factor in determining whether a positive response in H. armigera egg density was detected to an insect-attractant application at a Bollgard II1/pigeonpea boundary. Given the high attractiveness of pigeonpea, it is not surprising therefore that no further enhancement of H. armigera egg lay was detected. The strong positive stimuli from the pigeonpea appears to have swamped the stimuli coming from the applied insect-attractant, leading to no additional response over that to the pigeonpea alone. 4.5. General discussion Refuges are important for protecting toxins from the development of resistance in their target pests, whether the insects are exposed to externally applied toxins (pesticide sprays) or toxins within the plant (as in the case of Bollgard II1) (Roush, 1994; Tabashnik, 1994). Such refuges provide a source of individuals unexposed to the toxin and therefore unselected for any resistance mechanism that protects them from the toxin. Refuges invariably involve additional planting and management of a crop, often with no commercial value (as in the case of pigeonpea in Australia) with an associated cost to the grower. Any method that increases the productivity of the refuge, either by increasing the numbers of insects produced by the refuge or through decreasing the exposure of individuals to the toxins, may reduce this cost. Both the increase in productivity of the refuge and the decrease in selection pressure for resistance through reduced exposure to the Bt toxins can combine to effectively increase the protection against resistance. One approach to reducing this cost may be to plant a more attractive refuge crop that requires a smaller area to provide the same level of protection, as is done when pigeonpea is used as a refuge crop to protect Bollgard II1 instead of non-transgenic cotton. Pigeonpea is deemed to require half the area of nontransgenic cotton to provide the same level of refuge effect (Monsanto, 2004). However, based on the data reported here, there are no further gains in refuge effectiveness from combining insectattraction technology with pigeonpea. It is possible that incorporating an oviposition stimulant with a moth attractant may lead to higher levels of oviposition in the refuge, further increasing protection from resistance development. However, increased productivity in the refuge may also result in increased predation and parasitism there, concentrating this away from the low number of survivors in Bollgard II1. This could possibly reduce the overall increase in value of such a refuge strategy. Although the differences reported here from the use of insectattraction technology with non-transgenic cotton refuges are not great, it is clear that increasing egg densities in specific locations through the use of an attractant is possible. With a finite number of moths, one may extrapolate to suggest that if higher egg numbers
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are laid near the source of an attractant, lower numbers would be laid further away and that the balance of egg density could be shifted. If increasing egg density in the refuge crop increases the production of next generation moths there (assuming no change in the activity of parasitoids and predators in response to the increased egg densities), then the productivity of the refuge crop will have increased. In addition, if moths are being drawn from Bollgard II1 to the refuge by the attractant and some of their eggs are being laid in the refuge instead of the crop, then the lower egg numbers would result in fewer larvae emerging and thus fewer insects exposed to the Bt proteins in Bollgard II1. This will decrease the selection pressure for resistance to the Cry proteins and thus decrease the risk of resistance developing. The results reported here suggest a number of potentially useful avenues for further increasing refuge effectiveness, as well as confirming the effectiveness of refuge crops already in use and ruling out some refuge strategy combinations that were thought to be potentially useful. In conclusion, an effective H. armigera moth attractant has the potential to increase the level of protection against Cry resistance development that is offered by a non-transgenic cotton refuge through increasing refuge productivity. Using such a mechanism, perhaps in combination with an oviposition stimulant, may provide the opportunity to reduce the area of non-transgenic cotton used for a refuge without reducing the refuge efficacy and thus reducing the cost of providing the refuge to the grower. If, through such a mechanism, the refuge efficiency of non-transgenic cotton can be increased, then it may be more economical for growers to replace non-commercial refuge crops (such as pigeonpea) with cotton, simplifying the field management to a single crop and offering the potential of some additional cotton crop in low pest years. Acknowledgements Mr. Anthony Hawes, Ag Biotech Australia Pty Ltd. provided the Magnet moth attraction/feeding stimulant used in some of these studies. Dr John Rogers, Research Connections and Consulting, Brisbane, Australia assisted with the preparation of this paper, including the statistical analyses. The Darling Downs Cotton Grower’s Association Inc provided wind speed and direction data from their network of meteorological stations for this study. References Ag Biotech Australia, 2004. Magnet Insect Attraction Technology Technical Manual. Ag Biotech Australia, Richmond, Australia. Internet resource: http://www. agbiotech.com.au/resources/magnet-tech-manual.pdf (accessed 15 September 2009).
