东南沿海地区小菜蛾对Bt δ-内毒素和Bt制剂的抗性检测

采用浸叶法测定了2003年秋季、2004年春季采自广东惠州、福建福州、浙江杭州和江苏南京的小菜蛾Plutella xylostella田间种群对Cry1Aa、Cry1Ab、Cry1Ac和Cry2Aa以及Bt制剂kurstaki亚种 (Bacillus thuringiensis subsp. kurstaki, Btk）的抗性水平。与敏感品系PHI-S相比,广东惠州田间小菜蛾种群的抗性水平最高,其对Cry1Ab和Cry1Ac的抗性分别达到了168和120倍,均为高抗水平; 对Btk制剂的抗性有47倍,达到了中抗水平;对Cry1Aa和Cry2Aa具有低水平抗性 (分别为5.8和5.6倍)。福建福州、浙江杭州和江苏南京田间小菜蛾种群抗性水平相近,对Cry1Ab和Cry1Ac具有低至中等水平抗性 (8～28倍),对Btk制剂具有低水平抗性 (3.5～7倍),对Cry1Aa和Cry2Aa还没有产生明显抗性。因此,在我国东南沿海地区要注意Btk制剂与Bt其他亚种制剂或其他生物杀虫剂轮换使用,以减小Bt制剂对小菜蛾的选择压力,延缓小菜蛾对Bt抗性的发展。     

Characterization of resistance to Bacillus thuringiensis toxin Cry1Ac in Plutella xylostella from China

A field population (SZ) of Plutella xylostella, collected from the cabbage field in Shenzhen, Guangdong Province of China in 2002, showed 2.3-fold resistance to Cry1Aa, 110-fold to Cry1Ab, 30-fold to Cry1Ac, 2.1-fold to Cry1F, 5.3-fold to Cry2Aa and 6-fold resistance to Bacillus thuringiensis var. kurstaki (Btk) compared with a susceptible strain (ROTH). The SZBT strain was derived from the SZ population through 20 generations of selection with activated Cry1Ac in the laboratory. While the SZBT strain developed 1200-fold resistance to Cry1Ac after selection, resistance to Cry1Aa, Cry1Ab, Cry1F, and Btk increased to 31-, 1900-,>33- and 17-fold compared with the ROTH strain. However, little or no cross-resistance was detected to Cry1B, Cry1C and Cry2Aa in the SZBT strain. Genetic cross analyses between the SZBT and ROTH strains revealed that Cry1Ac-resistance in the SZBT strain was controlled by a single, autosomal, incompletely recessive gene. Binding studies with ¹²⁵I-labeled Cry1Ac showed that the brush border membrane vesicles (BBMVs) of midguts from the resistant SZBT insects had lost binding to Cry1Ac. Allelic complementation tests demonstrated that the major Bt resistance locus in the SZBT strain was same as that in the Cry1Ac-R strain which has “mode 1” resistance to Bt. An F₁ screen of 120 single-pair families between the SZBT strain and three field populations collected in 2008 was carried out. Based on this approach, the estimated frequencies of Cry1Ac-resistance alleles were 0.156 in the Yuxi population from Yunnan province, and 0.375 and 0.472 respectively in the Guangzhou and Huizhou populations from Guangdong province.     

Inheritance of resistance to Bacillus thuringiensis Cry1Ac toxin in a greenhouse-derived strain of cabbage looper (Lepidoptera: Noctuidae)

A population of cabbage looper, Trichoplusia ni (Hübner), collected from commercial greenhouses in the lower mainland of British Columbia, Canada, in 2001 showed a resistance level of 24-fold to Dipel, a product of Bacillus thuringiensis (Bt) subspecies kurstaki. This population was selected with Cry1Ac, the major Bt Cry toxin in Dipel, to obtain a homogenous population resistant to Cry1Ac. The resulting strain of T. ni, named GLEN-Cry1Ac, was highly resistant to Cry1Ac with a resistance ratio of approximately 1000-fold. The larvae from the GLEN-Cry1Ac strain could survive on Cry1Ac-expressing transgenic broccoli plants that were highly insecticidal to T. ni and diamondback moth, Plutella xylostella (L.). The inheritance of Cry1Ac resistance in this T. ni strain was autosomal and incompletely recessive. The degree of dominance of the resistance was -0.402 and -0.395, respectively, for the neonates in reciprocal crosses between the GLEN-Cry1Ac and a laboratory strain of T. ni. Using chi2 goodness-of-fit test, we demonstrated that the inhibition of larval growth resulting from testing 12 toxin doses in the progeny of the backcross fit the predicted larval responses based on a monogenic inheritance model. Therefore, we conclude that the inheritance of the resistance to Cry1Ac in the T. ni larvae is monogenic.     

