| The widespread use of chemical pesticides currently has caused increasinglyserious environmental and agricultural products pollution. Environmentally SafeBio-Pesticide has become a new pathway in the control of pests and plant disease.Antifungal activities of6H-Oxireno(e)(2)benzoxacyclotetradecin-6,12(7H)-dione（monocillinⅠ）is produced by Trichoderma harzianum, one excellent biocontrolagainst a range of plant pathogen. Whereas monocillinⅠ biosynthetic pathway hasbeen unclear and yields did not meet agricultural production needs. Developinghigh-yield strains of monocillin Ⅰ has important theoretical and practicalsignificance of the development and application of new biological pesticides.This study which based on detailed studies monocillin Ⅰ synthesis andimproving its production illustrates pkst-1and pkst-2genes in monocillin Ⅰsynthesis role. Furthermore, the feasibility of optimize of fermentation conditionsfor monocillinⅠ production by high-yield strains were discussed. The results of themain study were as follow.pkst-1gene silencing tranformant T2and pkst-2gene knockout tranformant T4were genetically stable by southern blot analysis. Based on analysis secondarymetabolites from T2, T4and wild-type T88strains, identified pkst-1and pkst-2geneencode key enzymes in the biosynthesis of monocillinⅠ, and suggests polyketidesynthase PKST-1is responsible for regulation of synthesis monocillinⅠ, whilePKST-2is responsible for regulating monocillinⅠmetabolic precursor.The pkst-1and pkst-2gene overexpression transformants T3and T5werestably inherited identified by southern blot analysis. Yields of monocillinⅠby T5was higher13.22%than it by wild type T88, and also was higher11.68%than it byT3（P<0.05）； The inhibition on the grouth of plant-root-rot pathogenRhizoctonia solani by T5fermentation broth was higher14.01%than it by wild-typeT88strains, but the yields of monocillinⅠ and the inhibition on the grouth ofR. solani by T3were the same as them by T88（P>0.05）. The results show that thepkst-2gene over expression strain T5can be used as the monocillinⅠproducingstrain and biocontrol against R. solani.Main biological characteristics of pkst-2gene overexpression transformant T5were studied. The results showed that optimal temperatures for mycelial growth、sporulation and monocillinⅠfermentation are25℃to30℃; An optimal pH valueof medium is slight acidity; light can stimulate sporulation significantly.Easily utilizable carbons, easily utilizable organic nitrogen and inorganic nitrogen as well as the high concentrations of potassium dihydrogen phosphate were fit formycelial growth and sporulation. While slowly utilizable carbons, slowly utilizableorganic nitrogen and the low concentrations of potassium dihydrogen phosphate canpromote monocillinⅠfermentation. In addition, pkst-1and pkst-2gene can expresshighly induced by plant pathogen using relative quantification Real-time PCR. Theresults of indoor antagonism experiment of T5strain to plant pathogen showed thatT5strain can inhibit the growth of plant pathogen by promoting mycelial rupture,atrophy using attachment, winding, parallel growth and piercing methods, and is thesame as wild type T88aganist pathogens as described in the literature.The high-yielding medium development by statistical methods can save a lot oftime and raw material, and the result is effective and reliable. Optimal temperature,initial pH of medium and inoculum concentration for monocillinⅠfermention by T5strain were given using single factor test and Central Composite design, andaccording to regression equation generated response surface. The best combinationfor monocillinⅠfermention by T5strain is temperature26℃, initial pH of medium5.88, and1.81×106spores/mL of inoculum concentration. The highest yield ofβ-Resorcylic acid lactones was18.14mg/L.Regression equation for the optimization of maltose, soy flour and potassiumdihydrogen phosphate for monocillinⅠby T5strain is given by orihogonal designand Plaekett-Burman design, and according to regression equation generatedresponse surface. The best combination is4.4267%of maltose,0.372%of soy flour,and0.04%of potassium dihydrogen phosphate. The highest monocillinⅠyields byT5strain in optimal medium is21.94mg/L, and that is44.73%higher than ininoriginal medium on wild type T. harzianum. |
PDF Full Text Request