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Drake, V.A., 1990. Methods for studying adult movement in Helicoverpa. In: Zalucki, M.P. (Ed.), Helicoverpa Research Methods and Prospects. Springer-Verlag, New York, pp. 109–121. Drake, V.A., Fitt, G.P., 1990. Studies of Heliothis mobility at Narrabri, summer 1998/ 90. In: Proceedings, 5th Australian Cotton Conference, 8–9 August 1990, Broadbeach, Queensland, Australia Cotton Growers Research Association, Brisbane, Queensland, pp. 295–305. Farrow, R.A., Daly, J.C., 1987. Long-range movement as an adaptive strategy in the genus Helicoverpa (Lepidoptera: Noctuidae): a review of its occurrence and detection in four pest species. Aust. J. Zool. 35, 1–24. Firempong, S., Zalucki, M.P., 1990. Host plant preferences of population of Helicoverpa armigera (Hu¨bner) (Lepidoptera: Noctuidae) from different geographic locations. Aust. J. Zool. 37, 665–673. Fitt, G.P., 1991. Host selection in Heliothinae. In: Bailey, W., Ridsdill-Smith, J. (Eds.), Reproductive Behavior in Insects: Individuals and Populations. Chapman & Hall, London, UK, pp. 173–201. Hedin, P.A., 1976. Seasonal variation in the emission of volatiles by cotton plants growing in the field. Environ. Entomol. 5, 1234–1238. Hopper, C.P., 1981. The Behavior and Population Dynamics of Helicoverpa armigera (Hb) (Lepidoptera: Noctuidae) in the Sudan Gezira. Ph.D. Thesis. Cranfield Institute of Technology. Jallow, M.F.A., 1998. Host-Plant Selection and Use by Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae): Individual Variation within and Among Populations. Ph.D. thesis. University of Queensland. Jallow, M.F.A., Zalucki, M.P., 1995. A technique for measuring intra-specific variation in oviposition preference of Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae). Aust. J. Zool. 34, 281–288. Jallow, M.F.A., Zalucki, M.P., 1996. Within and between-population variation in host-plant preference and specificity in Australian Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae). Aust. J. Zool. 44, 503–519. Jayaraj, S., 1982. Biological and ecological studies of Heliothis. In: Reed, W., Kumble, V. (Eds.), Proceedings, International Workshop on Heliothis management, ICRISAT, India, pp. 17–28. Monsanto, 2004. A Guide to the 2004/05 Bollgard II1 Resistance Management Plan. Monsanto, Melbourne, Australia. Office of the Gene Technology Regulator (OGTR), 2002. Risk Assessment and Risk Management Plan DIR 012/2002 for Commercial Release of Bollgard II1 and Bollgard II1/Roundup Ready1 Cotton. Office of the Gene Technology Regulator, Canberra, Australia. Internet resource: http://www.ogtr.gov.au/internet/ogtr/ publishing.nsf/Content/dir012-3/$FILE/dir012finalrarmp.pdf (accessed 15 September 2009). Office of the Gene Technology Regulator (OGTR), 2006. Risk Assessment and Risk Management Plan DIR 059/2005 for Commercial Release of Herbicide Tolerant (Roundup Ready Flex1 MON 88913) and Herbicide Tolerant/Insect Resistant (Roundup Ready Flex1 MON 88913/Bollgard II1) Cotton South of Latitude 22 South in Australia. Office of the Gene Technology Regulator, Canberra, Australia. Internet resource: http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/ Content/dir059-3/$FILE/dir059finalrarmp1.pdf (accessed 15 September 2009). Parsons, F.S., 1940. Investigations on cotton bollworm, Helicoverpa armigera. Part 3. Relationships between oviposition and flowering curves of food plants. Bull. Entomol. Res. 31, 147–177. Payne, R., Murray, D., Harding, S., Baird, D., Soutar, D., 2006. GenStat for Windows 9th Edition Introduction. VSN International, Hemel Hempstead, UK. Roush, R.T., 1994. Managing pests and their resistance to Bacillus thuringiensis: Can transgenic crops be better than sprays? Biocontrol Sci. Technol. 4, 501– 516. Tabashnik, B.E., 1994. Delaying insect adaptation for transgenic plants: seed mixtures and refugia revisited. Proc. R. Soc. Lond. Ser. B: Biol. Sci. 255, 7–12. United States Environmental Protection Agency (US EPA), 2007. Insect Resistance Management Fact Sheet for Bacillus thuringiensis (Bt) Cotton Products. US EPA. Internet resource: http://www.epa.gov/oppbppd1/biopesticides/pips/bt_ cotton_refuge_2006.htm (accessed 15 September 2009).
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Agriculture, Ecosystems and Environment journal homepage: www.elsevier.com/locate/agee
Enhancement of refuges for Helicoverpa armigera (Lepidoptera: Noctuidae) used in the resistance management plan for cotton (Gossypium hirsutum L.) containing Bollgard II1 traits Stewart J. Addison * Monsanto Australia, PO Box 92, Harlaxton, Queensland 4350, Australia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 4 February 2009 Received in revised form 22 October 2009 Accepted 22 October 2009 Available online 27 November 2009
Helicoverpa armigera (Hu¨bner) egg densities were sampled across interfaces between cotton containing Bollgard II1 traits and pigeonpea or unsprayed non-transgenic cotton refuges to determine the effectiveness of the refuge crops for Bollgard II1 resistance management plans (RMPs) against H. armigera. Additionally, a commercially available moth-attraction technology (Magnet1) was included at interfaces with both refuge crops to determine whether the use of such an attractant technology enhances the effectiveness of the refuges. Where the refuge crop was unsprayed non-transgenic cotton, there was no consistent change in the egg-density profile across the interface, suggesting that the Helicoverpa moths were not detecting and responding to the transition from Bollgard II1 to nontransgenic cotton. However wherever Magnet was applied, there was a consistent increase in egg density on or near the Magnet-treated cotton rows. This suggests that moth-attraction technology has potential to increase the efficiency of non-transgenic cotton refuges by increasing oviposition there so that the refuge produces more individuals unselected for Bt trait resistance. However, it is not yet known how best to deploy insect-attraction technology in non-transgenic cotton refuges. Pigeonpea was a very attractive refuge crop, with Helicoverpa egg densities an order of a magnitude higher than the adjacent Bollgard II1 crop and with no spillover of this increased oviposition into cotton. Application of moth attractant to the edge of the pigeonpea refuge did not alter the egg-density profile across the crop interface, suggesting that the combination of moth-attraction technology with pigeonpea refuges would not increase the effectiveness of the refuge. These studies identify avenues for improving the effectiveness of refuge crops for the management of Bt resistance in H. armigera. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Bacillus thuringiensis Magnet Transgenic crops Pigeonpea Oviposition Bt resistance Insect-attraction technology
1. Introduction The use of spatial and temporal refuges have long been advocated to delay insect adaptation to transgenic plants as part of resistance management plans (RMPs) (Roush, 1994; Tabashnik, 1994) and currently are a mandated part of the RMP for cotton (Gossypium hirsutum) containing the Bollgard II1 traits against Helicoverpa armigera (Lepidoptera: Noctuidae) in Australia (OGTR, 2002, 2006). The refuges are aimed to produce susceptible individuals that will mate with moths from Bollgard II1, including any individuals carrying alleles for resistance to the additional proteins produced from genes incorporated into the cotton genome from Bacillus thuringiensis (Bt) (which comprises the Bollgard II1 trait), thus reducing the probability of homozygousresistant individuals occurring in field populations.