Binding of Bacillus thuringiensis Cry1Ac Toxin to Aminopeptidase in Susceptible and Resistant Diamondback Moths (Plutella xylostella)

Bacillus thuringiensis Cry1Ac toxin bound to a 120-kDa protein isolated from the brush border membranes of both susceptible and resistant larvae of Plutella xylostella, the diamondback moth. The 120-kDa protein was purified by Cry1Ac toxin affinity chromatography. Like Cry1Ac-binding aminopeptidase N (EC 3.4.11.2) from other insects, this protein was eluted from the affinity column with 200 mM N-acetylgalactosamine. The purified protein had aminopeptidase activity and bound Cry1Ac toxin on ligand blots. Purified aminopeptidase was recognized by antibodies to the cross-reacting determinant found on phosphatidylinositol-specific phospholipase C-solubilized proteins. The results show that the presence of Cry1Ac-binding aminopeptidase in the brush border membrane is not sufficient to confer susceptibility to Cry1Ac. Furthermore, the results do not support the hypothesis that resistance to Cry1Ac was caused by lack of a Cry1Ac-binding aminopeptidase.     

Susceptibility of a Field-Derived, Bacillus thuringiensis-Resistant Strain of Diamondback Moth to In Vitro-Activated Cry1Ac Toxin

Resistant and susceptible populations of the diamondback moth (Plutella xylostella) were tested with crystalline, solubilized, and partially and fully activated forms of the Bacillus thuringiensis Cry1Ac δ-endotoxin. Fully activated toxin greatly reduced the resistance ratio (ratio of the 50% lethal concentration for the resistant population to that for the susceptible population) of the resistant population, suggesting that a defect in toxin activation is a major resistance mechanism.     

Binding and toxicity of Bacillus thuringiensis protein Cry1C to susceptible and resistant diamondback moth (Lepidoptera: Plutellidae)

We studied mechanisms of resistance to Bacillus thuringiensis insecticidal crystal protein Cry1C in the diamondback moth, Plutella xylostella (L.). Binding assays with midgut brush border membrane vesicles prepared from whole larvae showed no significant difference between resistant and susceptible strains in binding of radioactively-labeled Cry1C. These results indicate that reduced binding of Cry1C to midgut membrane target sites did not cause resistance to Cry1C. Thus, the mechanism of resistance to Cry1C differs from that observed in several previously reported cases of resistance to Cry1A toxins in diamondback moth. We tested Cry1C toxin and Cry1C crystalline protoxin against resistant and susceptible larvae using leaf disk bioassays. After adjusting for the size difference between Cry1C toxin and protoxin, we found that with resistant larvae, toxin was significantly more toxic than protoxin. In contrast, with susceptible larvae, no significant difference in toxicity occurred between Cry1C toxin and protoxin. The resistance ratios for Cry1C were 19 for toxin and 48 for protoxin. These results suggest that reduced conversion of Cry1C protoxin to toxin is a minor mechanism of resistance to Cry1C. Because neither reduced binding nor reduced conversion of protoxin to toxin appear to be major mechanisms, one or more other mechanisms are important in diamondback moth resistance to Cry1C.     

Cross-resistance and stability of resistance to Bacillus thuringiensis toxin Cry1C in diamondback moth.

