Mechanism And Application Of Trichoderma Atroviride D16 Inducing Tanshinones Biosynthesis In Salvia Miltiorrhiza

Salvia miltiorrhiza Bge.is a traditional medical plant in China and widely-used in clinic medicine.Due to excessive collection of wild plant resources and unstable cultivar,production and quality of cultivated S.miltiorrhiza can't meet the requirements of market.As researched,endophytic fungus is one kind of fungi resident in various plant tissues and organs for a period or the whole lifespan without causing any damage or hypersensitivity,which can promote medical plants growth and secondary metabolites accumulation.Start with endophytic fungi,my team previously isolated Trichoderma atroviride D16 from geo-authentic habitat Shangluo,Shanxi province in China,which could greatly promote S.miltiorrhiza hairy roots growth and tanshinones accumulation.The effects of Trichoderma atroviride D16 are significantly surpassing than other chemical and biological elicitors.And the active part of T.atroviride D16was confirmed as polysaccharide fraction?PSF?and mechanism of PSF promoting tanshinones accumulation was studied preliminarily through comparative transcriptomics.However,it keeps unclear why endophytic fungi could promote secondary metabolites accumulation,which restricts the practical application of endophytic fungi.The study separated and purified polysaccharide fraction of T.atroviride D16 to confirm the specific single polysaccharide PSF-W-1 and further confirm the character of PSF-W-1.Based on comparative transcriptomics,several signal molecules(H₂O₂,Ca²⁺,NO,SA,JA,ABA)and three key transcription factors were studied in the transduction network of D16_PSFSF inducing tanshinones accumulation.And finally,T.atroviride D16 was manufactured into fertilizer to confirm the effects on S.miltiorrhiza seedlings cultivation.The best application method and dose was confirmed via greenhouse and field experiments.The main results are as followed:1.Based on polysaccharide fraction?PSF?of T.atroviride D16,monosaccharide composition of PSF was analyzed and function of each monosaccharide was compared with PSF,resulting in best synergetic effect of PSF on tanshinones accumulation.The water fraction of D16_PSF was determined as the main active fraction through modern technique and biologic tests.And finally,PSF-W-1 was isolated and purified further from water fraction.Chemical structure of PSF-W-1 was analyzed and molecular weight was 36.13 kDa.The content of carbohydrate and uronic acid was determined to be 94.15%and 0.76%by the phenolsulfuric acid method and m-hydroxydiphenyl method,respectively.2.Based on analysis of comparative transcriptomics,several signal molecules were related to PSF inducing tanshinones accumulation,including H₂O₂,Ca²⁺,NO,SA,JA,ABA.Under the treatment of inhibitors and carriers,function of H₂O₂ and Ca²⁺was certified further during the process of PSF inducing tanshinones accumulation.As researched,H₂O₂ is the key transduction signal molecule and NO and ABA may play a significant role in the transduction network during PSF inducing tanshinones accumulation.A prediction was put forward that PSF triggered S.miltiorrhiza defense button and H₂O₂ as an upstream signal molecule passed the message to NO and ABA,which caused transcriptional factors expression and tanshinones synthesis.However,this prediction needs to be verified through further experiments.3.Based on further analysis of comparative transcriptomics,SmERF1B,SmGRF1 and SmMYB86 were identified as key transcription factors inducing tanshinones synthesis under the treatment of PSF.Full-length gene of three key transcription factors were cloned and analyzed,which showed SmERF1B,SmGRF1 and SmMYB86 were constituted by 164 amino acids?aa?,375aa and 318aa.SmERF1B contains AP2 domain and SmGRF1 contains QLQ and WRC domain,while SmMYB86 contains SANT domain.The function parts of three putative key transcription factors are targeted to the plant cell nucleus by subcellular localization analysis.The three key transcription factors overexpressed hairy roots system were constructed for the further study.4.Trichoderma atroviride D16 was manufactured into solid fertilizer and applied on S.miltiorrhiza seedlings compared with PSF liquid fertilizer in greenhouse.Compared to PSF liquid fertilizer,solid manure effects are better in the chamber experiment.The optimum application dose of Trichoderma atroviride D16 was preliminarily determined at 15 g per S.miltiorrhiza seedling in greenhouse;Considerring the effect of actual cultivation environment and input-output ratio,5 g solid manure per S.miltiorrhiza seedling is more suitable through further field experiment.