* Tel.: +61 7 4634 8300; fax: +61 7 4634 8500. E-mail address: [email protected]. 0167-8809/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.agee.2009.10.015
The Australian Bollgard II1 RMP has five key components which contribute to the establishment and maintenance of both spatial and temporal refuges for H. armigera in the cotton-growing regions of eastern Australia (Monsanto, 2004). These components are (1) use of refuge crops (either non-cotton crops or nonBollgard II1 cotton) to produce Bt-susceptible moths on-farm, (2) a defined planting window for Bollgard II1 to reduce the number of H. armigera generations potentially exposed each season, (3) end-of-season pupae destruction by cultivation (southern areas) and trap crops (central Queensland) to remove any Bt-selected individuals from the population between cotton seasons, (4) control of cotton volunteers and stub cotton to remove the risk of creating mixtures of Bollgard II1 and conventional cotton that may allow survival of H. armigera larvae heterozygous for Bt resistance genes, and (5) spray limitations, including no use of Bt sprays in refuges. Of these RMP components, the maintenance of effective refuges is the most costly for growers, requiring significant amounts of land close to Bollgard II1, scarce irrigation water, and on-going
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management input during the season to ensure that refuge plants are healthy and remain attractive to H. armigera moths. In contrast, production systems elsewhere in the world have lower refuge requirements because cropping systems involve Bollgard II1 in small areas within diverse multi-cropping systems involving crops attractive to Helicoverpa (such as in India), or natural refuge areas that produce large numbers of unselected moths (most of southern and south-eastern cotton areas in the USA (US EPA, 2007)). Therefore, any techniques that enhance the performance of refuges are of particular interest to the Australian cotton industry. In considering the role of refuges in management of Bt resistance for Bollgard II1, little use has been made of what is known of H. armigera moth host-selection behavior (Parsons, 1940; Hedin, 1976; Hopper, 1981; Farrow and Daly, 1987; Drake, 1990; Fitt, 1991; Fitt and Farrow, unpublished data cited in Fitt, 1991) and pre- and post-alighting responses (Jayaraj, 1982; Jallow and Zalucki, 1995, 1996; Jallow, 1998), or exploiting existing semiochemical technology (Ag Biotech Australia, 2004) that can influence H. armigera moth behavior. This paper evaluates approaches to enhancing refuge effectiveness for Bollgard II1 in Australia that exploit this knowledge and suggests options that may lead to improvements in refuge effectiveness. Taken together, these previous studies have two implications for refuge-enhancement studies. Firstly, H. armigera moths strongly prefer pigeonpea for oviposition over cotton (Jayaraj, 1982). At a pigeonpea/cotton interface, moths are highly likely to respond by turning back into the pigeonpea, as Fitt (1991) reports for a maize/cotton interface. Secondly, while there are differences between cotton cultivars in ovipositional preference under choice situations, this is mostly related to leaf trichome density, and cultivars with similar leaf hairiness characteristics are likely to have a similar ranking of oviposition preference (Jallow, 1998). This means that, at an interface between two cotton cultivars (such as occurs at the boundary of Bollgard II1 and unsprayed refuge cotton), H. armigera moths are unlikely to lay differential numbers of eggs on either side of the boundary if the cultivars have similar pubescence characteristics. These considerations lead to some specific predictions about what may occur at interfaces between pigeonpea and unsprayed cotton refuges and Bollgard II1, with and without the addition of a moth feeding-attractant. Firstly, at the interface between Bollgard II1 and non-transgenic refuge cottons, there should be no change in egg density where the insect-attractant was not applied because moths are unlikely to detect the cotton refuge/ cotton crop interface. However, there may be a significant change in egg numbers where the attractant was applied, resulting from attraction of moths to the treated rows. Secondly, at the boundary of Bollgard II1 and a pigeonpea refuge, there should be a marked change in H. armigera egg density. Thirdly, because of the strong moth turning-response recorded by Drake and Fitt (1990) and Fitt and Farrow (unpublished data, Fitt, 1991) when moths left an attractive crop, any increase in egg densities in Bollgard II1 adjacent to pigeonpea should be restricted to rows close to the crop interface. Finally, at a Bollgard II1/pigeonpea interface there may not be any additional response in egg density where moth attractant was applied (compared to where it was not applied) because of the strong positive stimuli coming from the pigeonpea. These specific predictions were tested by recording Helicoverpa egg densities across a series of crop interfaces between Bollgard II1 and either pigeonpea or non-transgenic cotton, with and without a moth attractant (Magnet1). The primary objective of the study was to evaluate the effectiveness of the alternate refuge crops, with the secondary aim being to assess the likely impact of H. armigera moth attractants on refuge effectiveness, using Magnet as an example of this type of product.
