Studies On Evolution Of Resistance To Bt Toxins In Ostrinia Furnacalis

The Asian com borer(ACB),Ostrinia furnacalis(Guenee),is one of the most important insect pests of maize in China.Transgenic Bacillus thuringiensis maize expressing Bt toxins efficiently in the whole development stages provide a new tool for control of the ACB.And transgenic Bt maize expressing Crylle toxin provides good levels of control against ACB as well as showing a great potential to deal with Cry1Ac resistant insects,such as cotton bollworm,Helicoverpa armigera,a major insect pest of cotton and maize.Over the past twenty years,production practices have shown that widespread adoption and prolonged use of single trait maize could rapidly lead to the evolution of resistance within target pest populations in North America,Western Europe and South America.In order to achieve the sustainable utilization of Bt maize and to delay resistance,it is necessary to develop effective resistance monitoring programs and appropriate resistance management strategies.It is essential to determine the baseline susceptibility of target insect populations and to understand the characteristic of resistance to Bt toxins for implementing appropriate resistance management strategies.Therefore,the objectives of the current study were 1)to monitor spacial and temporal variability in susceptibility of the ACB from geographically distinct sites across the major corn-growing areas of China to several Bt toxins;2)to study the evolution of resistance to Bt toxins in the ACB under laboratory conditions.Here we imitate three conditions:exposure to a single toxin,mixed bi-toxins(similar to pyramiding in a single plant),or two toxins in alternation(similar to Bt crop rotation).3)to do some research on resistance mechanism in Cry1F-selected ACB.The resuts are sa follows:1.Monitor spacial and temporal variability in susceptibility of the ACB to several Bt toxins.Collections of 14 populations of ACB were made through 2015 to 2017 from 7 provinces of corn planting sites to determine the susceptibility to purified Cry1Ab,Crylle,Cry1F,Cry1Ah and Cry 1 Ac proteins.The bioassays were performed by exposing neonates to the agar-free semi-artificial diet incorporated with toxins.The RR(resistance ratio)of Cry1Ab toxin ranged from 0.35 to 1.17.The RR of Cry1F toxin ranged from 0.15 to 1.57.The RR of Cry1le toxin ranged from 0.27 to 1.35.The RR of CrylAc toxin ranged from 0.36 to 2.9.The RR of Cry1Ah toxin ranged from 0.31 to 1.08 ?g/g.Although variation in susceptibility among ACB populations were observed,these differences were not caused by prior exposure to Bt insecticides.Instead,it may reflect natural variation.ACB field populations appear to remain susceptible to the tested toxins.Use the diagnostic dose to monitor resistant individuals among the field populations from 2015 to 2017,the diagnostic dose of each toxin are as follows:CrylAb(40.5 ?g/g and 94.19 ?g/g)?Cry1Ie(299 ?g/g)?Cry1F(425 ?g/g).In 2015 and 2016,the number of tested larvae was 96 for each field population.In 2017,the number of tested larvae was 960 for each field population.All the tested larva died in 2015 and 2016.Despite some survival in discriminating dose testing in 2017,growth inhibition data indicated that Bt toxins had significant effects on ACB growth and development,with growth inhibition rate ranged between 93.52%(Cry1Ie:Songyuan)and 98.06%(CrylAb:Luoyang).It is quite impotrant to determine the baseline susceptibility of ACB field populations to Bt toxins before the wide commercialization of Bt maize in China.And the data represent the first step toward the establishment of an effective monitoring programme for the ACB.2.Specific binding of Cry1F in resistant and susceptible strains of the ACB BBMVs.Homologous competition assays were performed incubating BBMV with Alexa-Cry1F and increasing concentrations of unlabeled Cry 1F,and the result indicated that binding of Cry1F is of high affinity on BBMV of ACB-BtS.Compare Cry1F receptor abundance and Cry 1F binding affinity between ACB-BtS and ACB-FR by varying the amount of BBMVs and by saturation binding of Cry1F,and the results indicated that Cry1F binding remains absent in ACB-FR BBMVs.Cry1F resistance in ACB-FR appears to be receptor-mediated.Study on the mechanism of ACB to CrylF protein will provide more reference information for the durable utilization of transgenic cry1F maize.3.Study on evolution of resistance to Bt plants expressing single toxin or expressing two or more toxins in the ACB.The initial susceptible strain ACB-BtS were collected from corn field in Yangling,Shaanxi Province,China,and maintained on artificial diet in the laboratory for several generations without exposure to Bt toxins.The larvae were chronically exposed throughout their development to Bt toxins with three regimes:(i)exposure to a single toxin:CrylAb,CrylF or Cry1Ie(similar to maize expressing one toxin continuous cropping);(ii)exposure to mixed bi-toxins:CrylAb and CrylF or Cryl Ab and Cry1Ie(similar to pyramiding in a single plant);(iii)exposure to two toxins in alternation:CrylAb to CrylF or Cry1Ie(similar to Bt crop rotation).The selection was at the concentration of each toxin's LC50 value,i.e.0.2 ?g/g,0.5 ?g/g and 2.0 ?g/g of CrylAb,CrylF and Crylle,respectively.During single toxin exposure resistance to each toxin did not evolve in ACB colonies during the first several generations of selection.After selected for 6 generations,CrylAb-selected ACB's resistance ratio(RR)was only 5.76,indicating robust tolerance to CrylAb despite chronic exposure at LC50 doses over multiple generations.In contrast to the robust endurance of CrylAb,resistance to CrylF and Crylle increased markedly with the RR 556.78 and 111.11,respectively.After mixing CrylAb and CrylF together,then selecting for survivors,the subsequent survivors showed resistance towards CrylF after 6 generations but did not show resistance towards Cry1Ab(RRs:239.54 vs 7.79).When insects were selected on diet containing only CrylF to a diet containing only CrylAb,over the course of multiple generations a similar pattern emerged after 5 generations:the insects showed tolerance to Cry1F but not to Cry1Ab(RRs:223.06 vs 15.77).Similar results were observed when CrylAb was combined with Crylle in either pyramided or rotation-mimicking schemes:after 6 generations,the insects showed tolerance to Crylle but not to CrylAb.When comparing CrylAb selection alone to combinations of CrylAb with CrylF or CrylAb with Cry1le,ACB evolved resistance to CrylAb quicker and to higher RR when another toxin was present.The result is important for establishing resistance monitoring programme and resistance management strategies which are suitable for our country.