| Taxus cuspidata are precious and endangered medicinal plant with abundant endophytic fungal resources.With the continuous increase of pathogens,it was imperative to develop new microbial pesticides.With the deepening of research,it is found that endophytic fungi can inhibit pathogens and reduce the severity of plant disease.Therefore,the first part of this study used the roots of Taxus cuspidata as the material and fungi isolated from the roots as the research object.The antagonistic activity experiments were used to screen antimicrobial strains from the roots.The extract,purify,and identify the antifungal metabolite secreted by root antimicrobial strain,and study the mechanism of action of antimicrobial metabolite.The main contents and results of this study are as follows:1.A total of 409 root fungi were isolated from the roots of Taxus cuspidata.The strain species were determined through morphological observation and molecular identification,mainly belonging to the genus Paraconiothyrium sp.,Aspergillus sp.,Alternaria sp.,Fusarium sp.,Hypocrea sp.,Microdiplodia sp.,Phomopsis sp.,Ilyonectria sp.,Talaromyces sp.,Diaporthe sp.It has been found that the dominant populations of root fungi in Taxus cuspidata are the genera Ilyonectria sp.,Paraconiothyrium sp.and Aspergillus sp.2.The antagonistic activity testing was conducted on the isolated root fungi,and it was found that three fungi had significant antagonistic activity,with the root strain R3 having the strongest antagonistic activity.And the antifungal active ingredients of the antimicrobial strain were extracted,purified,and identified by nuclear magnetic resonance,and the antifungal active ingredient of the antimicrobial strain was determined to be cyclopyazonic acid(CPA).Using CPA content and biomass as indicators,single factor optimization was carried out on the cultivation conditions of antimicrobial strain.The optimal culture medium was potato powder,3%lactose,and 1%ammonium sulfate;The optimal cultivation conditions were:cultivation time of 6 d,cultivation temperature of 28℃,and p H of 6.02.At the same time,we also optimized the cultivation conditions of the antimicrobial strain using response surface methodology.The optimal cultivation conditions were 6 d of cultivation time,28℃of cultivation temperature,and p H 6.4.3.The antimicrobial mechanism of the active ingredients was studied and it was found that CPA has an inhibitory effect on pathogens,with the strongest inhibitory effect on Rhizoctonia solani.And through microscopic observation,we found that CPA inhibits the mycelial growth of Rhizoctonia solani and affects the production of its conidia.We also measured the electrolysis rate and malondialdehyde content,and found that CPAhas strong antifungal activity by increasing electrolyte leakage in Rhizoctonia solani mycelium and enhancing lipid peroxidation in the cell membrane of Rhizoctonia solani.Therefore,CPAnot only inhibits the growth of fungal hyphae and the production of conidia,but also inhibits the growth of pathogen by destroying their cell membranes.In the first part of this study,we isolated,purified,and screened the antagonistic activity of the root fungi of Taxus cuspidata.We obtained excellent root resistant fungi,which laid the foundation for further development and utilization of root resistant fungi.We also extracted,purified,and identified the antimicrobial active components of the root resistant fungi,and studied the mechanism of action of the antimicrobial active components,laying a theoretical foundation for the subsequent development of microbial pesticides.However,the slow growth rate and extremely low paclitaxel content of Taxus cuspidata cannot meet the huge medical market.Some fungi could promote and improve the growth and development of plants,and promoted the accumulation of certain metabolites in plants.In the second part of study,fungi isolated from the rhizosphere soil of Taxus cuspidata were used as the research object.Selective mediums were used to screen rhizosphere growth promoting fungi.Rhizosphere growth promoting fungus were applied to the seedlings of Taxus cuspidata,and the growth indexes of Taxus cuspidata were measured.We also systematically studied the effects on physiological index(antioxidant enzyme activity)and the content of taxoids(paclitaxel,baccatin III,cephalomannine,and 10-deacetylpaclitaxel),and explored their molecular mechanisms through transcriptome sequencing.The main contents and results of this study are as follows:4.A total of 649 strains of rhizosphere