The Optimization Of Agrobacterium Tumefaciens Mediated Trichoderma Asperellum Transformation System And Analysis Of T-DNA Insertional Mutants

It is globally known that Trichoderma spp.is an important biocontrol agent against plant diseases.Now,the Trichoderma biocontrol factor acquires the highest share in international biological pesticide formulation.However,due to the increasingly environment problem,Trichoderma spp.is inevitably threatened by both biotic and abiotic stress factors when used in the field.And these stress factors affect both the growth and physiological state of Trichoderma spp.,resulting a loss of the ability of biocontrol.Therefore,it has great significance in building a Trichoderma spp.mutant library,laing a foundation for searching Trichoderma whieh are more adaptive and more conductive to prodction,studing the mechanism during Trichoderma spp.interaction with evironment,plants,as well as pathogenic,and knowing about Trichoderma spp.feature of resisting biotic and abiotic environmental conditions.We optimized the Agrobacterium tumefaciens mediated transformation system(ATMT)and constructed the T-DNA insertional mutant library of Trichoderma asperellum.The results indicated that 200?M 3'5'-Dimethoxy-4-Hydroxy Acetophenone(AS)contained both in induced-and co-cultivated(pH 5.0)medium,A.tumefaciens cultured 6h in induced medium,the conidia of T.asperellum is 106 eonidia per milliliter,which shown an optimiztion ATMT system on T.asperellum.And the transformantion efficiency of ATMT on T.asperellum is about 60 mutants per 106 conidia.After analyzed the genetic stabilities of the transformant,200 mutant strains were gained.The T-DNA insertional library for T.asperellum was preliminary developed,thus laying a foundation for searching the T.asperellum which were more adaptive and more conductive to prodction.Two strains of transformant T1-T200,T39 and T45 shown a greatly surpassed than their wild type strain in growth rate,and the T39 also has a increasingly tolerant to A.alternata fermentation liquid.While the T59 has a increasing NaCI tolerance,and the T3 and T5 have a increasing NaHCO3 tolerance.Under NaCI condition,T59 has an increasing antagonistic ability against pathogenic fungi,especially fr59 has the highest inhibition rate of 52.00%against A.alternate.Though T3 and T5 have a same antagonistic ability against both A.alternate and R.solani under NaHC03 condition,their antagortistic ability against F.oxysporum declines with a increase in the concentration of NaHCO3.Through the co-culturing test,T39 and T45 shown a much improved antagonistic ability than their wild type strain,especially T45 has the maximum inhibition rate of 80.76%against R,solani.Furthermore,we induced the Pop“lus davidiana×P.alba var.pyramidalis seedlings with T39,T45 and their wild type strain,respectively,the results shown that all three strains can promoting both the growth of Populus and the development of its root,moreover,the three strains can also increased the activity of the SOD,POD,CAT and PAL.Among them,T45 has distinct differences in promot the growth of seedlings and its enzyme activities.The growth rate is 14.28%compare with the control group;and the increased activity of SOD,POD,CAT and PAL were 2.63,2.45,12 and 6.35 times compare with the control group,respectively.Induce WT and its NaHCO3 resistant mutant strains T3 and T5 with the Populus davidianaxP.alba var.Pyramidalis seedlings which were treated in NaHCO3 of different concentrations.The results shown that NaHCO3 which concentration is under 0.2%has very small impact on the seedlings;0.2%NaHCO3 seriously harmed the growth of the seedlings,the seedlings is in dehydration and its root is seriously browning.In 0.3%NaHCO3,the Populus davidiana×P.alba var.Pyramidalis seedlings were seriously harmed and died after 3 days.But though induced with the three strains,the activity of SOD,CAT and POD in the Populus davidianaxP.alba var.pyramidalis leaves were efficiently improve as well as its tolerance to NaHCO3.However,all the three strains has almost the same impact on the activity of the SOD,CAT and POD in the Populus davidianaxP.alba var.pyramidalis leaves.This study laid a theoretical and material foundation for making the T.asperellum agent with more adaptability to the environment and more prominent biocontrol,and is also conducive to the study on the function of genes related to the antireversion force feature of T.asperellum.