Screening,Identification Of Multifunctional Trichoderma And Agrobacterium Tumefaciens Mediated Transformation Of Trichoderma Koaningii

Trichoderma,an important part of soil microbial flora which can control a variety of plant disease if increase its content in soil,is a kind of biological control fungi of wide application.By analyzing the genetic and functional diversity of Trichoderma,it can provide the theoretical foundation for developing multifunctional Trichoderma,expanding their application area in effectively controlling plant pathogens.It is possible to find Triehoderma strains of multifunction like phosphate solubilization,except for the ability to control plant pathogens.1.Functional properties of 60 isolates of Trichoderma from soil were studied.All of the 60 Trichoderma strains were detected in their capability to degrade insoluble-phosphate,insoluble-potassium and chlorpyrifos by solid plate culture method.The results showed that no isolate had obvious insoluble-phosphate solubilization and degrade chlorpyrifos ability,but transparent circle was on solid medium which had insoluble-potassium.Trichoderma were dually cultured with Botrytis cinerea which is a pathogen to capsicum,and 26 isolates were shown to inhibit Botrytis cinerea,of which 12 isolates showed better inhibitory ability than the others.Trichoderma were detected in liquid culture.The result indicated that some strains did not show the ability of solubilizing the insoluble-phosphate,whereas isolate T-404,T-440,T-497,T-403,T-400 showed stronger ability in solubilizing the insoluble-phosphate,in which isolate T-404(PO4-3 content,109.22 mg/L)was the strongest;no isolate showed strong ability in solubilizing insoluble-potassium in liquid culture.Most isolates could endure chlorpyrifos at a high concentration,but couldn't degrade it.Soaking seed of capsicum with zymolytic liquid of Trichoderma strains was studied on germinate ratio,germinate force and root length,the result showed that the effect of some isolates to germinate of seed was not evidence,and even had certain inhibition.1 and 50 times dilute zymolytic liquid of isolate T-450 had a relatively notable promote for germinate of seed.Isolate T-400 had an relatively notable promoting role to capsicum,which could promote the fresh weight.In view of the results above,five isolates were selected with multifunctional potential.They were isolate T-400,T-403,T-404,T-440 and T-450.2.5 Trichoderma strains were identified on their morphological characters and homologous analysis of ITS and 18S rDNA nucleotide sequence.Isolate T-403,T-404 and T-440 were classified as Trichoderma koningiopsis(T.koningiopsis);and isolate T-400,T-450 were classified as Trichoderma koningii(T.koningii).3.The factors influencing the transformantion efficiency of agrobacterium tumefaciens(A.tumefaciens)mediated transformation of T.koningii were studied.The results showed that the transformation efficiency was correlated with the 3'5'-Dimethoxy-4-Hydroxy Acetophenone(AS)concentration in induced medium,the induced time of A.tumefaciens,and the initial concentration of A.tumefaciens and the initial concentration of T.koningii conidia.According to these results,we established optimal conditions for transformation of T.koningii as follows:A.tumefaciens cultured to OD600 0.40?0.58 in induced medium with 200?M AS for 6 hours and then concentrated by 2 times;mixing the A.tumefaciens ferment broth with same volume T.koningii conidia suspension which concentration was 107 conidia per miilliter;spreading 400 ?L mixture on the co-cultivation medium(200 ?M AS)with a filter paper on it;incubating 48h at 26?.Under these conditions,the transformation efficiency of ATMT on T.koningii is 60-130 mutants per 107 conidia.We constructed a T-DNA insertional mutant library of T.koningii with 200 transformants.The quality of the mutant library was evaluated.All the randomly selected resistant colonies were proved to be stable through mitotic cell division.4.Mutants ware detected in their capability to degrade insoluble-phosphate.The result showed that mutants,named isolate T-87 of them was the best because it's phosphate-releasing capacity was 123.68 mg/L,inereased 76.57%compared with wild isolate T-400.