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Field evaluation of potato plants transgenic for a cry1Ac gene conferring resistance to potato tuber moth, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae)
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Crop Protection 25 (2006) 216–224 www.elsevier.com/locate/cropro
Field evaluation of potato plants transgenic for a cry1Ac gene conferring resistance to potato tuber moth, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae) M.M. Davidsona,, R.C. Butlera, S.D. Wrattenb, A.J. Connera,b a
New Zealand Institute for Crop & Food Research Limited, Private Bag 4704, Christchurch, New Zealand National Centre for Advanced Bio-Protection Technologies, Lincoln University, P.O. Box 84, Canterbury, New Zealand
b
Received 28 July 2004; accepted 20 April 2005
Abstract Cry1Ac-transgenic potato plants derived from Russet Burbank and Red Rascal cultivars were evaluated in field trials at Lincoln, Canterbury, New Zealand. Foliage of transgenic plants had fewer potato tuber moth (Phthorimaea operculella (Zeller)) mines, of which the majority were o200 mm2, than the non-transgenic control plants. Of the 800 transgenic tubers examined 5 months after harvesting the first field trial (1999/2000), 0.5% had potato tuber moth mine damage compared with 9% of non-transgenic tubers. After the second field trial (2000/2001) 0.06% of 5307 transgenic tubers had mine damage compared with damage to 25% of non-transgenic tubers. Mined foliage was collected during the first field trial, resulting in a total of 45 larvae from transgenic foliage and 269 from nontransgenic foliage. Two pupae and nine parasitoids emerged from larvae collected from and reared on transgenic foliage. Between 86% and 88% (Red Rascal or Russet Burbank cultivar, respectively) of larvae collected and reared on non-transgenic foliage either pupated or were parasitised. In the second field trial, three of the four transgenic lines evaluated produced yields comparable with their nontransgenic parent cultivar. These results suggest transgenic potato plants resistant to potato tuber moth could augment integrated pest management programmes. However, the potential of the target pest to develop resistance to such plants and effects on non-target insects must be evaluated before such plants can be recommended for potato tuber moth management. r 2005 Elsevier Ltd. All rights reserved. Keywords: Phthorimaea operculella; Cry1Ac; Resistance; Transgenic potato; Field trials
1. Introduction The potato tuber moth, Phthorimaea operculella (Zeller), is considered one of the most economically damaging pests of potato (Solanum tuberosum L.) crops in regions with warm temperate to tropical climates (Radcliff, 1982). Larvae mine into leaves, stems and tubers, and the latter damage results in rejection of tubers by growers and potato processors. Further, the level of P. operculella infestation in the foliage can influence infestation levels in tubers (Lagnaoui et al., 1996; Coll et al., 2000). Integration of management Corresponding author. Tel.: +64 3 325 6400; fax: +64 3 325 2074.
E-mail address: [email protected] (M.M. Davidson). 0261-2194/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2005.04.010
strategies (spray insecticides, cultural methods, biological control) can help to manage this pest in the field and storage (Hanafi, 1999). However, the development of resistant potato cultivars could increase the efficacy of cultural and biological methods and reduce the use of insecticides (Arnone et al., 1998). In New Zealand, populations of P. operculella larvae peak between February and April. P. operculella are particularly prevalent in hot, dry summers (Foot, 1979). They infest potato foliage before tubers are colonised, but as the foliage senesces the larvae become proportionally more common in the tubers, initially in green, exposed tubers (Foot, 1979). Soil moisture and tuber depth affect the ability of larvae to infest tubers (Foot, 1979). Management methods emphasise the use of cultural
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practices such as moulding, ‘seed’ depth, timing of planting and irrigation (Hanafi, 1999) to reduce tuber exposure to or increase mortality of P. operculella. However, P. operculella population can still reach levels where it is necessary to apply insecticides (Hanafi, 1999). Potato cultivars with resistance to P. operculella could eliminate the need for, or at least reduce the number of, insecticide applications to manage this pest. However, attempts to develop such cultivars using traditional breeding methods have not been successful (Arnone et al., 1998). Genetic engineering has become a viable method for developing insect-resistant transgenic plants. Transgenic potato plants resistant to P. operculella have been developed predominantly through the transfer of a single cry gene derived from Bacillus thuringiensis Berliner (Bt). The cry genes transferred into potato cultivars include cry1Ab (Jansens et al., 1995; Can˜edo et al., 1999), cry1Ac (Davidson et al., 2002a, 2004) and cry5 (Douches et al., 1998, 2002; Li et al., 1999). All of these cry genes are specific in their activity to Lepidoptera, except cry5, which is also active against Coleoptera and is therefore likely to affect a wider range of non-target insects. Potato plants transgenic for the cry1Ac gene provide a similar level of resistance to potato tuber moth as cry1Ab-transgenic plants (Jansens et al., 1995; Can˜edo et al., 1999), even though they exhibited lower Cry protein expression (Davidson et al., 2002a). We have focused our attention on the use of the cry1Ac gene for potato tuber moth resistance since the lower amount of Cry protein in transgenic potatoes to effect the desired trait may be more acceptable to consumers. We have developed potato plants transgenic for a cry1Ac gene under the transcriptional control of a CaMV35S promoter using Agrobacterium-mediated transformation (Davidson et al., 2002a, 2004). Greenhouse trials identified transgenic lines that appeared phenotypically normal relative to parental cultivars and that inhibited the growth of P. operculella larvae. These lines were, in turn, evaluated in small-scale field trials to screen for transgenic lines with the highest resistance and comparable yield to their non-transgenic parent cultivars (Davidson et al., 2002b). The present study reports on the performance of selected lines in scaled-up field trials using natural infestation by a wild population and artificial infestation by a laboratory colony of P. operculella. Potato tuber yield was assessed relative to non-transgenic parent cultivars.