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2. Materials and methods 2.1. Unsprayed non-transgenic cotton refuge – 2005 This section of the study used two large Bollgard II1 fields (areas 0.36 and 0.55 km2) with adjoining areas of unsprayed, irrigated non-transgenic cotton at Cecil Plains, Queensland (278350 S, 1518150 E). In Field 1 (Bollgard II1 cultivar Sicot 289B), rows were oriented 548 east of north (approximately north-east to south-west) with the cotton refuge of cultivar Sicot 80 located on the north-east side of the field, 50 m across a minor public road with no roadside vegetation between the field and the refuge. Six millilitres of Magnet insect-attractant were applied to the upper surface of a 1-m section of crop canopy on the end of each of 60 Bollgard II1 rows (60 m), i.e. 360 mL on 60 m of row, on the northeast boundary of the field weekly from 21 February 2005 (Julian Day 52) for 2 weeks using a fabricated sprayer with a ‘dribble’ nozzle. The Magnet and no-Magnet sample sites were approximately 350 m apart within the field, with two sampling transects in each area 40 m apart. Sampling points were at the row ends (0 m) and then 3, 10, 50 and 200 m into the Bollgard II1 crop along the rows. All Helicoverpa eggs were counted on a 1-m section of crop row at each sampling point prior to each Magnet application and again 3 days after spraying (DAS). In Field 2 (Bollgard II1 cultivar Sicot 80B), crop rows were oriented 368 west of north (approximately north-west to south-east and at right angles to rows in Field 1), with eight rows of unsprayed non-transgenic Sicot 80 refuge in the south-west side of the field, with the remainder of the field being Sicot 80B cotton. Threehundred and sixty milliliters of Magnet was applied to a 60 m section of the sixth non-transgenic row in from the south-western field edge (Row 0), as for Field 1 at this site. Eggs were sampled in Row 0 and 3, 10, 50 and 200 m (rows) from the treated row; thus Row 3 was the first Bollgard II1 row. The Magnet and no-Magnet sample areas in this field were approximately 300 m apart. Sampling sites in Fields 1 and 2 were 550 m apart at their closest point. Sampling transects in the two fields were oriented at 1808 to each other, and directly towards and away from the expected predominant wind direction (north-east). During the 2004/2005 growing season, approximately 75% of Heliothine eggs on the Darling Downs were H. armigera by February 2005 (D. Murray, personal communication). Fifteen-minute interval meteorological data for the experimental period were obtained from weather stations operated at Horrane (278320 17.7400 S, 1518140 4.5100 E), Norwin (278330 42.9000 S, 1518200 31.4400 E), and Tyunga (278400 12.9600 S, 1518170 38.7400 E) by the Darling Downs Cotton Grower’s Association. These weather stations triangulate the experimental site at distances of between 7.0 and 8.7 km over near-flat ground. Wind direction and speed data were plotted as polar scatter plots for the Helicoverpa flight period (17.45 (sunset) to midnight; Drake and Fitt, 1990) for the days between Magnet application on Julian Days 52 and 59 and the subsequent egg counts three days later. Direction was plotted as the deviation angle from north, and the speed as the distance from the centre of the graph. 2.2. Unsprayed non-transgenic cotton refuge – 2006 In February 2006, the effect of Magnet application on Helicoverpa egg distribution was assessed in three Bollgard II1 fields at the Cecil Plains site. Three Magnet applications were made to 100 m of crop row in each field at weekly intervals from 2 February 2006 (Julian Day 33), as previously. In each field, four sampling transects 20 m apart were established in each treated area and egg counts made 4, 16 and 400 m (rows) from the Magnet-treated row. Helicoverpa egg counts commenced 3 days after the first Magnet application and from then on were made
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prior to each Magnet application and again 3 days later. All Helicoverpa eggs were counted on a 1-m section of crop row at each sampling site. In 2006, Fields 1 and 2 were planted to the same Bollgard II1 cultivar throughout (Sicot 289BR) while the third field contained two cultivars (DP570B and Sicot 289B) with the 4 and 16 m samples being DP570B and the 400 m sample Sicot289B. These two cultivars were phenologically and morphologically extremely similar, including leaf pubescence characteristics. During the 2005/2006 growing season, approximately 70% of Heliothine eggs on the Darling Downs were H. armigera by January 2006 (D. Murray, personal communication). 2.3. Unsprayed pigeonpea refuge – 2005 This site was located at Dalby Agricultural College, Australia (278090 S, 1518170 E) in a Bollgard II1 (cultivar Sicot 71B) cotton field with an immediately adjacent unsprayed and irrigated pigeonpearefuge field. Weekly from 18 February 2005 (Julian Day 49) for 3 weeks, 360 mL of Magnet insect-attractant was applied over 60 m of the outside row of the pigeonpea refuge adjacent to the Bollgard II1 crop (Row 0) as for the 2005 cotton-refuge experiments. An untreated 60 m section of the crop interface approximately 250 m away was designated as a control area. In each area, two transects 60 m apart were established with sampling points in the treated row (Row 0) and then Row 3 (i.e. 2 rows into the Bollgard II1), and Rows 10, 50 and 200. All Helicoverpa spp. eggs were counted on a 1m section of crop row at each sampling site prior to each Magnet application and again 3 days later. 2.4. Data structure and statistical analysis Magnet is a patented mixture of floral volatiles, terpenoids and green leaf volatiles that attracts moths of H. armigera but the attractive effect of the plant volatiles disappears within 4–6 days of application (Ag Biotech Australia, 2004). In these experiments, Magnet was applied at weekly intervals for several weeks, with egg counts done just prior to Magnet application and then again 3 days later. The egg counts made on the day of Magnet application reflect the distribution of ovipositing Helicoverpa moths over the previous few days while the 3-days post-treatment egg count quantifies moth responses to the Magnet application in a repeated-measures data structure. Data sets were analyzed utilizing this data structure in a restricted maximum likelihood (REML) analysis with the pretreatment counts as a covariate and the random model that minimized deviance using GenStat Version 9 (Payne et al., 2006). The test statistic was the Wald statistic divided by its degrees of freedom, with the statistic being distributed approximately as the chi-square distribution (Payne et al., 2006). This gives tests of