We tested toxins of Bacillus thuringiensis against larvae from susceptible, Cry1C-resistant, and Cry1A-resistant strains of diamondback moth (Plutella xylostella). The Cry1C-resistant strain, which was derived from a field population that had evolved resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai, was selected repeatedly with Cry1C in the laboratory. The Cry1C-resistant strain had strong cross-resistance to Cry1Ab, Cry1Ac, and Cry1F, low to moderate cross-resistance to Cry1Aa and Cry9Ca, and no cross-resistance to Cry1Bb, Cry1Ja, and Cry2A. Resistance to Cry1C declined when selection was relaxed. Together with previously reported data, the new data on the cross-resistance of a Cry1C-resistant strain reported here suggest that resistance to Cry1A and Cry1C toxins confers little or no cross-resistance to Cry1Bb, Cry2Aa, or Cry9Ca. Therefore, these toxins might be useful in rotations or combinations with Cry1A and Cry1C toxins. Cry9Ca was much more potent than Cry1Bb or Cry2Aa and thus might be especially useful against diamondback moth.     

Inheritance of Resistance to the Bacillus thuringiensis Toxin Cry1C in the Diamondback Moth

Laboratory selection increased resistance to the Bacillus thuringiensis toxin Cry1C in a strain of diamondback moth (Plutella xylostella). The selected strain was derived from a field population that had evolved high levels of resistance to Bacillus thuringiensis subsp. kurstaki and moderate resistance to Cry1C. Relative to the responses of a susceptible strain of diamondback moth, the resistance to Cry1C of the selected strain increased to 62-fold after six generations of selection. The realized heritability of resistance was 0.10. Analysis of F(inf1) hybrid progeny from reciprocal crosses between the selected strain and a susceptible strain showed that resistance to Cry1C was autosomally inherited. The dominance of resistance to Cry1C depended on the concentration; inheritance was increasingly dominant as the concentration decreased. Responses of progeny from single-pair families showed that resistance to Cry1C and resistance to Cry1Ab were inherited independently, which enhances opportunities for managing resistance. However, compared with projections based on previously reported recessive inheritance of resistance to Cry1A toxins, the potentially dominant inheritance of resistance to Cry1C observed here could accelerate evolution of resistance.     

小菜蛾对Bt毒素Cry1Ac和Bt制剂抗性的选育及其抗性种群的生物学适应性

用Bt制剂和Bt毒素Cry1Ac分别对源自深圳田间的小菜蛾Plutella xylostella在室内进行抗性种群选育,获得相对抗性倍数分别为24.36、38.16倍的抗性种群DBM.1Ac-R30和DBM.Bt-R46。对这2个抗性种群及其敏感种群（DBM.Bt-S）的生长发育、存活及繁殖特征进行了详细地观察与比较,并以甘蓝为饲喂材料构建了2个抗性实验种群的生命表。结果表明,DBM.1Ac-R30和DBM.Bt-R46种群较DBM.Bt-S种群的产卵量和孵化率下降,幼虫发育历期延长,雌雄比显著降低,雌成虫数量、寿命减少。DBM.1Ac-R30和DBM.Bt-R46种群相对于DBM.Bt-S种群的相对适合度分别为0.75和0.65,抗性种群在繁殖能力上存在明显的生存劣势。     

小菜蛾对九种杀虫剂的抗药性

采用叶片药膜法测定了9种杀虫剂对海南、山东、广东、湖北武汉和襄樊等5个地区小菜蛾Plutella xylostella(L.)田间种群的毒力,和室内相对敏感品系比较。结果显示,5个地区的小菜蛾种群都对氯氰菊酯产生了10倍以上的抗性,广东和山东种群达到30倍以上的抗性。对阿维菌素的抗性山东小菜蛾种群达到135倍,广东种群达到30倍,其他地区均在10倍以下。但是,作用机制与阿维菌素类似的药剂,氟虫腈的抗性5个地区均在5倍或5倍以下。对昆虫生长调节剂定虫隆和氟虫脲的抗性除广东种群分别在10倍和20倍以上外,其他地区均在5倍以下。广东种群对多杀菌素的抗性为7.8倍,其他地区的种群没有产生抗性,多杀菌素和杀虫双均为作用于乙酰胆碱受体的药剂,但是作用的位点不同。杀虫双对5个种群的LC50值均是比较大的(>800 mg/L),尽管没有明显的抗性,不适宜用于小菜蛾田间防治。对呼吸链电子转移抑制剂溴虫腈的抗性均在10倍以下。对辛硫磷的抗性在2~5倍。     