2. Materials and methods 2.1. Field trial and design Transgenic lines derived from potato cultivars Russet Burbank and Red Rascal that had been selected from
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previous small-scale field trials (Davidson et al., 2002b) were evaluated in the field over the summers of 1999/ 2000 and 2000/2001 at Lincoln, Canterbury, New Zealand (Environmental Risk Management Authority approval GMF98008). In 1999, the field trial was established from transgenic and non-transgenic plants micro-propagated in tissue culture. Tissue culture plantlets were transplanted into seedling trays, grown in a containment greenhouse for 3–4 weeks, and then moved to a screened enclosure to harden off for 2–3 weeks before planting in the field on 1 December 1999. In the following year, tubers from the previous year’s field trial of the transgenic and non-transgenic potato plants were planted in the field on 7 December 2000. Four replicate plots in 1999 and six plots in 2000, each containing 50 plants of each of the four transgenic lines and two non-transgenic parent cultivars (experimental controls), were planted in a randomised block layout in the field. There were five rows (0.76 m apart) per plot, each with 10 plants spaced 0.3 m apart within a row. Plots were separated by 1 m within a row. The experimental plots were completely surrounded by plots from trials of other transgenic potatoes or three buffer rows of non-transgenic potato plants (breeding line 2390, Urenika V431-1) to prevent edge effects during the trial. Weeding was done by hand and the potato plants were subjected to overhead irrigation as required. Rows were mounded once, 4 weeks after planting, at a shallower depth than normal (approximately 20 cm) to encourage tuber infestation by P. operculella larvae, to allow appropriate assessment of tuber resistance. 2.2. Field cage experiments In the 1999/2000 field trial, cages (120 60 50 cm3) or mesh sleeves (35 cm long 15 cm diameter) were placed within the plots in the first week of February. A cage covered several plants, while a mesh sleeve enclosed a single shoot of a plant. A cage was positioned over 3–4 plants of an outer row for two of the four plots of each transgenic or non-transgenic line (12 cages in total). Two mesh sleeves were placed on randomly selected shoots of plants in the outer rows of each plot of transgenic or non-transgenic line (48 sleeves in total). On 8 February 2000, 400 or 500 eggs laid on filter paper by laboratory-reared females were placed on plants within cages of transgenic or non-transgenic lines derived from Russet Burbank or Red Rascal, respectively. The plants in the cages were examined for mines 2 weeks later and once a week thereafter. Leaves with mine areas of 4500 mm2 were collected and taken to the laboratory to recover larvae before they pupated. The field cages were examined until no mined leaves were found. Thirty eggs were placed in each mesh sleeve on 8 February 2000. Based on development of tuber moth in the cages, the mesh sleeves and shoots covering
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non-transgenic foliage were removed on 24 March, while those on transgenic lines were removed on 7 April 2000, when no more mines had been found in the cages. The sleeves and shoots were taken to the laboratory where the number of pupae collected from each sleeve was recorded. 2.3. Foliage surveys Surveys for PTM larval mines (occupied and unoccupied mines) involved examining the foliage of four (1999/2000) or five (2000/2001) randomly chosen plants within each plot once a week between 24 February and 31 March 2000, and 5 March and 3 April 2001 (inclusive). On the final survey date in the second field trial (3 April 2001) the foliage of the Russet Burbank transgenic and non-transgenic plants had senesced, so only the transgenic and non-transgenic Red Rascal plants were examined. The surface area of each mine was recorded using five categories: o25, 25–100, 100–200, 200–500 and 4500 mm2. A pheromone trap (DeSIRes, HortResearch, Auckland, New Zealand) was set up in the centre of each field trial to capture male moths, providing an indication of P. operculella activity. Moths were counted every 1–2 weeks. Sticky bases were replaced at least once each month or when more than 20 moths were caught. Pheromone caps were replaced once each month. In 1999/2000, for each survey date all mined leaves from a given potato line were placed in plastic bags and taken to a laboratory. Larvae recovered from the foliage were reared to pupation on field-grown foliage of the potato line from which they had been collected. The larvae were reared individually in 50 ml plastic specimen containers containing a 100 mm2 piece of filter paper (Whatmans, No. 1) and an excised leaflet (30–60 mm long) collected from the appropriate potato line. Leaflets were replaced every 2 days. The number of P. operculella that pupated was recorded. 2.4. Harvest and tuber survey Tubers were harvested on 3–5 May 2000 and 24–25 April 2001 for the first and second field trial, respectively. The tubers from each row within a plot were placed in paper bags and stored at 11 1C. The number of plants per row was recorded (some plants did not establish) and tubers from plants within the same row of a plot were collected into paper bags. Yield analysis involved counting and weighing tubers greater than 50 g for each row within a plot. The yield from the three inner rows of each replicate plot only was used for the analysis. In the second field trial, some of the transgenic and non-transgenic plants belonging to the Russet Burbank cultivar exhibited obvious signs of viral
infection. Consequently, tubers from these plants were eliminated from the yield analysis. Five months after harvesting the first field trial, tubers were examined for P. operculella larval mines. For each plot, 50 tubers (25 tubers from each of two inner rows) were examined and scored for the presence or absence of mines. At the time of yield analysis of the second field trial, all of the tubers from two inner rows from each plot were examined for the presence of mines. The presence or absence of mines was recorded within the categories of green (exposed to sunlight) or non-green tubers for two size classes, tubers less than or greater than 50 g.
2.5. Statistical analysis The total number of mines of all sizes, and the total numbers 4200 mm2, were calculated for each plot for each sample date. The totals were analysed using a loglinear model (McCullagh and Nelder, 1989). These two data sets were used to explore changes over sample times. In addition, the total number of mines over all survey dates in each size class was calculated for each plot. These were analysed similarly, to assess the overall distribution of mine sizes found for each line. Comparisons between lines, dates or sizes were made as part of the analyses using analysis of deviance, in a similar way to that used for analysis of variance. For presentation, the estimated mean numbers per plot from the analyses were converted into mean numbers of mines per plant. Data for all potato lines, including some not reported in the current study, were used in the analyses of yield data. Only data from the middle three rows of each plot from the 2000/2001 field trial were used. Individual tuber weights were calculated for data from each row as weight per row/number per row. Weight and numbers of tubers per plant were also calculated by dividing the total for each row by the number of plants. There was a strong trend across the rows as well as between the blocks of plots. Thus, a straightforward analysis of variance for a randomised block experiment may give biased results. Therefore, a mixed model analysis was used, fitted with residual maximum likelihood methods (REML) (Gilmour et al., 1997). This included random effects adjustments of plot to plot and rows within plot differences, as well as fixed effects for differences between the lines. Comparisons between the transgenic lines and the control for the same cultivar were made using contrasts included in the analysis. All analyses of larval mine and yield data were carried out with GenStat (GenStat Committee, 2002), and a level of 5% was used throughout to determine significance. All analyses included transgenic and nontransgenic lines from other cultivars not relevant to the current study.
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3. Results 3.1. Field cage experiments No P. operculella larvae or pupae were recovered from cages or mesh sleeves enclosing plants or shoots of the transgenic line D61, derived from Russet Burbank (Table 1). One pupa out of 240 eggs and 23 larvae out of 800 eggs were recovered from foliage of transgenic line D64 (derived from Russet Burbank) that had been covered with mesh sleeves or cages, respectively. A high percentage of larvae were recovered (72%) from the 800 eggs placed on non-transgenic plants of Russet Burbank, but only a low percentage of pupae (18%) were recovered from the 240 eggs placed in mesh sleeves. Likewise, in the cages covering non-transgenic plants of Red Rascal, a high percentage (93%) of larvae were recovered from 1000 eggs placed in the cages, but only around 30% of the eggs placed in mesh sleeves resulted in recovered pupae. No larvae were found on plants from cages enclosing plants of line D02, derived from Red Rascal, but three pupae out of 240 eggs were recovered from mesh sleeves. Three larvae were found in mines from foliage of transgenic plants from line D53 enclosed in cages, but no pupae were recovered from mesh sleeves.
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In 1999/2000, more mines in total and a greater number of large (4200 mm2) mines were found on nontransgenic foliage than on transgenic foliage (F ¼ 120 and 164, respectively; df ¼ 1, 90; Po0:001) (Fig. 2). This pattern was even stronger in the 2000/2001 trial (F ¼ 487 and 4008, respectively; df ¼ 1, 135; Po0:001) (Fig. 2). During the survey period of each trial the changes between sample dates varied significantly between the lines (Po0:05 for the date by line interactions) for the total number of mines. The total number of mines recorded on non-transgenic plants increased (1999/2000) or remained high (2000/2001) throughout the survey period whereas the number of mines recorded on transgenic foliage generally remained low (1999/2000) or decreased (2000/2001) (Fig. 2). There were some smaller differences (Po0:01) between the transgenic lines in both trials, with slightly more mines per plant for D64 than for D61 in both trials, and slightly more mines for D53 than for D02 in 2000/2001.