significance for the fixed effects that are equivalent to the F-tests in ANOVA but with an optimal random model fitted to the covariance patterns in the repeated-measures structure. In 2006, there were no pre-treatment counts for the first Magnet treatment but there were for the second and third applications. Additionally, in 2006 there was only one treatment, i.e. Magnet, in each field, so all fields were included in a single REML analysis that considered Fields, Distance from Magnet and Julian Date. Two alternative data structures were investigated for this year’s data. Firstly, the three sets of 3 days after spraying data were analyzed without pre-treatment count as a covariate. Secondly, this analysis was repeated for the second and third application dates with their respective pre-treatment counts as covariates, as was done for the 2005 data. The 2005 pigeonpea-refuge data and the 2006 data were transformed by the H(x + 0.5) and log10 (x + 1) transformations, respectively, because residuals were not normally distributed. Where data were transformed and there were pre-treatment covariate data, the pre-treatment data were transformed onto the same scale as the post-treatment data for analysis. Because of the scale on which Magnet affects moth behavior, the Magnet and no-Magnet areas were located some hundreds of metres apart in each field. Consequently, the treatment main effects (i.e. Magnet vs. no-Magnet) were inevitably confounded with ‘position in the field’ effects and so were not used to assess the impact of the treatments. Instead, treatment effects were sought in the Treatment Distance interaction term. That is, different egg-density profiles where Magnet was, and was not, applied were used as evidence that Magnet was affecting moth distribution and oviposition. Additionally, given the random variations in moth distribution in the absence of any attractive treatments, the Distance response in no-Magnet areas was expected to be variable over time and space. In contrast, if Magnet is having an effect then the Distance response in Magnet areas should be consistent over time and space. Additionally, differential variation of moth numbers over the field could lead to a Treatment Time interaction, but this is another component of the confounding of treatment and field position discussed previously. Finally, if the unsprayed refuge of non-transgenic cotton or pigeonpea was having an effect on the egg distribution in the Bollgard II1 field, then a Distance main effect was expected regardless of whether Magnet was applied or not. 3. Results 3.1. Unsprayed non-transgenic cotton refuge – Field 1, 2005 At this site, there were differences between the Magnettreated and untreated areas and a significant Julian Day TreatTreatment interaction (Table 1) that reflect differences in moth
Table 1 Wald tests for the REML analysis of Helicoverpa egg count data from Bollgard II1 adjacent to unsprayed non-transgenic cotton and pigeonpea, 2005. Fixed term in REML analysis
Pre-treatment egg count Julian Day Treatment Distance Julian Day Treatment Julian Day Distance Treatment Distance Julian Day Treatment Distance *
Cotton refuge (Field 1)
Cotton refuge (Field 2)
Pigeon pea refuge
d.f.
Wald statistic/d.f.
d.f.
Wald statistic/d.f.
d.f.
Wald statistic/d.f.
1 1 1 4 1 4 4 4
8.08** 19.58*** 4.16* 0.80NS 11.77*** 0.82NS 3.76** 1.21NS
1 1 1 4 1 4 4 4
113.76*** 79.72*** 3.76NS 1.69NS 13.63*** 1.39NS 5.05*** 1.53NS
1 2 1 4 2 8 4 8
115.37*** 31.42*** 0.02NS 24.54*** 7.64*** 4.52*** 1.78NS 0.50NS
Wald statistic/degrees of freedom test significant at P < 0.05. Wald statistic/degrees of freedom test significant at P < 0.01. Wald statistic/degrees of freedom test significant at P < 0.001. NS Not significant (P > 0.05). **
***
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in egg density in Rows 0–3 (average of 9.8 and 7.6 eggs m1 on Days 55 and 62, respectively) compared to the rest of the field (average of 2.8 and 2.3 eggs m1 across the rest of the field on the same two sampling dates) (Fig. 1A and B). The overall egg density in Row 0 was significantly higher than in Rows 10, 50 and 200 (all P < 0.05) while the non-Magnet area showed the opposite pattern, with Row 0 with lower egg densities than Rows 10, 50 and 200 (all P < 0.05).
activity in different parts of the field over the duration of the experiment. Specifically, the part of the field that did not receive Magnet in Row 0 on Julian Day 52 had 2.5 times the number of eggs 3 days later compared to the area where the Magnet was applied in Row 0; however, 3 days after the Day 59 treatment the egg densities were approximately the same. These differences are not interpreted as indicating actual Treatment effects, but are rather artifacts of the experimental layout imposed by Magnet’s mode of action. There were large differences between sampling dates, with oviposition pressure falling markedly between Julian Day 55 and Julian Day 62. As indicated previously, from the refuge-enhancement perspective the Distance main effect term and the interaction term involving Treatment and Distance are the critical lines of the REML table (Table 1). The absence of a Distance main effect across the cotton refuge/Bollgard II1 interface indicates that the Helicoverpa moths are not detecting and responding to the crop boundary, in contrast to the very clear response at the pigeonpea/Bollgard II1 interface reported later in this paper. This suggests that there is not any difference in attractiveness/apparency of the two types of cotton in this experiment. In the area where no-Magnet was applied, the pattern of egg densities across the field was markedly different on the two 3-DAS sampling dates (Fig. 1). On Day 55, egg counts rose sharply from the refuge edge (Row 0 – 4.8 eggs m1) to be 13–20 eggs m1 for the rest of the field (Fig. 1A). On Day 62, egg counts were much lower (mean = 4.2 eggs m1), and rose gradually from Row 0 (2.6 eggs m1) to reach 7.1 eggs m1 at Row 200 (Fig. 1B). In contrast, the area where the Magnet was applied in Row 0 showed identical patterns on the two sampling dates with sharp increases
In this field, there were no statistical differences between the Magnet-treated and untreated areas (P = 0.053) but there was a significant Julian Day Treatment interaction (Fig. 2), again reflecting differences in moth activity in different parts of the field over the duration of the experiment. As for Field 1, there was no Distance main effect, again suggesting that the moths did not respond to the cotton refuge/cotton crop interface in the way they responded to the pigeonpea/cotton crop boundary. Again there was a large difference between the two 3-DAS sampling dates, with oviposition pressure in Field 2 falling between Julian Day 55 and Julian Day 62 (Fig. 2). For the Treatment and Treatment Distance effects, the pattern of significant differences for this field is the same as recorded in the other field with an unsprayed non-transgenic cotton refuge, and the opposite of that recorded where pigeonpea was the refuge crop (Table 1). That is, there were higher egg densities close to the site of the Magnet application than further into the field (Fig. 2), with egg density in sampling Row 3 (i.e. the first Bollgard II1 row) significantly higher than further in the field (P < 0.05).