Integrative model for binding of Bacillus thuringiensis toxins in susceptible and resistant larvae of the diamondback moth (Plutella xylostella)

Insecticidal crystal proteins from Bacillus thuringiensis in sprays and transgenic crops are extremely useful for environmentally sound pest management, but their long-term efficacy is threatened by evolution of resistance by target pests. The diamondback moth (Plutella xylostella) is the first insect to evolve resistance to B. thuringiensis in open-field populations. The only known mechanism of resistance to B. thuringiensis in the diamondback moth is reduced binding of toxin to midgut binding sites. In the present work we analyzed competitive binding of B. thuringiensis toxins Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F to brush border membrane vesicles from larval midguts in a susceptible strain and in resistant strains from the Philippines, Hawaii, and Pennsylvania. Based on the results, we propose a model for binding of B. thuringiensis crystal proteins in susceptible larvae with two binding sites for Cry1Aa, one of which is shared with Cry1Ab, Cry1Ac, and Cry1F. Our results show that the common binding site is altered in each of the three resistant strains. In the strain from the Philippines, the alteration reduced binding of Cry1Ab but did not affect binding of the other crystal proteins. In the resistant strains from Hawaii and Pennsylvania, the alteration affected binding of Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F. Previously reported evidence that a single mutation can confer resistance to Cry1Ab, Cry1Ac, and Cry1F corresponds to expectations based on the binding model. However, the following two other observations do not: the mutation in the Philippines strain affected binding of only Cry1Ab, and one mutation was sufficient for resistance to Cry1Aa. The imperfect correspondence between the model and observations suggests that reduced binding is not the only mechanism of resistance in the diamondback moth and that some, but not all, patterns of resistance and cross-resistance can be predicted correctly from the results of competitive binding analyses of susceptible strains.     

Different cross-resistance patterns in the diamondback moth (Lepidoptera: Plutellidae) resistant to Bacillus thuringiensis toxin Cry1C.

Two strains of the diamondback moth, Plutella xylostella (L.), were selected using Cry1C protoxin and transgenic broccoli plants expressing a Cry1C toxin of Bacillus thuringiensis (Bt). Both strains were resistant to Cry1C but had different cross-resistance patterns. We used 12 Bt protoxins for cross-resistance tests, including Cry1Aa, Cry1Ab, Cry1Ac, Cry1Bb, Cry1C, Cry1D, Cry1E, Cry1F, Cry1J, Cry2Ab, Cry9Aa, and Cry9C. Compared with the unselected sister strain (BCS), the resistance ratio (BR) of one strain (BCS-Cry1C-1) to the Cry1C protoxin was 1,090-fold with high level of cross-resistance to Cry1Aa, Cry1Ab, Cry1Ac, Cry1F, and Cry1J (RR > 390-fold). The cross-resistance to Cry1A, Cry1F, and Cry1J in this strain was probably related to the Cry1A resistance gene(s) that came from the initial field population and was caused by intensive sprayings of Bt products containing Cry1A protoxins. The neonates of this strain can survive on transgenic broccoli plants expressing either Cry1Ac or Cry1C toxins. The other strain (BCS-Cry1C-2) was highly resistant to Cry1C but not cross-resistant to other Bt protoxins. The neonates of this strain can survive on transgenic broccoli expressing Cry1C toxin but not Cry1Ac toxin. The gene(s) conferring resistance to Cry1C segregates independently from Cry1Ac resistance in these strains. The toxicity of Cry1E and Cry2Ab protoxins was low to all of the three strains. The overall progress of all work has resulted in a unique model system to test the stacked genes strategy for resistance management of Bt transgenic crops.     

