| Transgenic crops producing insecticidal protein toxins from Bacillus thuringiensis (Bt) have been adopted worldwide. Planting of Bt crops have provided effective control of target pests, and also reduced the use of chemical insecticides. Bt cotton expressing Cry1Ac protein has been adopted in China since1997, now it accounts for nearly70%of all cotton. The large-scale planting of Bt cotton has been very successful so far not only in controlling Helicoverpa armigera on Bt cotton designed to resist this pest but also in reducing its population density on other host crops. Some pest populations have evolved resistance to Bt crops in the field, and decreased susceptibilty to Cry1Ac in field populations of H. armigera from northern China was associated with intensive Bt cotton planting. The evolution of resistance in field populations of H. armigera to Bt cotton is a major threat to the continued efficacy of Bt cotton in China."High dose/refuge" and "pyramiding" are two major resistance management strategies designed for delaying insect resistance development to Bt crops. These two strategies are mainly based on knowledge of Bt resistance from the laboratory-selected strains with resistance to Bt. However, resistance evolution to Bt crops is much more complex in field populations than in laboratory-selected strains. It will be rational to develop resistance detection tactics and resistance management strategies according to resistance nature of field-derived resistant populations. In order to gain a timely understanding of the evolution of resistance of H. armigera to Cry1Ac after continuous cultivation of Bt cotton, we detected resistance alleles to Cry1Ac in two field populations of H. armigera collected respectively from Yellow River Region and Yangtze River Region by using a single pair mating technique (or F1screen). Diverse cadherin alleles conferring Cry1Ac resistance were identified from these two field populations. Two different resistance alleles were detected from five Cry1Ac-resistant strains isolated from field populations. Cry2Ab is an important candidate Bt toxin gene pyramided with CrylAc in the second generation Bt cotton. A Cry2Ab-resistant strain of H. armigera was selected in the laboratory, and cross resistance pattern and inheritance mode of this resistant strain was evaluated. The results from the present study will have a great significance in designing resistance monitoring and management tactics for Bt cotton in China.1. Diverse cadherin mutations conferring resistance to CrylAc in a field population of H. armigera from Jiangpu of Jiangsu ProvincePrevious results revealed3null alleles (r1-r3) of a cadherin gene (Ha_BtR) conferring CrylAc resistance in H. armigera. An F1screen of123single pair families was conducted between a Cry1Ac-resistant strain (the SCD-rl strain, homozygous for the r1allele of Ha_BtR) and field-derived insects from Jiangpu population (Jiangsu province, China) in2008. Five new null alleles of Ha_BtR (r4-r8) were identified in six candidate single-pair families. These null alleles were created through either an insertion or a point mutation. Interestingly, intact alleles of Ha_BtR were found in two field-derived insects from another two candidate single-pair families. It suggests that these two field-derived insects may carry novel resistance alleles of Ha_BtR, with missense mutations resulting in a non-functional cadherin protein, or a major dominant mutation at a locus other than cadherin. The resistance allele frequency of Ha_BtR was detected at an appreciable level (0.024) in the Jiangpu population of H. armigera in2008. Together with previous findings, a total of eight different resistance alleles of Ha_BtR were identified from three Chinese strains ofH. armigera. Mutational diversity of Ha_BtR could impair DNA screening for Bt resistance allele frequency in the field, and an F1screen should be used routinely for monitoring cadherin-based resistance allele frequencies in H. armigera.2. Detection of resistance allele frequency and identification of cadherin mutations in a field population of H. armigera from Anyang of Henan ProvinceUsing an F1screen method, we detected the frequency of alleles conferring resistance to the CrylAc in Anyang population of H. armigera in2009. The field parents of34single-pair families may carry resistance alleles in the215single-pair families tested, and the resistance allele frequency was estimated to be0.091. Two new types of mutational modes of cadherin were detected, namely deletion at the cytoplasmic domain and mis-splicing of cadherin. However, intact alleles of cadherin were present in field-derived insects from most of the candidate single-pair families. It suggests that cadherin can confer resistance to Cry1Ac through amino acid substitutions in cadherin of H. armigera.Among the29lines generated by crossing survivors from the F1screen with the susceptible SCD strain, bioassay results at the diagnostic concentration of CrylAc showed that for Anyang population,86%of the resistance alleles were recessive cadherin alleles and14%were non-recessive alleles at any locus. Using two methods to estimate genotype frequencies,49to80%of resistant individuals had at least one non-recessive resistance allele. The results here suggest that non-recessive alleles make much more contribution to the resistant individual frequency in Anyang population than the recessive cadherin alleles. Adaptive resistance management strategies are urgently needed to cope with non-recessive resistance alleles in field-selected populations of H. armigera.3. Inheritance modes and complementation tests with cadherin locus of CrylAc-resistant alleles of H. armigera derived from Fi screensFive Cryl Ac-resistant strains of H. armigera were established from part of positive single-families of F1screens. The resistance strains Jp14, Anl, An29, An357and An156had128-,215-,41-,881-and121-fold resistance to CrylAc compared respectively to the susceptible SCD strain. All the five strains showed no cross-resistance to Cry2Ab. Through reciprocal crosses between resistant strains and SCD, we found that CrylAc resistance in all five strains was autosomal and controlled by a single locus. CrylAc resistance in Anl, An29, An357and An156was non-recessive, but CrylAc resistance in Jp14was recessive. Genetic complementation tests showed that CrylAc resistance in Jpl4, Anl, An29and An357was cadherin-based, and CrylAc resistance in An156was not cadherin-based. From these results, we concluded:(1) F1screen can detect both recessive resistance alleles and non-recessive resistance alleles in H. armigera;(2) Diverse mutations of cadherin may confer different levels of magnitude and dominance of CrylAc resistance;(3) More efforts are recommended to detect, characterize and design strategies to counter non-recessive resistance alleles of H. armigrea to Bt cotton.4. Cross resistance and genetics of Cry2Ab resistance in a laboratory-selected strain of H. armigeraLaboratory selection with Cry2Ab against a field population from Anyang produced high levels of resistance to Cry2Ab in An2Ab strain of H. armigera. When An2Ab was selected at the11th generation (G11), it developed262-fold resistance to Cry2Ab and37-fold cross-resistance to Cry1Ac compared to the susceptible progenitor strain (An). Reciprocal crosses between the An and An2Ab at G11showed that resistance to Cry1Ac and Cry2Ab in the An2Ab strain was partially dominant. Backcrossing between the F1progeny and An showed that resistance to Cry2Ab and Cry1Ac in the An2Ab strain was controlled by more than one genes. Resistance to Cry2Ab increased to636-fold at the20th generation (G20) of selection, and cross resistance to Cry1Ac also elevated to60-fold compared to An. This is the first report that Cry2Ab-resistant strain of H. armigera has cross-resistance to Cry1Ac. Although we have not determined if An2Ab strain can survive on the Bt cotton expressing both CrylAc and Cry2Ab toxins, cross resistance potential between Cry2Ab and CrylAc reminds us we should use dual-Bt cotton (Cry2Ab+CrylAc) cautiously as a resistance management tactic in China. |
PDF Full Text Request