Fig. 1. Helicoverpa egg density (SEM) on Bollgard II1 adjacent to an unsprayed nontransgenic cotton refuge in Field 1 with and without Magnet application in Row 0, 2005, 3 days after Magnet application on (A) Julian Day 52, and (B) Julian Day 59.
Fig. 2. Helicoverpa egg density (SEM) on Bollgard II1 adjacent to an unsprayed nontransgenic cotton refuge in Field 2 with and without Magnet application in Row 0, 2005, 3 days after Magnet application on (A) Julian Day 52, and (B) Julian Day 59.
3.2. Unsprayed non-transgenic cotton refuge – Field 2, 2005
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Fig. 4. Helicoverpa egg density (SEM) on Bollgard II1 3 days after Magnet applications, 2006. For each field, means with the same letter are not significantly different (P < 0.05). Fig. 3. Pooled wind direction and speed data around the 2005 non-transgenic cotton refuge experimental site (Cecil Plains district, Queensland) during the Helicoverpa oviposition period (sunset–midnight) for the six post-treatment nights. The direction the wind is blowing from is plotted as the deviation angle from north and the speed as the distance from the centre of the graph.
3.3. Wind direction data, Cecil Plains – 2005 The wind direction during the Helicoverpa oviposition period was consistent for both experimental periods at the three meteorological station sites that triangulated the experimental site, and so was pooled (Fig. 3). Over these six evenings, the wind blew from the north-east to east for 82% of the moth flight period (average angle = 698 east of north) at speeds that averaged 13 km h1 and ranged up to 28 km h1. 3.4. Unsprayed non-transgenic cotton refuge – 2006 The treatment effects for this experiment were examined at the interaction level rather than the main-effect level because there were significant interactions in the REML analysis (Table 2). There was a Julian Day Field interaction, reflecting differing changes in egg counts over the three dates in the three fields, i.e. the natural variation that existed in Helicoverpa egg pressure. The Julian Day Distance interaction was not significant, indicating that the changes in egg density with distance from the Magnet application were the same over the three sampling dates. This consistency suggests that the changes with respect to distance were not due to some random fluctuation in egg density, but rather reflect a consistent change with respect to the treatment of interest, i.e. distance from the Magnet application. The important interaction was Field Distance (Fig. 4). Examination of the two-way table,
Table 2 Wald tests for the REML analysis of Helicoverpa egg count data from Bollgard II1 adjacent to unsprayed non-transgenic cotton, 2006. Fixed term in REML analysis
d.f.
Wald statistic/d.f.
Julian Day Field Distance Julian Day Field Julian Day Distance Field Distance Julian Day Field Distance
2 2 2 4 4 4 8
147.12*** 282.54*** 12.39*** 2.95* 1.96NS 7.36*** 1.92NS
* ***
Wald statistic/degrees of freedom test significant at P < 0.05. Wald statistic/degrees of freedom test significant at P < 0.001. Not significant (P > 0.05).
NS
and t-tests between distances for each Field, indicate that distances of 4 and 16 m were not different in Field 1 (P > 0.05) but were significantly different in both of the other two fields (P < 0.05). Additionally, the relationships between the 4 and 16 m distances and the 400 m distance were different in the different fields, with the 400 m distance having variously more, equal or fewer eggs than both the 4 m and 16 m distances. This indicates that the scale of Magnet effects on Helicoverpa egg distribution is relatively small-scale when Magnet application is restricted to a single crop row. The analysis of data from the second and third Magnet applications with pre-treatment counts as a covariate showed the same relationships at the Field Distance level as the analysis with three dates but without pre-treatment counts as a covariate (data not shown). That is, Distances 4 and 16 were not different in Field 1 (P > 0.05) but were significantly different in both of the other two fields (P < 0.05). 3.5. Unsprayed pigeonpea refuge – 2005 At this site, there was a significant Julian Day main effect and significant Julian Day Treatment interaction (Table 1). These results indicate that (a) the Helicoverpa ovipositional pressure varied through time (with oviposition pressure falling markedly during the experimental period (Fig. 5)), and (b) this change in oviposition pressure through time was different in different sections of the field. Note also that the pattern of significant differences is the opposite of that recorded where the refuge crop was non-transgenic cotton (Table 1). That is, when the refuge crop was unsprayed non-transgenic cotton there was no significant Distance main effect but a significant Treatment Distance interaction, but for the pigeonpea refuge the opposite pattern occurred. There was a highly significant Distance main effect and Julian Day Distance interaction, but no Treatment Distance interaction. Fig. 5 provides an understanding of these effects. There was the same Distance profile for both Treatments on each of the three sampling dates with a significant increase in Row 0 compared to all other Distances (P < 0.05). However, the increase in Row 0 was numerically largest when the oviposition pressure was highest (Julian Day 52) (Fig. 5A). The spike of oviposition at the margin of the pigeonpea refuge resulted in the highly significant Distance main effect while the change in response in Row 0 as egg pressure declined during the experiment led to the significant Julian Day Distance interaction.