3.2. Foliage survey More male P. operculella moths were caught in the pheromone trap during the survey period in the second field trial (2000/2001), than during the survey period of the first field trial (1999/2000) (Fig. 1). This is reflected in the numbers of mines, where fewer mines per plant were recorded on foliage of non-transgenic plants throughout the survey period of the first field trial (1.5–10 mines per plant) compared with the second field trial (6–15 mines per plant) (Fig. 2).
Fig. 1. Average daily number of male P. operculella caught in a pheromone trap for 1999/2000 and 2000/2001 field trials.
Table 1 Number of P. operculella from the first field trial (1999/2000); recovered as larvae or pupae from artificially infested cages or mesh sleeves, respectively, and number of larvae collected from naturally infested transgenic or control (non-transgenic) foliage, and subsequent number that pupated or generated parasitoids Potato cultivar
Line
Artificially infested
Naturally infested
No. of larvaea
No. of pupaeb
No. of larvae
No. of pupae
No. of parasitoidsc
Russet Burbank
Control D61 D64
576 0 23
43 0 1
153 8 13
37 1 1
98 1 4
Red Rascal
Control D02 D53
930 0 3
73 3 0
116 10 14
27 0 0
73 3 1
a
Number of larvae recovered from cages out of 800 or 1000 eggs for Russet Burbank or Red Rascal lines, respectively. For more details see Section
2. b c
Number of pupae recovered from mesh sleeves from a total of 240 eggs for all lines. For more details see Section 2. Most of the parasitoids to emerge were Apanteles subandinus Blanchard, the remainder were Diadegma spp.
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Fig. 2. Mean numbers per plant of P. operculella larval mines (occupied and unoccupied) recorded from foliage examined in the 1999/2000 and 2000/ 2001 field trials on different sample dates for Russet Burbank non-transgenic plants (K) and transgenic lines, D64 (m), D61 (’); and Red Rascal non-transgenic plants (~) and transgenic lines, D02 (.), D53 ( ). Closed symbols are for all mine sizes, open symbols are for mine sizes 4200 mm2.
However, these differences were markedly smaller than the difference between the transgenic lines and the controls. Summarised over all survey dates, the distribution of mine sizes varied significantly between the transgenic and non-transgenic lines (Po0:05) in both trials. More than half the mines recorded on transgenic foliage in 1999/2000 were less than 100 mm2 whereas the majority of mines recorded on the non-transgenic plants were 4200 mm2 (Fig. 3). This effect was even stronger in 2000/2001. On average, less than one mine per plant was greater than 200 mm2 for any of the transgenic lines in either trial, whereas there were up to seven mines per plant of this size for the control lines in 1999/2000, and more than 30 in 2000/ 2001. More larvae were collected from non-transgenic foliage (269 larvae) than from transgenic foliage (45 larvae) throughout the survey period in the first field trial (Table 1). Of the larvae recovered from transgenic foliage, two pupated (one from each of the transgenic lines derived from Russet Burbank) and nine were parasitised by Apanteles subandinus Blanchard or Diadegma spp. Approximately 25% of the larvae collected from the non-transgenic foliage of both cultivars pupated, and over 60% were parasitised, mostly by A. subandinus, but a few were parasitised by Diadegma spp.
3.3. Harvest and tuber survey The tuber weight per plant and number of tubers produced per plant of each transgenic line did not differ significantly from their respective parent cultivars (P40:05) (Table 2). However, the individual tuber weights of transgenic lines D61 and D64 were slightly lower (90% or 82%, respectively) than for the control plants. The transgenic lines derived from Red Rascal did not differ from the control plants (P40:1) for any of the measurements presented in Table 2. Four transgenic tubers of 800 (0.5%) examined from the first field trial (1999/2000) had at least one larval mine. Thirty-seven non-transgenic tubers of 400 examined (9.3%) had mine damage. In the second field trial (2000/2001), three transgenic tubers out of 5307 examined (0.06%) had at least one P. operculella mine while 695 control (non-transgenic) tubers of 2777 examined (25%) had mine damage (Table 3). The three mined transgenic tubers were in the ‘green’ category (exposed to sunlight in the field). Of the infested control tubers, a higher percentage of green tubers was mined compared with non-green tubers and this was more apparent in Russet Burbank than in Red Rascal (Table 3). Tuber size had less effect than the extent of greening on mining, with a slightly greater percentage of small (o50 g) tubers being mined than large (X50 g) tubers.
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Fig. 3. Mean number of P. operculella larval mines recorded per plant for each of the mine surface area size classes (mm2) summarised over survey dates, in the 1999/2000 and 2000/2001 field trials for Russet Burbank derived transgenic lines (D61, D64) and non-transgenic line (RB) and Red Rascal derived transgenic lines (D02, D53) and non-transgenic line (RR).
4. Discussion The cry1Ac-transgenic potato plants tested in the current study produced similar yields to those of nontransgenic parent cultivars (Table 2). They were also more resistant to P. operculella, particularly D61, derived from the Russet Burbank cultivar and the two
lines derived from the Red Rascal cultivar, D02 and D53 (Figs. 2 and 3; Tables 1 and 3). The results of this study further establish the value of using genetic engineering to develop transgenic potato plants with improved resistance to larvae of P. operculella. Efforts to breed potato cultivars for resistance to P. operculella have had limited success (Arnone et al., 1998). Within a
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Table 2 Yield of transgenic and control (non-transgenic) potato lines from the second field trial (2000/2001) Potato cultivar
Line
No. of tubers per row
Tuber weight per plant (kg)
No. of tubers per plant
Individual tuber weight (g)a
Russet Burbank
Control D61 D64
57 60 56
0.682 0.621 0.594
6.8 6.9 7.3
100 90 82
Red Rascal
Control D02 D53
56 63 59 8
0.635 0.685 0.647 0.104
5.6 6.3 5.9 0.8
113 110 109 9
Mean LSDb a
These values were calculated from individual row data and include adjustments for spatial trends so are not necessarily equal to the weight per plant/number per plant. b Mean LSD for all comparisons between lines and the control for the cultivar, averaged over the two cultivars. LSDs are approximate (df ¼ 35), as exact degrees of freedom cannot be obtained. Table 3 Percentages of tubers for transgenic and control (non-transgenic) lines from the second field trial (2000/2001) with P. operculella larval mine damage and percentage of green and non-green tubers, less than and greater than 50 g, with mine damage Potato cultivar
Line
n
% Mined tubers
% Green, mined tubers
% Mined tubers
o50 g (n)
450 g (n)
o50 g (n)
450 g (n)
Russet Burbank
Control D61 D64
1272 1267 1100
27 0.08 0.18
87 (82) 0 (123) 3.7 (54)
75 (28) 1 (55) 0 (33)
24 (509) 0 (435) 0 (360)
20 (652) 0 (654) 0 (653)
Red Rascal
Control D02 D53
1506 1467 1473
23 0 0
49 (208) 0 (133) 0 (172)
31 (45) 0 (34) 0 (52)
20 (615) 0 (563) 0 (614)
17 (638) 0 (737) 0 (635)
short period we have developed potatoes transgenic for a cry gene with proven resistance under relatively strong natural infestation pressure (average of up to 15 mines per non-transgenic plant) from a field population and high artificial infestation pressure from a laboratory colony of P. operculella. The current field trials of cry1Ac-transgenic potato plants produced results comparable to those obtained with cry1Ab or cry5 transgenic potato plants resistant to P. operculella (Can˜edo et al., 1999; Douches et al., 2002). In greenhouse experiments, Jansens et al. (1995) reported similar results of P. operculella mine damage on foliage of cry1Ab-transgenic potato plants compared with controls. They recorded only a few mines greater than 80 mm2 and no mines 4320 mm2 on cry1Abtransgenic foliage. In field trials on cry1Ab-transgenic potato lines, no P. operculella larvae were found in foliage or tubers, but high numbers were recorded on the non-transgenic controls (Can˜edo et al., 1999). Likewise, Douches et al. (2002) reported that feeding damage on cry5 transgenic potato tubers was negligible (between 0% and 2%), compared with on nontransgenic tubers (6–27%). The only transgenic tubers with mine damage in the 2000/2001 field trial of the present study were green, indicating exposure to light and their presence therefore on or near the soil surface,