Genetic and biochemical characterization of field-evolved resistance to Bacillus thuringiensis toxin Cry1Ac in the diamondback moth, Plutella xylostella

The long-term usefulness of Bacillus thuringiensis Cry toxins, either in sprays or in transgenic crops, may be compromised by the evolution of resistance in target insects. Managing the evolution of resistance to B. thuringiensis toxins requires extensive knowledge about the mechanisms, genetics, and ecology of resistance genes. To date, laboratory-selected populations have provided information on the diverse genetics and mechanisms of resistance to B. thuringiensis, highly resistant field populations being rare. However, the selection pressures on field and laboratory populations are very different and may produce resistance genes with distinct characteristics. In order to better understand the genetics, biochemical mechanisms, and ecology of field-evolved resistance, a diamondback moth (Plutella xylostella) field population (Karak) which had been exposed to intensive spraying with B. thuringiensis subsp. kurstaki was collected from Malaysia. We detected a very high level of resistance to Cry1Ac; high levels of resistance to B. thuringiensis subsp. kurstaki Cry1Aa, Cry1Ab, and Cry1Fa; and a moderate level of resistance to Cry1Ca. The toxicity of Cry1Ja to the Karak population was not significantly different from that to a standard laboratory population (LAB-UK). Notable features of the Karak population were that field-selected resistance to B. thuringiensis subsp. kurstaki did not decline at all in unselected populations over 11 generations in laboratory microcosm experiments and that resistance to Cry1Ac declined only threefold over the same period. This finding may be due to a lack of fitness costs expressed by resistance strains, since such costs can be environmentally dependent and may not occur under ordinary laboratory culture conditions. Alternatively, resistance in the Karak population may have been near fixation, leading to a very slow increase in heterozygosity. Reciprocal genetic crosses between Karak and LAB-UK populations indicated that resistance was autosomal and recessive. At the highest dose of Cry1Ac tested, resistance was completely recessive, while at the lowest dose, it was incompletely dominant. A direct test of monogenic inheritance based on a backcross of F1 progeny with the Karak population suggested that resistance to Cry1Ac was controlled by a single locus. Binding studies with 125I-labeled Cry1Ab and Cry1Ac revealed greatly reduced binding to brush border membrane vesicles prepared from this field population.     

Broccoli plants with pyramided cry1Ac and cry1C Bt genes control diamondback moths resistant to Cry1A and Cry1C proteins

This study was undertaken to determine the effects of pyramiding two Bacillus thuringiensis (Bt) genes in the same plant on the production of Bt proteins and the control of diamondback moths (DBM, Plutella xylostella) resistant to one or the other protein. Broccoli lines carrying both cry1Ac and cry1C Bt genes were produced by sexual crosses of cry1Ac- and cry1C-transgenic plants. Plants containing both genes were selected by tests for resistance to kanamycin and hygromycin, and confirmed by PCR analysis for the Bt genes. Both cry1Ac and cry1C mRNAs were detected in the hybrid lines, and Cry1Ac and Cry1C proteins were stably produced at levels comparable to the parental plants. Plants producing both Cry1Ac and Cry1C proteins caused rapid and complete mortality of DBM larvae resistant to Cry1A or Cry1C, and suffered little or no leaf damage. These plants, in combination with the resistant DBM populations available, will allow greenhouse or field studies of resistance management strategies involving gene pyramiding.     

Development and characterization of diamondback moth resistance to transgenic broccoli expressing high levels of Cry1C

A field-collected colony of the diamondback moth, Plutella xylostella, had 31-fold resistance to Cry1C protoxin of Bacillus thuringiensis. After 24 generations of selection with Cry1C protoxin and transgenic broccoli expressing a Cry1C protein, the resistance that developed was high enough that neonates of the resistant strain could complete their entire life cycle on transgenic broccoli expressing high levels of Cry1C. After 26 generations of selection, the resistance ratios of this strain to Cry1C protoxin were 12,400- and 63,100-fold, respectively, for the neonates and second instars by a leaf dip assay. The resistance remained stable until generation 38 (G38) under continuous selection but decreased to 235-fold at G38 when selection ceased at G28. The Cry1C resistance in this strain was seen to be inherited as an autosomal and incompletely recessive factor or factors when evaluated using a leaf dip assay and recessive when evaluated using Cry1C transgenic broccoli. Saturable binding of (125)I-Cry1C was found with brush border membrane vesicles (BBMV) from both susceptible and Cry1C-resistant strains. Significant differences in Cry1C binding to BBMV from the two strains were detected. BBMV from the resistant strain had about sevenfold-lower affinity for Cry1C and threefold-higher binding site concentration than BBMV from the susceptible strain. The overall Cry1C binding affinity was just 2.5-fold higher for BBMV from the susceptible strain than it was for BBMV from the resistant strain. These results suggest that reduced binding is not the major mechanism of resistance to Cry1C.     