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Fitt, 1991). The short-range movement takes place close to the ground (1–4 m) (Drake and Fitt, 1990) and is the class of movement of most interest to refuge enhancement. Within the short-range movement category, not much is known about the behavior of Helicoverpa moths with respect to field interfaces between more and less attractive crops. However, this is an important issue for refuge enhancement. It has been often suggested (e.g. Parsons, 1940; Hedin, 1976; Hopper, 1981) that volatiles emanating from flowering cotton and maize may act as long-range attractants or arrestants for Helicoverpa females, thus allowing moths to concentrate within areas of flowering hosts. Drake and Fitt (1990) and Fitt and Farrow (unpublished data, cited in Fitt, 1991) observed moth behavior at cotton–maize crop boundaries as well as at edges of cotton fields bounded by fallow land. They indicated that up to 60% of H. armigera moths flying out of the maize turned back rather than fly over adjacent cotton crops. This turning behavior occurred at both downwind and upwind sides of the crop although moths tended to travel further from the maize edge before turning on the downwind side than on the upwind side. The moths were clearly able to perceive the patch boundaries and to respond very quickly. Similar turning behavior was observed to a lesser extent at the boundary of cotton and fallow ground. Additionally, once in the attractive crop they tend not to leave but rather remain there, presumably to feed and lay eggs. From a refuge-enhancement perspective, this behavior underpins the use of refuge crops such as pigeonpea, maize and sorghum as an option in Bollgard II1 RMPs (Monsanto, 2004). 4.1. Unsprayed non-transgenic cotton refuges, 2005
Fig. 5. Helicoverpa egg density (SEM) on Bollgard II1 adjacent to an unsprayed pigeonpea refuge with and without Magnet application in Row 0, 2005, 3 days after Magnet application on Julian Days 49, 56 and 63.
Importantly, there was no Treatment Distance interaction. This indicates that the change in egg density with respect to the pigeonpea was the same whether Magnet was applied in Row 0 or not. From this, it can be concluded that where the adjacent refuge crop was pigeonpea, the addition of Magnet at the edge of the refuge field did not change the response of the Helicoverpa moths to the refuge crop, as measured by the post-treatment egg density. 4. Discussion Studies of H. armigera moth movement indicate that this species undertakes both short-range movement (up to few kilometres) and long-range movement (hundreds of kilometres) at various times of the year (Farrow and Daly, 1987; Drake, 1990;
There were no consistent changes in H. armigera egg density across the Bollgard II1/cotton interfaces in the absence of Magnet. This result is consistent with the predictions based on the studies of moth ovipositional behaviour by Jallow (1998). However, where Magnet was applied at the Bollgard II1/cotton interface, there was a consistent increase in egg density at or close to the interface. Because Magnet insect-attraction technology (Ag Biotech Australia, 2004) contains plant volatiles that are responsible for the attractiveness of various plants to Helicoverpa moths seeking nectar prior to oviposition, moth reactions to such insectattractant applications would be expected to mimic moth responses to an attractive plant, and where the insect-attractant was applied to non-transgenic cotton close to a Bollgard II1/nontransgenic cotton boundary, it would be expected that H. armigera moths would respond to this application by spending more time in, and laying more eggs on and near, the treated rows. This is what occurred (Figs. 1 and 2). The wind during the oviposition periods of these trials was of a direction and strength (Fig. 3) that would have carried moths to the west–south–west, i.e. into the sampled Bollgard II1 crop in Field 1 and away from the sampled crop in Field 2. At any crop interface that elicited the turning-response observed by Drake and Fitt (1990) and Fitt and Farrow (unpublished data, cited in Fitt, 1991), this may result in a shift in the position of any peak of oviposition downwind. In the 2005 trials, a more marked increase in egg density close to the Magnet-treated strip in Field 1 was recorded than in Field 2 (Figs. 1 and 2) and this is consistent with the displacement of the Magnet-induced egg peak away from the sampled area in Field 2 and into the adjacent cotton refuge. Sampling on both sides of the narrow Magnet-treated strip would have resolved this and should be incorporated into future trial designs. In addition, this result suggests that when laying out nontransgenic cotton refuges on which narrow strips of insectattractant technology are to be used, the refuge areas should be positioned so they are downwind of Bollgard II1 during the
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evening Helicoverpa oviposition period. Existing meteorological data could be used to determine whether wind direction during the period from sunset to midnight is consistent enough during the cotton-growing season to implement this option. Alternatively, applying the strips of insect-attractant technology some distance in from the edge of the refuge would avoid displacement of the oviposition peak by the wind into Bollgard II1 regardless of the wind direction. Such approaches to the placement of the insectattractant technology would increase the probability that moths are more likely to be ‘arrested’ by the insect-attractant within the non-transgenic cotton and remain there to lay eggs, thus avoiding increasing egg lay in Bollgard II1 crops as opposed to on the refuges and so increasing refuge effectiveness. 4.2. Unsprayed non-transgenic cotton refuges, 2006 The Helicoverpa oviposition pressure across Field 1 in 2006 was very high (50–60 eggs m1) while it was only 6–19 eggs m1 in the other two fields (Fig. 4). That the response to the insect-attraction technology was different between the fields with high and more moderate egg-pressure suggests that when Helicoverpa egg pressure is very high, attractant technology may not affect egg distribution but that it may at more moderate oviposition pressures. Magnet is designed to attract moths from relatively short distances at times when a moth is seeking a nectar source (Ag Biotech Australia, 2004), rather than as an oviposition stimulant per se. Perhaps at high moth densities there was interference between females that masks the attraction response to this particular insect-attraction technology. 4.3. Insect-attraction technology to increase effectiveness of refuges One difference that is apparent between the cotton-refuge fields and the pigeonpea-refuge field is that, in the former, the application of the insect-attraction technology at the refuge/crop interface significantly altered the Helicoverpa oviposition response at the crop/refuge interface, but this did not occur when the refuge crop was pigeonpea. It seems likely that the attractiveness of the Magnet volatiles was swamped by the volatiles/stimuli being detected from the pigeonpea, but Magnet was clearly detectable by Helicoverpa moths when the refuge crop was unsprayed nontransgenic cotton. This points to there being potential to enhance the effectiveness of unsprayed non-transgenic cotton refuges, but not pigeonpea refuges, through the use of insect-attraction technology by increasing the attractiveness/‘apparency’ of the refuge compared to Bollgard II1. What is not known at this stage is the optimal application pattern of the attraction technology to achieve this effect. Possible options include spraying of refuge edge rows, or alternating sprayed and unsprayed rows in the refuge so that attraction of Helicoverpa moths is maximized while minimizing cost. 4.4. Pigeonpea refuges The egg-density profiles across these crop interfaces showed up to an order of magnitude higher egg density in pigeonpea than in the adjacent Bollgard II1 crop. Oviposition-choice studies have shown that pigeonpea is much more attractive than cotton for oviposition by H. armigera (Jayaraj, 1982) while cotton was ranked in the mid-range by Firempong and Zalucki (1990) along with crops such as soybean and lucerne. Jallow and Zalucki (1995, 1996) and Jallow (1998) tested a range of host plants and found that most female moths ranked maize, sorghum, and tobacco highest, followed by cotton, with the least preferred plants being cowpea and lucerne. These studies make clear that while cotton is susceptible to H. armigera damage, it is not a preferred plant for
