Using F₂ Screening Method To Monitor Resistance To Transgenic Bt Cotton In Field Population Of Helicoverpa Armigera (Hübner)

Transgenic Bacillus thuringiensis(Bt) cotton producing Cry1Ac protein has been widely planted in northern China for a long time.The high degree of effectiveness and low environmental risk of Bt crops have encouraged Bt cotton producers to rapidly adopt this technology.The rapid and extensive commercialization of Bt cotton could increase the risk of resistance development in target insects.The studies showed that Helicoverpa armigera have been selected for high levels of resistance to Cry1Ac toxins or transgenic Bt cotton in labs.In Qiuxian County(Hebei,China),Bt cotton has been commercially planted since 1998. Previous study using F₂ screen in 1999 found that the frequency of resistance alleles to Bt cotton in field populations of Helicoverpa armigera was 0.0058.Considering the high selection pressures in this region,we conducted another F₂ screen,high-dose bioassay, F₁screen and DNA-based method to monitor the Bt resistance in the field populations of H. armigera from 2003 to 2007 as Bt cotton acreage continued to increase over the years.The resistance monitoring program is to be used in a proactive way for resistance management, and the results of resistance monitoring will be used to ensuring the long-term durability of Bt plantsThe results from the F₂ screen showed that 20 out of 340 isofemale lines during 2003 to 2005 were identified to carry resistance alleles to Bt cotton in Qiuxian County(2003 to 2005) and Weixian County(2004).The resistance allele frequency in field populations of H. armigera ranged from 0.0119 to 0.0297.The data analysis showed an overall frequency of 0.0146 and a 95%confidence interval of 0.0084-0.0225 for the 3-year period in Qiuxian County.This value is significantly greater than the value reported from 1999(p=0.042).The F₂ screen for detecting Bt resistance alleles was successfully conducted for 105,42 and 131 lines with an average of 372.4±16.0,328.1±9.5 and 314.0±9.0 F₂ neonates were screen on Bt cotton plants, respectively from 2003 to 2005.The results showed that 4,4 and 7lines were considered to be true positive lines for Bt resistance,respectivelyand thus the resistance allele frequency for the population collected during 2003 to 2005was estimated as 0.0119 with a 95%CI of 0.0039-0.0243,0.0297 with a 95%CI of 0.0099-0.0606 and 0.0154 with a 95%CI of 0.0067-0.0277,respectively.The average detection powers of F₂ screen(1-P_NO) for all the lines screened were 96%,97%and 94%,respectively.In 2004 in Weixian County,F₂ screen for detecting Bt resistance alleles was successfully conducted for 62 lines,and an average of 334.7±8.6 F₂ neonates were screened on Bt cotton plants.The results showed that 4 lines were considered to be true positive lines for Bt resistance and thus the resistance allele frequency for the population collected during 2004 was estimated as 0.0243 with a 95%CI of 0.0091-0.0471.The F₂ screen had＞80%detection power for 96%of the lines screened with an average detection power of 95.1%.Comparing the lab strain of Bt-resistance(YCR) and Bt-susceptible(YCS) H. armigera on Bt cotton leaf for 5d,the result showed that the resistant and susceptible H. armigera had significant difference in larval surviving rate and growth rate on Bt cotton leaf.The susceptible strain(ss) was unable to survive on Bt cotton.Only 2-6% heterozygous individuals(r₁s) survived,the survivors could not reach stage of mid-2^nd instar and body weight of≥0.6mg.The resistant individuals(r₁r₁) had considerably higher survival rate(75%),and a majority of survivors(65%among survivors) reached≥0.6 mg and at least mid-2^nd instar.Therefore,the survivors reached≥0.6 mg and beyond mid-2^nd instar on Bt cotton leaf for 5 days were categorized as resistant individuals.In 2006 to 2007,we used Bt cotton leaves as screen media to monitor the resistance allele frequency of field population of H.armigera in Qiuxian County.In 2006 and 2007,F₂ screen for detecting Bt resistance alleles was successfully conducted for 134 and 137lines, and an average of 187.9±3.4 and 105.1±1.5F₂ neonates were screened on Bt cotton plants, respectively.The resistance allele frequency for the population collected during 2006 and 2007 were estimated as 0.1135with a 95%CI of 0.0862-0.1449 and 0.0745 with a 95%CI of 0.0532-0.0995,respectively.The average detection powers of F₂ screen(1-P_NO) for all the lines screened were 95%and 90%,respectively.In 2007,F₁ screen for detecting Bt resistance alleles was successfully conducted for 135 lines,and an average of 165.2±5.2 F₁ neonates were screened on Bt cotton plants.The results showed that 29 lines were considered to be true positive lines for Bt resistance and thus the resistance allele frequency for the population collected during 2007 was estimated as 0.107 with a 95%CI of 0.055-0.159.Dose-response bioassay is used to monitor Bt resistance in field population of H. armigera in Hebei Province from 2003 to 2007.The results showed that the RF to Cry1Ac toxin were 15.3,12.9,12.5,6.7,16.5 and 11.5.In 2006 to 2007,the DNA-based method was used to detect the mutation of Cadherin gene in the field resistance H.armigera individuals.The results showed that the primers designed by YCR and YCS could not be used to detect r₁ resistance gene in filed resistance individuals.Our results suggest that significant shift in resistance allele frequency had occurred in the field populations of H.armigera in Qiuxian and Weixian County.It confirmed that after intinial resistance allele frequencies reaches 0.005;Bt resistance in field population will develop more fast.It is necessary to focuse on the development and refinement of cost-effective methods for Bt resistance decetion and monitoring.It also need to introduce Bt cotton that expressing multi-Bt toxins and integrate the Bt cotton technology with biological,chemical,and cultural practices for management of this key cotton pest.