| 1.The selection of resistance strain YCR and detecting freqency of resistance alleleThe resistant strain(YCR) was selected with Bt cotton leaves from 73 to 88 generation,and resistance rate to Cry1Ac protoxin was increased from 2523-fold of 73 generation to 7311-fold of 88 gerenation.The YCR strain was subsequently selected to 104 generation in the laboratory and maintained the extremely high level of resistance to Bt. thuringiensis.In addition,using PASA(PCR amplification of specific alleles) method,we identified the deletion mutation gene of cadhern-like(r1) in YCR and detected the frequency of the resistance allele,the result showed that the frequency of resistance allele (r1)in YCR strain approached 1.2.Establishing of standard of detection resistant cotton bollwormComparing wtih the Bt-resistance and Bt-susceptible cotton bollworm on Bt cotton leaves for 5d,the result showed that Bt-susceptible and Bt-resistant H.armigera had significantly difference in larval surviving rate and growth rate on Bt cotton leaves. Resistant individuals(r1r1)had considerably higher survival rate(75%),and a majority of survivors(65%of the total or 87%of the survivors)reached≥0.6mg/larvae and at least mid-2nd instar.Whereas the surviving rate of reciprocal crosses of YCR and YCS strain,and YCS strain were 6%(0.1-0.5mg/larvae weight,from first instar to early of second instar), 2%(0.1-0.3mg/larvae weight,first instar)and 0%,respectively.Therefore,individuals which reached＞0.6 mg/larvae and beyond mid-2nd instar on Bt cotton leaves for 5 days, were considered as the homozygous resistant individual standard.3.The growth rate of H.armigera on Bt cotton leavesComparing with the development of Bt-resistant and Bt-susceptible bollworm(YCR and YCS)on Bt cotton leaves for 5 days,both the surviving rates of YCR on Bt cotton and non-Bt cotton leaves were 80%,which was not obviously different.6.7%survivors could developed adults by continuously feeding with Bt cotton leaves,and the development period of Bt-resistant larvae on Bt cotton leaves was delayed for 6 days than on non-Bt cotton.The pupation rate,pupa weight and emergence rate were lower than on non-Bt cotton,which indicated that the Bt-resistant insect suffered a survival disadvantage on Bt cotton leaves relative to its non-Bt counterpart.The asynchrony between susceptible and resistant populations and the surviving ability on Bt cotton under long-term selection pressure would influence the random mating between a lot of susceptible moths from refuge and few resistant moths from Bt cotton field,and weaken the efficiency of refuge strategy.4.Resistance inheritance and linkage analysis of resistance to Cry1Ac in H.armigeraUsing diet containing Bt toxin method,Logit regression analysis of Bt-resistant strain (YCR),Bt-susceptible strain(YCS)and reciprocal crosses between them(F1 and F'1) of H. armigera indicated that LC50 of the progenies from F1 and F'1 were 12.031(95%CI: 7.972-18.263)μg/mL and 6.617(95%CI:4.337-9.960)μg/mL for 21%MVPII WP; 61.659(95%CI:43.048-90.552)μg/mL and 98.217(95%CI:76.726-123.645)μg/mL for 20%MVPII SC,respectively,which there were no significantly difference between the two LC50s.These results showed the gene that confers resistance to Bt toxin was on autosome. According to the formula of Stone(1968),the dominance degree(D) of F1 and F'1 were -0.51and -0.69 for 21%MVPII WP(-0.61 and -0.44 for 20%MVPII SC),respectively.The result indicated that the resistance to Cry1Ac toxin was inhetited as an incompeletely recessive trait(-1＞D＞0).The genetype of progenies of two backcrosses BC1(♀F1×♂YCR) and BC2 and(♂F1×♀YCR) on Bt cotton leaves for 5d had 1:1 separation following the Mendel's law,at same time,they were verified by using PASA method,results showed that the inheritance of resistance was on autosome too,and the truncated cadherin-like gene(r1) in YCR strain was tightly linked to Cry1Ac resistance.5.Resistance monitoring of field populations of H.armigeraDuring 2005-2007,we estimated frequencies of alleles conferring resistance to Bt cotton by using the F1 screen method in Qiuxian and Weixian(2005) counties.By feeding Bt cotton plants or leaves for 5 d,F1 offspring from each single-pair line established in 2005-2007 were screened for resistance alleles based on larval growth,development,and surviving rate.8,24 and 29 collected-field male moths out of 86,127 and 135 lines were detected to carry resistance alleles in Qiuxian,and the frequencies of resistance alleles were estimated as 0.047(95%CI:0.015-0.079),0.094(0.044-0.145)and 0.107(0.055-0.159), respectively;1 collected-field male moth out of 32 lines were detected to carry resistance allele in Weixian,and the frequency of resistant allele Weixian was estimated as 0.016 (0-0.0595).In 2005,7 collected-field female moths out of 131 iso-lines were detected to carry resistance alleles by using F2 screen method in Qiuxian,and the frequency of resistance alleles was estimated as 0.01539(0.0067-0.0277),the detection power of F2 screen(1-PNO) for lines screened was 93.8%.The results from the F1 screen and F2 screen methods in 2005 were consistant during detecting resistance of cotton bollworm population in Qiuxian,which indicated that the resistant herozygosis with rare and recessive gene can be detected by both F1 screen and F2 screen.Comparing with F2 screen,the F1 screen was more effective,applicative and low-cost method for monitoring resistance in field population.In 2007,the results from dose/response method showed that there was 13.14-fold resistance to 20%MVPII SC which means that there was a middle level resistance in this region.The results from several methods comfirmed that the resistance in field population had been increasing.Long-term use of Bt cotton expressing single Cry1Ac toxin and the lack of proper resistance management practice might accelerated the resistant evolution in field populations of the cotton bollworm.It is necessary to establish and implement effective resistance management strategy in this region as soon as possible. |
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