which most likely increased their exposure to P. operculella (Foot, 1979). Under existing cultural practices, such as the moulding or mounding of soil around potato plants, exposure of tubers to P. operculella would be minimised. In the present study, the mean number of mines recorded on transgenic plants was lower than that on non-transgenic plants throughout the survey periods (Fig. 2). Although some larvae were present on these cry1Ac9-transgenic plants, most of the mines found were less than 200 mm2 (Fig. 3). This would suggest that even though larvae were hatching from eggs and feeding on foliage of these transgenic lines, few larvae were able to develop through to pupae since the size of the mine reflects the size of the mining larva (Jansens et al., 1995). In the current work, the low number of larvae or pupae from transgenic foliage in the artificially infested cage experiments or from naturally infested foliage (Table 1) further illustrates the inability of P. operculella larvae to survive on this foliage. The high level of resistance and comparable yield of cry1Ac-transgenic potato lines D61 (derived from Russet Burbank), D02, or D53 (derived from Red Rascal) suggests that these lines could be deployed using a high dose/refuge strategy. Shelton et al. (2000) conducted an experiment on the optimal refuge size
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for Bt-broccoli plants resistant to Plutella xylostella (L.) and found that a separate refuge, 20% the size of the transgenic field, was the most effective at preventing an increase in frequency of resistance alleles in the insect pest population. However, the size of a refuge will be determined by the extent of acceptable crop loss by the grower. For example, the US Environmental Protection Agency recommended that growers of Bt cotton use refuges of either 5% the size of the total crop, separate to or planted within the Bt crop, or a separate refuge 20% the size of the total crop that is treated with non-Bt insecticide, for the 2001 growing season (US EPA, 2000). Shelton et al. (2000) showed that spraying the refuge reduced its potential to dilute the frequency of resistance alleles in the insect population. Where the target insect can move between plants during the larval stage, separate refuges are better than seed mixtures at delaying resistance (Shelton et al., 2002). A high dose/refuge strategy, when incorporated into an integrated pest management programme, is likely to be appropriate for managing P. operculella when using transgenic potatoes similar to those used in the present study. To help determine the size and placement of the refuge, knowledge of the movement of larvae within and between plants, and of adult dispersal behaviour is required. Cameron et al. (2002) found that larvae could form mines on leaflets from two different plants when these leaflets are in contact with one another. They also established that 15% of P. operculella moths dispersed from a release site, moving up to 30 m into new crops with a small percentage dispersing up to 250 m away. Cameron et al. (2002) concluded that a refuge should be placed immediately adjacent to transgenic crops, but further information on the proportion of moths moving between crops is needed to determine the size of the refuge. The potential of P. operculella to develop resistance to such transgenic plants also requires further research. The potential effect of a Bt toxin produced by a transgenic plant on natural enemies can be via a direct toxicological response or indirectly through a reduction in the prey or host population. Davidson et al. (2002a) observed that P. operculella larvae could survive up to 10 days on the transgenic foliage; it could have therefore been possible for parasitoids to emerge before their host died. This was found not to be the case. Further research is required before any conclusions can be made regarding the lower number of parasitoids to emerge from P. operculella larvae reared on transgenic foliage than on non-transgenic foliage observed in the present study.
Acknowledgments We thank Andrew MacLachlan for assistance with statistical analysis, Jill Reader for help with maintaining
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plants in the field, and Crop & Food Research staff for assisting with planting and harvesting the field trial. A Technology for Industry Fellowship from the Foundation for Research, Science and Technology with Lincoln University and Alex McDonald (Merchants) Ltd. provided financial support for M.M. Davidson.
References Arnone, S., Musmeci, S., Bacchetta, L., Cordischi, N., Pucci, E., Cristofaro, M., Sonnino, A., 1998. Research in Solanum spp. of sources of resistance to the potato tuber moth, Phthorimaea operculella (Zeller). Potato Res. 41, 39–49. Cameron, P.J., Walker, G.P., Penny, G.M., Wigley, P.J., 2002. Movement of potato tuber moth Phthorimaea operculella (Lepidoptera: Gelechiidae) within and between crops, and some comparisons with diamondback moth Plutella xylostella (Lepidoptera: Plutellidae). Environ. Entomol. 31, 65–75. Can˜edo, V., Benavides, J., Golmirzaie, A., Cisneros, F., Ghislain, M., Lagnaoui, A., 1999. Assessing Bt-transformed potatoes for potato tuber moth, Phthorimaea operculella (Zeller) management. CIP Program Report 1997–98, Lima, Peru, pp. 161–170. Coll, M., Gavish, S., Dori, I., 2000. Population biology of the potato tuber moth, Phthorimaea operculella (Lepidoptera: Gelechiidae), in two potato cropping systems in Israel. Bull. Entomol. Res. 90, 309–315. Davidson, M.M., Jacobs, J.M.E., Reader, J.K., Butler, R.C., Frater, C.M., Markwick, N.P., Wratten, S.D., Conner, A.J., 2002a. Development and evaluation of potatoes transgenic for a cry1Ac9 gene conferring resistance to potato tuber moth. J. Am. Soc. Hortic. Sci. 127, 590–596. Davidson, M.M., Takla, M.F.G., Reader, J.K., Butler, R.C., Wratten, S.D., Conner, A.J., 2002b. Evaluation of field grown potato lines transgenic for a cry1Ac9 gene conferring resistance to potato tuber moth. N. Z. Plant Prot. 55, 405–410. Davidson, M.M., Takla, M.F.G., Jacobs, J.M.E., Butler, R.C., Wratten, S.D., Conner, A.J., 2004. Transformation of potato cultivars with a cry1Ac9 gene confers resistance to potato tuber moth. N. Z. J. Crop Hortic. Sci. 32, 39–50. Douches, D.S., Westedt, A.L., Zarka, K.A., Schroeter, B., Grafius, E.J., 1998. Potato transformation to combine natural and engineered resistance for controlling tuber moth. HortScience 33, 1053–1056. Douches, D.S., Li, W., Zarka, K.A., Coombs, J., Pett, W., Grafius, E.J., El-Nasr, T., 2002. Development of Bt-cry5 insect-resistant potato lines ‘Spunta-G2’ and ‘Spunta-G3’. HortScience 37, 1103–1107. Foot, M.A., 1979. Bionomics of the potato tuber moth, Phthorimaea operculella (Lepidoptera: Gelechiidae), at Pukekohoe. N. Z. J. Zool. 6, 623–636. GenStat Committee, 2002. GenStat Release 6.1, Reference Manual, Parts 1–3. VSN International. Gilmour, A.R., Cullis, B.R., Verbyla, A.P., 1997. Accounting for natural and extraneous variation in the analysis of field experiments. J. Agric. Biol. Environ. Stat. 2, 269–293. Hanafi, A., 1999. Integrated pest management of the potato tuber moth in field and storage. Potato Res. 42, 373–380. Jansens, S., Cornelissen, M., de Clercq, R., Reynaerts, A., Peferoen, M., 1995. Phthorimaea operculella (Lepidoptera: Gelechiidae) resistance in potato by expression of the Bacillus thuringiensis CryIA(b) insecticidal crystal protein. J. Econ. Entomol. 88, 1469–1476.