Shared binding sites in Lepidoptera for Bacillus thuringiensis Cry1Ja and Cry1A toxins.

Bacillus thuringiensis toxins act by binding to specific target sites in the insect midgut epithelial membrane. The best-known mechanism of resistance to B. thuringiensis toxins is reduced binding to target sites. Because alteration of a binding site shared by several toxins may cause resistance to all of them, knowledge of which toxins share binding sites is useful for predicting cross-resistance. Conversely, cross-resistance among toxins suggests that the toxins share a binding site. At least two strains of diamondback moth (Plutella xylostella) with resistance to Cry1A toxins and reduced binding of Cry1A toxins have strong cross-resistance to Cry1Ja. Thus, we hypothesized that Cry1Ja shares binding sites with Cry1A toxins. We tested this hypothesis in six moth and butterfly species, each from a different family: Cacyreus marshalli (Lycaenidae), Lobesia botrana (Tortricidae), Manduca sexta (Sphingidae), Pectinophora gossypiella (Gelechiidae), P. xylostella (Plutellidae), and Spodoptera exigua (Noctuidae). Although the extent of competition varied among species, experiments with biotinylated Cry1Ja and radiolabeled Cry1Ac showed that Cry1Ja and Cry1Ac competed for binding sites in all six species. A recent report also indicates shared binding sites for Cry1Ja and Cry1A toxins in Heliothis virescens (Noctuidae). Thus, shared binding sites for Cry1Ja and Cry1A occur in all lepidopteran species tested so far.     

Examination of the F2 screen for rare resistance alleles to Bacillus thuringiensis toxins in the diamondback moth (Lepidoptera: Plutellidae)

A synthetic laboratory population of the diamondback moth, Plutella xylostella (L.), was used to test the F2 screen developed for detecting the frequency of rare resistance alleles to Cry1Ac and Cry1C toxins of Bacillus thuringiensis (Bt). Of the 120 single-pair matings set up, 106 produced enough F2 families for screening of Cry1Ac or Cry1C resistance alleles using both transgenic broccoli and an artificial diet overlay assay with a diagnostic dose. When using Bt broccoli plants as the F2 screen method, only one F2 family was detected for Cry1Ac resistance and no family was detected for Cry1C resistance. Six families were detected for either Cry1Ac or Cry1C resistance using the diet assay. The survivors in the diagnostic diet assay were crossed with the resistant individuals to confirm their resistance genotypes. Four F2 families were confirmed to contain one copy of an allele resistant to Cry1Ac in the original single-pairs and four other F2 families contained an allele resistant to Cry1C. Our results suggest that using transgenic plants expressing a high level of a Bt toxin in an F2 screen may underestimate the frequency of resistance alleles with high false negatives, or fail to detect true resistance alleles. The diagnostic diet assay was a better F2 screen method to detect alleles, especially for the Cry1Ac resistance with monogenic inheritance in the diamondback moth. The estimated probabilities of false positives and false negatives were 33 and 1%, respectively, for detecting Cry1Ac resistance at the allele frequency of 0.012 using the diagnostic diet assay. Careful validation of the screening method for each insect-crop system is necessary before the F2 screen can be used to detect rare Bt resistance alleles in field populations.     

