Effects And Mechanisms Of Arbuscular Mycorrhizal Fungi On Transgenic Bt Maize And Armyworm Mythimna Separata Under Elevated CO₂

The promotion and application of transgenic Bt resistant-insect crops provides a new approach for the prevention and control of lepidopteran pests,and effectively relieves the environmental pressure resulted by the large amount usage of chemical pesticides on the environment.In the meantime,the global atmospheric CO2 concentration has increased from 280 ?L/L before the industrial revolution to 408.52 ?L/L in 2018.Elevated atmospheric CO2 concentration(ab.elevated CO2)can significantly reduce the content of exogenous Bt-toxin protein of Bt crops while increasing crop yield and biomass accumulation,and in turn,leads to the ecological risk of reducing the target insect resistance.On the other hand,arbuscular mycorrhizal fungi(ab.AMF)are important beneficial microorganism that can form a mutually beneficial symbiotic relationship with ?80%of terrestrial plants in the world.AMF obtain carbon sources from host plants,and at the same time provide host plants with mineral elements(such as N and P,etc),thereby increasing the nitrogen absorption efficiency of the host plant roots,which is expected to improve the nitrogen metabolism physiology of transgenic Bt crops,and then promote the expression and synthesis of exogenous Bt-toxin protein.Defining this change and the internal mechanisms of transgenic Bt crops caused by elevated CO2 will be helpful on the ecological sustainable use of transgenic Bt crops under climate change.In this study,transgenic Bt maize was selected and combined with the hotspot problem of global climate change with atmospheric CO2 concentration arising,through inoculation with a species of AMF(Glomus caledonium)to research the plant growth and yield of transgenic Bt maize(serial number:IE09S034,exogenous gene:CryIIe)and its parental line of non-transgenic maize(ab.non-Bt maize;cv.Xianyu 335),and explore the effects of different treatments on the growth,development and reproduction of pest by using the main pest-armyworm that can survive and affected fed on Bt maize under ambient CO2(about 375 ?L/L)and elevated CO2(750 ?L/L).The main findings are shown as follows:1.Effects of the AMF inoculation on AMF colonization and soil physicochemical properties of transgenic Bt maize and its parental line of non-Bt maize grown under elevated CO2Transgenic Bt maize and its parental line of non-Bt maize were selected to investigate the effects of elevated CO2 on the AMF colonization situation in maize roots,and the impacts of the AMF inoculation on the physical and chemical properties of rhizosphere soil of maize plants.The results showed that:(1)Elevated CO2 and AMF inoculation both significantly increased the AMF colonization and the AMF-PLFA content in rhizosphere soil of transgenic Bt maize and its parental line of non-Bt maize.(2)CO2 level,AMF inoculation and transgenic treatment did not significantly affect pH values or the contents of total nitrogen and total phosphorus in the rhizosphere soil of maize plants.However,the inoculation with AMF at elevated CO2 significantly reduced organic carbon content in the rhizosphere soil of transgenic Bt maize and its parental line of non-Bt maize.(3)There were different effects of elevated CO2 and AMF inoculation on available nitrogen content in rhizosphere soil of maize plants,just showing opposite impacts on ammonium nitrogen and nitrate nitrogen,that was,elevated CO2 reduced the available nitrogen content while the inoculation of AMF increased the available nitrogen content in the rhizosphere soil of transgenic Bt maize and its parental line of non-Bt maize.Moreover,elevated CO2 and AMF inoculation both significantly reduced the ammonium nitrogen content and significantly increased the nitrate nitrogen content in the rhizosphere soil of transgenic Bt maize and its parental line of non-Bt maize.(4)Elevated CO2 and AMF inoculation significantly increased the contents of available phosphorus and available potassium in the rhizosphere soil of transgenic Bt maize and its parental line of non-Bt maize.So elevated CO2 is beneficial for the infestation of AMF on maize roots,and it can improve plant growth of transgenic Bt maize and its parental line of non-Bt maize by increasing the available elements contents in rhizosphere soil or promoting the absorption of soil nutrients by plants.2.Effects of the AMF inoculation on plant growth and nitrogen metabolism of transgenic Bt maize and its parental line of non-Bt maize grown under elevated CO2Transgenic Bt maize and and its parental line of non-Bt maize were used to explore the effects of AMF inoculation on the nitrogen metabolism,plant nutrition and growth of transgenic Bt maize and its parental maize grown under elevated CO2.The results indicated that:(1)Elevated CO2 and AMF inoculation both significantly increased the activity of alkaline protease and urease in rhizosphere soil of transgenic Bt maize and its parental line of non-Bt maize.(2)AMF inoculation significantly increased the activity of nitrate reductase,glutamate synthetase and glutamine synthetase in leaves of transgenic Bt maize and its parental line of non-Bt maize;in addition,elevated CO2 significantly increased the glutamine synthetase activity in the leaves of transgenic Bt maize and its parental line of non-Bt maize,and significantly reduced the nitrate reductase activity in the leaves of transgenic Bt maize.(3)AMF inoculation significantly reduced the total carbon content and significantly increased the contents of total nitrogen,total phosphorus and total potassium in the leaves of transgenic Bt maize and its parental line of non-Bt maize;in addition,elevated CO2 significantly increased the total carbon and total phosphorus contents,and significantly reduced the total nitrogen content in the leaves of transgenic Bt maize and its parental line of non-Bt maize without the inoculation of AMF.Moreover,elevated CO2 significantly increased the total nitrogen content in the leaves of transgenic Bt maize and its parental line of non-Bt maize both inoculated with AMF,and just significantly increased the total potassium content in the leaves of transgenic Bt maize inoculated with AMF.