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Lagnaoui, A., Ben Salah, H., El-Bedewy, R., 1996. Integrated management to control potato moth in North Africa and the Middle East. CIP Circular, June ’96, pp. 10–15. Li, W.B., Zarka, K.A., Douches, D.S., Coombs, J.J., Pett, W.L., Grafius, E.J., 1999. Coexpression of potato PVY coat protein and cryV-Bt genes in potato. J. Am. Soc. Hortic. Sci. 124, 218–223. McCullagh, P., Nelder, J.A., 1989. Generalised Linear Models. Chapman & Hall, London. Radcliff, E.B., 1982. Insect pests of potato. Ann. Rev. Entomol. 27, 173–204.
Shelton, A.M., Tang, J.D., Roush, R.T., Metz, T.D., Earle, E.D., 2000. Field tests on managing resistance to Bt-engineered plants. Nat. Biotechnol. 18, 339–342. Shelton, A.M., Zhao, J.-Z., Roush, R.T., 2002. Economic, ecological, food safety, and social consequences of the deployment of Bt transgenic plants. Ann. Rev. Entomol. 47, 845–881. US EPA, 2000. Bt cotton refuge requirements for the 2001 growing season. http://www.epa.gov/oppbppd1/biopesticides/pips/bt_cotton_refuge_2001.htm.
Crop Protection 25 (2006) 216–224 www.elsevier.com/locate/cropro
Field evaluation of potato plants transgenic for a cry1Ac gene conferring resistance to potato tuber moth, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae) M.M. Davidsona,, R.C. Butlera, S.D. Wrattenb, A.J. Connera,b a
New Zealand Institute for Crop & Food Research Limited, Private Bag 4704, Christchurch, New Zealand National Centre for Advanced Bio-Protection Technologies, Lincoln University, P.O. Box 84, Canterbury, New Zealand
b
Received 28 July 2004; accepted 20 April 2005
Abstract Cry1Ac-transgenic potato plants derived from Russet Burbank and Red Rascal cultivars were evaluated in field trials at Lincoln, Canterbury, New Zealand. Foliage of transgenic plants had fewer potato tuber moth (Phthorimaea operculella (Zeller)) mines, of which the majority were o200 mm2, than the non-transgenic control plants. Of the 800 transgenic tubers examined 5 months after harvesting the first field trial (1999/2000), 0.5% had potato tuber moth mine damage compared with 9% of non-transgenic tubers. After the second field trial (2000/2001) 0.06% of 5307 transgenic tubers had mine damage compared with damage to 25% of non-transgenic tubers. Mined foliage was collected during the first field trial, resulting in a total of 45 larvae from transgenic foliage and 269 from nontransgenic foliage. Two pupae and nine parasitoids emerged from larvae collected from and reared on transgenic foliage. Between 86% and 88% (Red Rascal or Russet Burbank cultivar, respectively) of larvae collected and reared on non-transgenic foliage either pupated or were parasitised. In the second field trial, three of the four transgenic lines evaluated produced yields comparable with their nontransgenic parent cultivar. These results suggest transgenic potato plants resistant to potato tuber moth could augment integrated pest management programmes. However, the potential of the target pest to develop resistance to such plants and effects on non-target insects must be evaluated before such plants can be recommended for potato tuber moth management. r 2005 Elsevier Ltd. All rights reserved. Keywords: Phthorimaea operculella; Cry1Ac; Resistance; Transgenic potato; Field trials
1. Introduction The potato tuber moth, Phthorimaea operculella (Zeller), is considered one of the most economically damaging pests of potato (Solanum tuberosum L.) crops in regions with warm temperate to tropical climates (Radcliff, 1982). Larvae mine into leaves, stems and tubers, and the latter damage results in rejection of tubers by growers and potato processors. Further, the level of P. operculella infestation in the foliage can influence infestation levels in tubers (Lagnaoui et al., 1996; Coll et al., 2000). Integration of management Corresponding author. Tel.: +64 3 325 6400; fax: +64 3 325 2074.
E-mail address: [email protected] (M.M. Davidson). 0261-2194/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2005.04.010
strategies (spray insecticides, cultural methods, biological control) can help to manage this pest in the field and storage (Hanafi, 1999). However, the development of resistant potato cultivars could increase the efficacy of cultural and biological methods and reduce the use of insecticides (Arnone et al., 1998). In New Zealand, populations of P. operculella larvae peak between February and April. P. operculella are particularly prevalent in hot, dry summers (Foot, 1979). They infest potato foliage before tubers are colonised, but as the foliage senesces the larvae become proportionally more common in the tubers, initially in green, exposed tubers (Foot, 1979). Soil moisture and tuber depth affect the ability of larvae to infest tubers (Foot, 1979). Management methods emphasise the use of cultural
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practices such as moulding, ‘seed’ depth, timing of planting and irrigation (Hanafi, 1999) to reduce tuber exposure to or increase mortality of P. operculella. However, P. operculella population can still reach levels where it is necessary to apply insecticides (Hanafi, 1999). Potato cultivars with resistance to P. operculella could eliminate the need for, or at least reduce the number of, insecticide applications to manage this pest. However, attempts to develop such cultivars using traditional breeding methods have not been successful (Arnone et al., 1998). Genetic engineering has become a viable method for developing insect-resistant transgenic plants. Transgenic potato plants resistant to P. operculella have been developed predominantly through the transfer of a single cry gene derived from Bacillus thuringiensis Berliner (Bt). The cry genes transferred into potato cultivars include cry1Ab (Jansens et al., 1995; Can˜edo et al., 1999), cry1Ac (Davidson et al., 2002a, 2004) and cry5 (Douches et al., 1998, 2002; Li et al., 1999). All of these cry genes are specific in their activity to Lepidoptera, except cry5, which is also active against Coleoptera and is therefore likely to affect a wider range of non-target insects. Potato plants transgenic for the cry1Ac gene provide a similar level of resistance to potato tuber moth as cry1Ab-transgenic plants (Jansens et al., 1995; Can˜edo et al., 1999), even though they exhibited lower Cry protein expression (Davidson et al., 2002a). We have focused our attention on the use of the cry1Ac gene for potato tuber moth resistance since the lower amount of Cry protein in transgenic potatoes to effect the desired trait may be more acceptable to consumers. We have developed potato plants transgenic for a cry1Ac gene under the transcriptional control of a CaMV35S promoter using Agrobacterium-mediated transformation (Davidson et al., 2002a, 2004). Greenhouse trials identified transgenic lines that appeared phenotypically normal relative to parental cultivars and that inhibited the growth of P. operculella larvae. These lines were, in turn, evaluated in small-scale field trials to screen for transgenic lines with the highest resistance and comparable yield to their non-transgenic parent cultivars (Davidson et al., 2002b). The present study reports on the performance of selected lines in scaled-up field trials using natural infestation by a wild population and artificial infestation by a laboratory colony of P. operculella. Potato tuber yield was assessed relative to non-transgenic parent cultivars.