Common, but complex, mode of resistance of Plutella xylostella to Bacillus thuringiensis toxins Cry1Ab and Cry1Ac

A field collected population of Plutella xylostella (SERD4) was selected in the laboratory with Bacillus thuringiensis endotoxins Cry1Ac (Cry1Ac-SEL) and Cry1Ab (Cry1Ab-SEL). Both subpopulations showed similar phenotypes: high resistance to the Cry1A toxins and little cross-resistance to Cry1Ca or Cry1D. A previous analysis of the Cry1Ac-SEL showed incompletely dominant resistance to Cry1Ac with more than one factor, at least one of which was sex influenced. In the present study reciprocal mass crosses between Cry1Ab-SEL and a laboratory susceptible population (ROTH) provided evidence that Cry1Ab resistance was also inherited as incompletely dominant trait with more than one factor, and at least one of the factors was sex influenced. Analysis of single pair mating indicated that Cry1Ab-SEL was still heterogeneous for Cry1Ab resistance genes, showing genes with different degrees of dominance. Binding studies showed a large reduction of specific binding of Cry1Ab and Cry1Ac to midgut membrane vesicles of the Cry1Ab-SEL subpopulation. Cry1Ab-SEL was found to be more susceptible to trypsin-activated Cry1Ab toxin than protoxin, although no defect in toxin activation was found. Present and previous results indicate a common basis of resistance to both Cry1Ab and Cry1Ac in selected subpopulations and suggest that a similar set of resistance genes are responsible for resistance to Cry1Ab and Cry1Ac and are selected whichever toxin was used. The possibility of an incompletely dominant trait of resistant to these toxins should be taken into account when considering refuge resistance management strategies.     

Diamondback moth resistance to Bacillus thuringiensis transgenic canola: evaluation of refugia size with non-recessive resistant insects

Abstract:  Current recommendations to delay the evolution of resistance to Bacillus thuringiensis crops are that a minimum of 5–50% of a crop-growing region should include non- B. thuringiensis varieties as refuges. These recommendations are based in part on the assumption that resistance will be inherited as a recessive trait. Laboratory microcosm experiments are described with transgenic canola expressing Cry1Ac and a non-recessive Cry1Ac resistant population of the diamondback moth Plutella xylostella , in which the effect of different sizes of refugia (0%, 20% and 50%) on resistance was compared over five generations. The LC₅₀ values for Cry1Ac increased markedly in the P. xylostella sub-populations with 0% (>100-fold) and 20% refugia (>35-fold) but showed little change (less than fivefold increase) with 50% refugia. The results support the idea that relatively high levels of refugia (non- B. thuringiensis varieties) may be required where resistance is not functionally recessive at the level of toxin expressed in the B. thuringiensis crop.     

Geographical variation in larval susceptibility of the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae) to Bacillus thuringiensis spore-crystal mixtures and purified crystal proteins and associated resistance development in India

The susceptibility of larvae of the diamondback moth, Plutella xylostella Linnaeus to purified crystal proteins and spore-crystal preparations of Bacillus thuringiensis was investigated for 13 populations from seven states in India. The LC50 (microg ml(-1), 48 h) values of Cry proteins for different populations of P. xylostella ranged from 0.14-3.74 (Cry1Aa), 0.007-1.25 (Cry1Ab), 0.18-2.47 (Cry1Ac) and 0.12-3.0 (Cry1C). The LC50 (mg (ai) l(-1), 48 h) of spore-crystal preparations ranged from 0.02-0.98 (HD-1) and 0.06-2.14 (HD-73). Significantly higher LC50 values for all tested toxins and strains were obtained with populations collected from Iruttupallam and Ottanchathiram in the southern state of Tamil Nadu, whereas some of the populations collected from the northern part of India were more susceptible than the susceptible IARI 17-65 population. The high levels of resistance in the Iruttupallam and Ottanchathiram populations to Cry1Ab suggested selection pressure by Cry1Ab, which is the predominant toxin in B. thuringiensis formulations used in India. Cry1Ab was found to be more toxic than the other toxins. The population from Iruttupallam showed increased resistance following selection with Cry1Ab in the laboratory (LC50 from 1.25 to 4.31 microg ml(-1) over two generations) and also showed cross resistance to CrylAa and CrylAc. The resistance to Biobit in the field population from Iruttupallam declined slowly; requiring c. 33 generations for an overall 10-fold decline in LC50 when the insects were reared in the laboratory without exposure to B. thuringensis.