oviposition. The strong preference for pigeonpea over cotton documented here is consistent with these results. Additionally, based on the strong turning behavior observed by Drake and Fitt (1990) and Fitt and Farrow (unpublished data, Fitt, 1991) when moths leave a highly attractive crop, a narrow zone of effect of the pigeonpea into the Bollgard II1 may have been expected. In these trials, the higher egg density in the Bollgard II1 was restricted to the row closest to the pigeonpea. This data indicates that once flying Helicoverpa moths have been attracted to/‘arrested by’ (sensu stricto Fitt, 1991) the pigeonpea, they do not then move into adjacent Bollgard II1 and lay eggs. This suggests that pigeonpea can be a source of large numbers of unselected individuals without inadvertently increasing the numbers of individuals exposed to the Bollgard II1 genes in the adjacent cotton. The relative strengths of the attractive stimuli coming from the pigeonpea and the moth attractant would appear to be the key factor in determining whether a positive response in H. armigera egg density was detected to an insect-attractant application at a Bollgard II1/pigeonpea boundary. Given the high attractiveness of pigeonpea, it is not surprising therefore that no further enhancement of H. armigera egg lay was detected. The strong positive stimuli from the pigeonpea appears to have swamped the stimuli coming from the applied insect-attractant, leading to no additional response over that to the pigeonpea alone. 4.5. General discussion Refuges are important for protecting toxins from the development of resistance in their target pests, whether the insects are exposed to externally applied toxins (pesticide sprays) or toxins within the plant (as in the case of Bollgard II1) (Roush, 1994; Tabashnik, 1994). Such refuges provide a source of individuals unexposed to the toxin and therefore unselected for any resistance mechanism that protects them from the toxin. Refuges invariably involve additional planting and management of a crop, often with no commercial value (as in the case of pigeonpea in Australia) with an associated cost to the grower. Any method that increases the productivity of the refuge, either by increasing the numbers of insects produced by the refuge or through decreasing the exposure of individuals to the toxins, may reduce this cost. Both the increase in productivity of the refuge and the decrease in selection pressure for resistance through reduced exposure to the Bt toxins can combine to effectively increase the protection against resistance. One approach to reducing this cost may be to plant a more attractive refuge crop that requires a smaller area to provide the same level of protection, as is done when pigeonpea is used as a refuge crop to protect Bollgard II1 instead of non-transgenic cotton. Pigeonpea is deemed to require half the area of nontransgenic cotton to provide the same level of refuge effect (Monsanto, 2004). However, based on the data reported here, there are no further gains in refuge effectiveness from combining insectattraction technology with pigeonpea. It is possible that incorporating an oviposition stimulant with a moth attractant may lead to higher levels of oviposition in the refuge, further increasing protection from resistance development. However, increased productivity in the refuge may also result in increased predation and parasitism there, concentrating this away from the low number of survivors in Bollgard II1. This could possibly reduce the overall increase in value of such a refuge strategy. Although the differences reported here from the use of insectattraction technology with non-transgenic cotton refuges are not great, it is clear that increasing egg densities in specific locations through the use of an attractant is possible. With a finite number of moths, one may extrapolate to suggest that if higher egg numbers
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are laid near the source of an attractant, lower numbers would be laid further away and that the balance of egg density could be shifted. If increasing egg density in the refuge crop increases the production of next generation moths there (assuming no change in the activity of parasitoids and predators in response to the increased egg densities), then the productivity of the refuge crop will have increased. In addition, if moths are being drawn from Bollgard II1 to the refuge by the attractant and some of their eggs are being laid in the refuge instead of the crop, then the lower egg numbers would result in fewer larvae emerging and thus fewer insects exposed to the Bt proteins in Bollgard II1. This will decrease the selection pressure for resistance to the Cry proteins and thus decrease the risk of resistance developing. The results reported here suggest a number of potentially useful avenues for further increasing refuge effectiveness, as well as confirming the effectiveness of refuge crops already in use and ruling out some refuge strategy combinations that were thought to be potentially useful. In conclusion, an effective H. armigera moth attractant has the potential to increase the level of protection against Cry resistance development that is offered by a non-transgenic cotton refuge through increasing refuge productivity. Using such a mechanism, perhaps in combination with an oviposition stimulant, may provide the opportunity to reduce the area of non-transgenic cotton used for a refuge without reducing the refuge efficacy and thus reducing the cost of providing the refuge to the grower. If, through such a mechanism, the refuge efficiency of non-transgenic cotton can be increased, then it may be more economical for growers to replace non-commercial refuge crops (such as pigeonpea) with cotton, simplifying the field management to a single crop and offering the potential of some additional cotton crop in low pest years. Acknowledgements Mr. Anthony Hawes, Ag Biotech Australia Pty Ltd. provided the Magnet moth attraction/feeding stimulant used in some of these studies. Dr John Rogers, Research Connections and Consulting, Brisbane, Australia assisted with the preparation of this paper, including the statistical analyses. The Darling Downs Cotton Grower’s Association Inc provided wind speed and direction data from their network of meteorological stations for this study. References Ag Biotech Australia, 2004. Magnet Insect Attraction Technology Technical Manual. Ag Biotech Australia, Richmond, Australia. Internet resource: http://www. agbiotech.com.au/resources/magnet-tech-manual.pdf (accessed 15 September 2009).
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