2. Materials and methods 2.1. Field trial and design Transgenic lines derived from potato cultivars Russet Burbank and Red Rascal that had been selected from
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previous small-scale field trials (Davidson et al., 2002b) were evaluated in the field over the summers of 1999/ 2000 and 2000/2001 at Lincoln, Canterbury, New Zealand (Environmental Risk Management Authority approval GMF98008). In 1999, the field trial was established from transgenic and non-transgenic plants micro-propagated in tissue culture. Tissue culture plantlets were transplanted into seedling trays, grown in a containment greenhouse for 3–4 weeks, and then moved to a screened enclosure to harden off for 2–3 weeks before planting in the field on 1 December 1999. In the following year, tubers from the previous year’s field trial of the transgenic and non-transgenic potato plants were planted in the field on 7 December 2000. Four replicate plots in 1999 and six plots in 2000, each containing 50 plants of each of the four transgenic lines and two non-transgenic parent cultivars (experimental controls), were planted in a randomised block layout in the field. There were five rows (0.76 m apart) per plot, each with 10 plants spaced 0.3 m apart within a row. Plots were separated by 1 m within a row. The experimental plots were completely surrounded by plots from trials of other transgenic potatoes or three buffer rows of non-transgenic potato plants (breeding line 2390, Urenika V431-1) to prevent edge effects during the trial. Weeding was done by hand and the potato plants were subjected to overhead irrigation as required. Rows were mounded once, 4 weeks after planting, at a shallower depth than normal (approximately 20 cm) to encourage tuber infestation by P. operculella larvae, to allow appropriate assessment of tuber resistance. 2.2. Field cage experiments In the 1999/2000 field trial, cages (120 60 50 cm3) or mesh sleeves (35 cm long 15 cm diameter) were placed within the plots in the first week of February. A cage covered several plants, while a mesh sleeve enclosed a single shoot of a plant. A cage was positioned over 3–4 plants of an outer row for two of the four plots of each transgenic or non-transgenic line (12 cages in total). Two mesh sleeves were placed on randomly selected shoots of plants in the outer rows of each plot of transgenic or non-transgenic line (48 sleeves in total). On 8 February 2000, 400 or 500 eggs laid on filter paper by laboratory-reared females were placed on plants within cages of transgenic or non-transgenic lines derived from Russet Burbank or Red Rascal, respectively. The plants in the cages were examined for mines 2 weeks later and once a week thereafter. Leaves with mine areas of 4500 mm2 were collected and taken to the laboratory to recover larvae before they pupated. The field cages were examined until no mined leaves were found. Thirty eggs were placed in each mesh sleeve on 8 February 2000. Based on development of tuber moth in the cages, the mesh sleeves and shoots covering
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non-transgenic foliage were removed on 24 March, while those on transgenic lines were removed on 7 April 2000, when no more mines had been found in the cages. The sleeves and shoots were taken to the laboratory where the number of pupae collected from each sleeve was recorded. 2.3. Foliage surveys Surveys for PTM larval mines (occupied and unoccupied mines) involved examining the foliage of four (1999/2000) or five (2000/2001) randomly chosen plants within each plot once a week between 24 February and 31 March 2000, and 5 March and 3 April 2001 (inclusive). On the final survey date in the second field trial (3 April 2001) the foliage of the Russet Burbank transgenic and non-transgenic plants had senesced, so only the transgenic and non-transgenic Red Rascal plants were examined. The surface area of each mine was recorded using five categories: o25, 25–100, 100–200, 200–500 and 4500 mm2. A pheromone trap (DeSIRes, HortResearch, Auckland, New Zealand) was set up in the centre of each field trial to capture male moths, providing an indication of P. operculella activity. Moths were counted every 1–2 weeks. Sticky bases were replaced at least once each month or when more than 20 moths were caught. Pheromone caps were replaced once each month. In 1999/2000, for each survey date all mined leaves from a given potato line were placed in plastic bags and taken to a laboratory. Larvae recovered from the foliage were reared to pupation on field-grown foliage of the potato line from which they had been collected. The larvae were reared individually in 50 ml plastic specimen containers containing a 100 mm2 piece of filter paper (Whatmans, No. 1) and an excised leaflet (30–60 mm long) collected from the appropriate potato line. Leaflets were replaced every 2 days. The number of P. operculella that pupated was recorded. 2.4. Harvest and tuber survey Tubers were harvested on 3–5 May 2000 and 24–25 April 2001 for the first and second field trial, respectively. The tubers from each row within a plot were placed in paper bags and stored at 11 1C. The number of plants per row was recorded (some plants did not establish) and tubers from plants within the same row of a plot were collected into paper bags. Yield analysis involved counting and weighing tubers greater than 50 g for each row within a plot. The yield from the three inner rows of each replicate plot only was used for the analysis. In the second field trial, some of the transgenic and non-transgenic plants belonging to the Russet Burbank cultivar exhibited obvious signs of viral
infection. Consequently, tubers from these plants were eliminated from the yield analysis. Five months after harvesting the first field trial, tubers were examined for P. operculella larval mines. For each plot, 50 tubers (25 tubers from each of two inner rows) were examined and scored for the presence or absence of mines. At the time of yield analysis of the second field trial, all of the tubers from two inner rows from each plot were examined for the presence of mines. The presence or absence of mines was recorded within the categories of green (exposed to sunlight) or non-green tubers for two size classes, tubers less than or greater than 50 g.
2.5. Statistical analysis The total number of mines of all sizes, and the total numbers 4200 mm2, were calculated for each plot for each sample date. The totals were analysed using a loglinear model (McCullagh and Nelder, 1989). These two data sets were used to explore changes over sample times. In addition, the total number of mines over all survey dates in each size class was calculated for each plot. These were analysed similarly, to assess the overall distribution of mine sizes found for each line. Comparisons between lines, dates or sizes were made as part of the analyses using analysis of deviance, in a similar way to that used for analysis of variance. For presentation, the estimated mean numbers per plot from the analyses were converted into mean numbers of mines per plant. Data for all potato lines, including some not reported in the current study, were used in the analyses of yield data. Only data from the middle three rows of each plot from the 2000/2001 field trial were used. Individual tuber weights were calculated for data from each row as weight per row/number per row. Weight and numbers of tubers per plant were also calculated by dividing the total for each row by the number of plants. There was a strong trend across the rows as well as between the blocks of plots. Thus, a straightforward analysis of variance for a randomised block experiment may give biased results. Therefore, a mixed model analysis was used, fitted with residual maximum likelihood methods (REML) (Gilmour et al., 1997). This included random effects adjustments of plot to plot and rows within plot differences, as well as fixed effects for differences between the lines. Comparisons between the transgenic lines and the control for the same cultivar were made using contrasts included in the analysis. All analyses of larval mine and yield data were carried out with GenStat (GenStat Committee, 2002), and a level of 5% was used throughout to determine significance. All analyses included transgenic and nontransgenic lines from other cultivars not relevant to the current study.