(4)Elevated CO2 and AMF inoculation both promoted maize plant biomass and economic yield of transgenic Bt maize and its parental line of non-Bt maize,respectively.So the inoculation of AMF can enhance the nitrogen metabolism of transgenic Bt maize and its parental line of non-Bt maize grown under elevated CO2,promote the growth of maize plants,thereby increasing the biomass and economic yield of maize,and is expected to achieve the decrease in fertilization supply and the increase in fertilization usage efficiency in maize production under climate change.Compared with CO2 level and AMF inoculation,the effects of transgenic treatment on plant growth and nitrogen metabolism physiology are marginal and slight.3.Effects and mechanism of the AMF inoculation on growth and development of the armyworm fed on transgenic Bt maize and its parental line of non-Bt maize under elevated CO2The effects of AMF inoculation on the growth,development and reproduction of armyworm,Mythimna separata fed on transgenic Bt maize and its parental non-Bt maize grown under ambient and elevated CO2 were studied,as well as the effects on the foliar content of exogenous Bt-toxin protein and endogenous secondary defense substances,including of jasmonic acid(ab.JA)and salicylic acid(ab.SA),and the transcriptive expression levels of the respectively regulatory genes in the leaves of transgenic Bt maize.The results showed that:(1)Transgenic Bt treatment had adverse effects on the feeding and utilization of M.separata larvae.Moreover,the AMF inoculation had positive impacts on the feeding and utilization of armyworm larvae fed on non-Bt maize,while had adverse influences on the feeding and utilization of army worm larvae fed on transgenic Bt maize.(2)Transgenic Bt treatment and elevated CO2 both delayed the development duration of armyworm larvae and pupae,and reduced the pupal weight and fecundity fed on transgenic Bt maize and its parental line of non-Bt maize inoculated with and without AMF,which was unfavorable for the population occurrence of armyworm,M.separata.However,the AMF inoculation had positive effects on the growth,development and reproduction of M.separata fed on the non-Bt maize,and simultaneously improved the resistance ability of transgenic Bt maize against the main insect pest of M.separata through delaying the development,and reducing the pupal weight and fecundity M.separata.(3)Elevated CO2 significantly reduced the foliar content of exogenous Bt-toxin protein and the transgene expression level of transgenic Bt maize inoculated without AMF,while significantly increased the content of exogenous Bt-toxin protein of transgenic Bt maize inoculated with AMF.In addition,AMF inoculation significantly increased the foliar content of exogenous Bt-toxin protein and the transgene expression level of transgenic Bt maize grown under ambient and elevated CO2 respectively.(4)AMF inoculation significantly increased the foliar content of JA and the expression levels of LOX and AOS which were key genes in JA pathway,as well as the foliar content of SA and the expression levels of PAL and NPR1 which were key genes in SA pathway for transgenic Bt maize and its parental line of non-Bt maize grown under ambient and elevated CO2.Moreover,elevated CO2 significantly increased the foliar content of JA and the expression levels of LOX and AOS,as well as the foliar content of SA and the expression level of PAL for transgenic Bt maize and its parental line of non-Bt maize inoculated with and without AMF.So elevated CO2 has adverse effects on the expression of Bt-transgene and the content of exogenous Bt-toxin protein in the leaves of transgenic Bt maize,resulting of the ecological risk of reducing its target resistance to insect pests.However,AMF inoculation greatly increases the expression of Bt-transgene and the content of exogenous Bt-toxin protein in the leaves of transgenic Bt maize grown under elevated CO2,thereby increasing its target resistance to insect pests.Furthermore,AMF inoculation and elevated CO2 both promote the production of endogenous secondary defense substances in transgenic Bt maize by increasing the expression levels of key genes in the JA and SA pathways,thereby improving its endogenous secondary defense ability.In summary,elevated CO2 and AMF inoculation are both conducive to enhance the contents of soil nutrient elements,as well as improve the absorption and utilization efficiency of nutrient elements by transgenic Bt maize and its parental line of non-Bt maize,and finally increase the biomass and economic yield of maize crops.The foliar content of exogenous Bttoxin protein of transgenic Bt maize without AMF inoculation has a marked decline under elevated CO2,while AMF inoculation not only largely relieves the risk of reducing the target insect resistance caused by the decrease of exogenous Bt-toxin protein for transgenic Bt maize grown under elevated CO2,but also greatly enhances the nitrogen metabolism level and significantly increases the expression level of Bt-transgene and the foliar exogenous Bttoxin protein of transgenic Bt maize.Combined with the significant increases of the endogenous secondary defense substances(JA,SA)and the key gene expression levels in the leaves of transgenic Bt maize,the control effects of transgenic Bt maize on the main lepidopteran pest of armyworm,M.separata are largely enhanced.Therefore,this study confirm that the AMF(G.caledonium)used to infect transgenic Bt maize can enhance the nitrogen utilization efficiency of crops and promote the expression levels of its endogenous and exogenous secondary defense substances,and the plant growth of transgenic Bt maize grown under elevated CO2,and finally achieve the optimal management of fertilizers for Bt crops that reduce the fertilizers application and enhance the fertilizers efficiency and the effects of resistance of transgenic Bt crops to insect pests,which will help ensure the ecological production safety and sustainable utilization of transgenic Bt crops under climate change.