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3. Results 3.1. Field cage experiments No P. operculella larvae or pupae were recovered from cages or mesh sleeves enclosing plants or shoots of the transgenic line D61, derived from Russet Burbank (Table 1). One pupa out of 240 eggs and 23 larvae out of 800 eggs were recovered from foliage of transgenic line D64 (derived from Russet Burbank) that had been covered with mesh sleeves or cages, respectively. A high percentage of larvae were recovered (72%) from the 800 eggs placed on non-transgenic plants of Russet Burbank, but only a low percentage of pupae (18%) were recovered from the 240 eggs placed in mesh sleeves. Likewise, in the cages covering non-transgenic plants of Red Rascal, a high percentage (93%) of larvae were recovered from 1000 eggs placed in the cages, but only around 30% of the eggs placed in mesh sleeves resulted in recovered pupae. No larvae were found on plants from cages enclosing plants of line D02, derived from Red Rascal, but three pupae out of 240 eggs were recovered from mesh sleeves. Three larvae were found in mines from foliage of transgenic plants from line D53 enclosed in cages, but no pupae were recovered from mesh sleeves.
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In 1999/2000, more mines in total and a greater number of large (4200 mm2) mines were found on nontransgenic foliage than on transgenic foliage (F ¼ 120 and 164, respectively; df ¼ 1, 90; Po0:001) (Fig. 2). This pattern was even stronger in the 2000/2001 trial (F ¼ 487 and 4008, respectively; df ¼ 1, 135; Po0:001) (Fig. 2). During the survey period of each trial the changes between sample dates varied significantly between the lines (Po0:05 for the date by line interactions) for the total number of mines. The total number of mines recorded on non-transgenic plants increased (1999/2000) or remained high (2000/2001) throughout the survey period whereas the number of mines recorded on transgenic foliage generally remained low (1999/2000) or decreased (2000/2001) (Fig. 2). There were some smaller differences (Po0:01) between the transgenic lines in both trials, with slightly more mines per plant for D64 than for D61 in both trials, and slightly more mines for D53 than for D02 in 2000/2001.
3.2. Foliage survey More male P. operculella moths were caught in the pheromone trap during the survey period in the second field trial (2000/2001), than during the survey period of the first field trial (1999/2000) (Fig. 1). This is reflected in the numbers of mines, where fewer mines per plant were recorded on foliage of non-transgenic plants throughout the survey period of the first field trial (1.5–10 mines per plant) compared with the second field trial (6–15 mines per plant) (Fig. 2).
Fig. 1. Average daily number of male P. operculella caught in a pheromone trap for 1999/2000 and 2000/2001 field trials.
Table 1 Number of P. operculella from the first field trial (1999/2000); recovered as larvae or pupae from artificially infested cages or mesh sleeves, respectively, and number of larvae collected from naturally infested transgenic or control (non-transgenic) foliage, and subsequent number that pupated or generated parasitoids Potato cultivar
Line
Artificially infested
Naturally infested
No. of larvaea
No. of pupaeb
No. of larvae
No. of pupae
No. of parasitoidsc
Russet Burbank
Control D61 D64
576 0 23
43 0 1
153 8 13
37 1 1
98 1 4
Red Rascal
Control D02 D53
930 0 3
73 3 0
116 10 14
27 0 0
73 3 1
a
Number of larvae recovered from cages out of 800 or 1000 eggs for Russet Burbank or Red Rascal lines, respectively. For more details see Section
2. b c
Number of pupae recovered from mesh sleeves from a total of 240 eggs for all lines. For more details see Section 2. Most of the parasitoids to emerge were Apanteles subandinus Blanchard, the remainder were Diadegma spp.
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Fig. 2. Mean numbers per plant of P. operculella larval mines (occupied and unoccupied) recorded from foliage examined in the 1999/2000 and 2000/ 2001 field trials on different sample dates for Russet Burbank non-transgenic plants (K) and transgenic lines, D64 (m), D61 (’); and Red Rascal non-transgenic plants (~) and transgenic lines, D02 (.), D53 ( ). Closed symbols are for all mine sizes, open symbols are for mine sizes 4200 mm2.
However, these differences were markedly smaller than the difference between the transgenic lines and the controls. Summarised over all survey dates, the distribution of mine sizes varied significantly between the transgenic and non-transgenic lines (Po0:05) in both trials. More than half the mines recorded on transgenic foliage in 1999/2000 were less than 100 mm2 whereas the majority of mines recorded on the non-transgenic plants were 4200 mm2 (Fig. 3). This effect was even stronger in 2000/2001. On average, less than one mine per plant was greater than 200 mm2 for any of the transgenic lines in either trial, whereas there were up to seven mines per plant of this size for the control lines in 1999/2000, and more than 30 in 2000/ 2001. More larvae were collected from non-transgenic foliage (269 larvae) than from transgenic foliage (45 larvae) throughout the survey period in the first field trial (Table 1). Of the larvae recovered from transgenic foliage, two pupated (one from each of the transgenic lines derived from Russet Burbank) and nine were parasitised by Apanteles subandinus Blanchard or Diadegma spp. Approximately 25% of the larvae collected from the non-transgenic foliage of both cultivars pupated, and over 60% were parasitised, mostly by A. subandinus, but a few were parasitised by Diadegma spp.
3.3. Harvest and tuber survey The tuber weight per plant and number of tubers produced per plant of each transgenic line did not differ significantly from their respective parent cultivars (P40:05) (Table 2). However, the individual tuber weights of transgenic lines D61 and D64 were slightly lower (90% or 82%, respectively) than for the control plants. The transgenic lines derived from Red Rascal did not differ from the control plants (P40:1) for any of the measurements presented in Table 2. Four transgenic tubers of 800 (0.5%) examined from the first field trial (1999/2000) had at least one larval mine. Thirty-seven non-transgenic tubers of 400 examined (9.3%) had mine damage. In the second field trial (2000/2001), three transgenic tubers out of 5307 examined (0.06%) had at least one P. operculella mine while 695 control (non-transgenic) tubers of 2777 examined (25%) had mine damage (Table 3). The three mined transgenic tubers were in the ‘green’ category (exposed to sunlight in the field). Of the infested control tubers, a higher percentage of green tubers was mined compared with non-green tubers and this was more apparent in Russet Burbank than in Red Rascal (Table 3). Tuber size had less effect than the extent of greening on mining, with a slightly greater percentage of small (o50 g) tubers being mined than large (X50 g) tubers.
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Fig. 3. Mean number of P. operculella larval mines recorded per plant for each of the mine surface area size classes (mm2) summarised over survey dates, in the 1999/2000 and 2000/2001 field trials for Russet Burbank derived transgenic lines (D61, D64) and non-transgenic line (RB) and Red Rascal derived transgenic lines (D02, D53) and non-transgenic line (RR).
4. Discussion The cry1Ac-transgenic potato plants tested in the current study produced similar yields to those of nontransgenic parent cultivars (Table 2). They were also more resistant to P. operculella, particularly D61, derived from the Russet Burbank cultivar and the two
lines derived from the Red Rascal cultivar, D02 and D53 (Figs. 2 and 3; Tables 1 and 3). The results of this study further establish the value of using genetic engineering to develop transgenic potato plants with improved resistance to larvae of P. operculella. Efforts to breed potato cultivars for resistance to P. operculella have had limited success (Arnone et al., 1998). Within a
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Table 2 Yield of transgenic and control (non-transgenic) potato lines from the second field trial (2000/2001) Potato cultivar
Line
No. of tubers per row
Tuber weight per plant (kg)
No. of tubers per plant
Individual tuber weight (g)a
Russet Burbank
Control D61 D64
57 60 56
0.682 0.621 0.594
6.8 6.9 7.3
100 90 82
Red Rascal
Control D02 D53
56 63 59 8
0.635 0.685 0.647 0.104
5.6 6.3 5.9 0.8
113 110 109 9
Mean LSDb a
These values were calculated from individual row data and include adjustments for spatial trends so are not necessarily equal to the weight per plant/number per plant. b Mean LSD for all comparisons between lines and the control for the cultivar, averaged over the two cultivars. LSDs are approximate (df ¼ 35), as exact degrees of freedom cannot be obtained. Table 3 Percentages of tubers for transgenic and control (non-transgenic) lines from the second field trial (2000/2001) with P. operculella larval mine damage and percentage of green and non-green tubers, less than and greater than 50 g, with mine damage Potato cultivar
Line
n
% Mined tubers
% Green, mined tubers
% Mined tubers
o50 g (n)
450 g (n)
o50 g (n)
450 g (n)
Russet Burbank
Control D61 D64
1272 1267 1100
27 0.08 0.18
87 (82) 0 (123) 3.7 (54)
75 (28) 1 (55) 0 (33)
24 (509) 0 (435) 0 (360)
20 (652) 0 (654) 0 (653)
Red Rascal
Control D02 D53
1506 1467 1473
23 0 0
49 (208) 0 (133) 0 (172)
31 (45) 0 (34) 0 (52)
20 (615) 0 (563) 0 (614)
17 (638) 0 (737) 0 (635)
short period we have developed potatoes transgenic for a cry gene with proven resistance under relatively strong natural infestation pressure (average of up to 15 mines per non-transgenic plant) from a field population and high artificial infestation pressure from a laboratory colony of P. operculella. The current field trials of cry1Ac-transgenic potato plants produced results comparable to those obtained with cry1Ab or cry5 transgenic potato plants resistant to P. operculella (Can˜edo et al., 1999; Douches et al., 2002). In greenhouse experiments, Jansens et al. (1995) reported similar results of P. operculella mine damage on foliage of cry1Ab-transgenic potato plants compared with controls. They recorded only a few mines greater than 80 mm2 and no mines 4320 mm2 on cry1Abtransgenic foliage. In field trials on cry1Ab-transgenic potato lines, no P. operculella larvae were found in foliage or tubers, but high numbers were recorded on the non-transgenic controls (Can˜edo et al., 1999). Likewise, Douches et al. (2002) reported that feeding damage on cry5 transgenic potato tubers was negligible (between 0% and 2%), compared with on nontransgenic tubers (6–27%). The only transgenic tubers with mine damage in the 2000/2001 field trial of the present study were green, indicating exposure to light and their presence therefore on or near the soil surface,
which most likely increased their exposure to P. operculella (Foot, 1979). Under existing cultural practices, such as the moulding or mounding of soil around potato plants, exposure of tubers to P. operculella would be minimised. In the present study, the mean number of mines recorded on transgenic plants was lower than that on non-transgenic plants throughout the survey periods (Fig. 2). Although some larvae were present on these cry1Ac9-transgenic plants, most of the mines found were less than 200 mm2 (Fig. 3). This would suggest that even though larvae were hatching from eggs and feeding on foliage of these transgenic lines, few larvae were able to develop through to pupae since the size of the mine reflects the size of the mining larva (Jansens et al., 1995). In the current work, the low number of larvae or pupae from transgenic foliage in the artificially infested cage experiments or from naturally infested foliage (Table 1) further illustrates the inability of P. operculella larvae to survive on this foliage. The high level of resistance and comparable yield of cry1Ac-transgenic potato lines D61 (derived from Russet Burbank), D02, or D53 (derived from Red Rascal) suggests that these lines could be deployed using a high dose/refuge strategy. Shelton et al. (2000) conducted an experiment on the optimal refuge size
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for Bt-broccoli plants resistant to Plutella xylostella (L.) and found that a separate refuge, 20% the size of the transgenic field, was the most effective at preventing an increase in frequency of resistance alleles in the insect pest population. However, the size of a refuge will be determined by the extent of acceptable crop loss by the grower. For example, the US Environmental Protection Agency recommended that growers of Bt cotton use refuges of either 5% the size of the total crop, separate to or planted within the Bt crop, or a separate refuge 20% the size of the total crop that is treated with non-Bt insecticide, for the 2001 growing season (US EPA, 2000). Shelton et al. (2000) showed that spraying the refuge reduced its potential to dilute the frequency of resistance alleles in the insect population. Where the target insect can move between plants during the larval stage, separate refuges are better than seed mixtures at delaying resistance (Shelton et al., 2002). A high dose/refuge strategy, when incorporated into an integrated pest management programme, is likely to be appropriate for managing P. operculella when using transgenic potatoes similar to those used in the present study. To help determine the size and placement of the refuge, knowledge of the movement of larvae within and between plants, and of adult dispersal behaviour is required. Cameron et al. (2002) found that larvae could form mines on leaflets from two different plants when these leaflets are in contact with one another. They also established that 15% of P. operculella moths dispersed from a release site, moving up to 30 m into new crops with a small percentage dispersing up to 250 m away. Cameron et al. (2002) concluded that a refuge should be placed immediately adjacent to transgenic crops, but further information on the proportion of moths moving between crops is needed to determine the size of the refuge. The potential of P. operculella to develop resistance to such transgenic plants also requires further research. The potential effect of a Bt toxin produced by a transgenic plant on natural enemies can be via a direct toxicological response or indirectly through a reduction in the prey or host population. Davidson et al. (2002a) observed that P. operculella larvae could survive up to 10 days on the transgenic foliage; it could have therefore been possible for parasitoids to emerge before their host died. This was found not to be the case. Further research is required before any conclusions can be made regarding the lower number of parasitoids to emerge from P. operculella larvae reared on transgenic foliage than on non-transgenic foliage observed in the present study.
Acknowledgments We thank Andrew MacLachlan for assistance with statistical analysis, Jill Reader for help with maintaining
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plants in the field, and Crop & Food Research staff for assisting with planting and harvesting the field trial. A Technology for Industry Fellowship from the Foundation for Research, Science and Technology with Lincoln University and Alex McDonald (Merchants) Ltd. provided financial support for M.M. Davidson.
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