fungi were isolated from the rhizosphere soil of Taxus cuspidata using the spread plate method on PDA medium.The species of the strains were determined through morphological observation and molecular identification,mainly including Hypocrea sp.,Ilyonectria sp.,Aspergillus sp.,Fusarium sp.,Alternaria sp.,Phomopsis sp.,Syncephalastrum sp.,Actinomucor sp.,Nectria sp.,Talaromyces sp.,Diaporthe sp.,Colletotrichum sp.,Coprinopsis sp.And Hypocrea sp.,Aspergillus sp.,Phomopsis sp.and Syncephalastrum sp.are the dominant populations of rhizosphere fungi in Taxus cuspidata.5.Through in vitro growth promotion screening of isolated rhizosphere soil fungi,this study found that 10 strains of rhizosphere soil fungi had nitrogen fixation potential and 8 strains of rhizosphere soil fungi had phosphorus dissolving potential.And,6 strains of rhizosphere soil fungi secreted indole-3-acetic acid and 12 strains of rhizosphere soil fungi produced protease.The rhizosphere soil fungus GS16 has the strongest ability to secrete indole-3-acetic acid,and has the potential for nitrogen fixation and phosphorus dissolution.The growth promoting activity of rhizosphere soil fungus GS16 on three year old seedlings of Taxus cuspidata was studied.The study found that the seedling height and root length of Taxus cuspidata treated with 1×10~5cfu/m L GS16were significantly increased compared to the control group.The number of lateral roots of Taxus cuspidata seedlings treated with 1×10~5cfu/m L GS16 significantly increased,compared with the control.These results indicate that rhizosphere soil fungus GS16 promotes the growth and development of seedlings.In general,rhizosphere soil fungus GS16 not only has growth promoting activity in vitro,but also has growth promoting activity in vivo.6.In order to explore the effects of rhizosphere soil fungus GS16 treatment on the physiological level of Taxus cuspidata,the activities of antioxidant enzymes(including POD,SOD,and CAT)in Taxus cuspidata seedlings were tested.This study found that the antioxidant enzyme activity of Taxus cuspidata seedlings treated with 1×10~5cfu/m L GS16 and IAA increased first and then decreased with the increase of time.And the antioxidant enzyme activity of 1×10~5cfu/m L GS16treatment was not only higher than that in 1×10~6cfu/m L,1×10~7cfu/m L and 1×10~8cfu/m L GS16treatments,but also almost higher than that of IAA treatment.At the same time,we found that the highest content of antioxidant enzyme activity in Taxus cuspidata treated with 1×10~5cfu/m L GS16mainly occurred at 35 d.In general,rhizosphere soil fungus GS16 increases the activity of antioxidant enzymes in seedlings of Taxus cuspidata,which will providean important guiding significance for the efficient utilization of Taxus cuspidata resources.7.In order to investigate the effect of rhizosphere soil fungus GS16 on the accumulation of target active components in Taxus cuspidata seedlings,the content of taxoids in Taxus cuspidata seedlings was detected.The study found that the accumulation of taxoids in seedlings of Taxus cuspidata treated with 1×10~5cfu/m L GS16 and IAA increased first and then decreased.Moreover,the accumulation of taxoids in 1×10~5cfu/m L GS16 treatment was not only higher than that in IAA treatment,but also higher than that in 1×10~6cfu/m L,1×10~7cfu/m L and 1×10~8cfu/m L GS16treatments.At the same time,we found that the highest accumulation of taxoids in Taxus cuspidata treated with 1×10~5cfu/m L GS16 was mainly concentrated at 35 d.In general,rhizosphere soil fungus GS16 increases the accumulation of taxoids in seedlings of Taxus cuspidata,which will provide an important theoretical basis for the efficient utilization of Taxus cuspidata resources.8.In order to explore the effect of rhizosphere soil growth promoting fungi on taxol biosynthesis pathway genes by promoting the accumulation of taxanes,transcriptome sequencing was carried out.Through differential gene expression analysis,it was found that most of the annotated differentially expressed genes in 1×10~5cfu/m L GS16 and IAA treated Taxus cuspidata were distributed in metabolic and cellular processes.At the same time,we also found that the GO classification enrichment pathway of differentially expressed genes in T.cuspidata treated with IAA is very similar to that of differentially expressed genes in T.cuspidata treated with 1×10~5cfu/m L GS16.We analyzed that the possible reason is that the rhizosphere soil promoting fungus GS16 can self secrete IAA,which is likely to regulate the accumulation of secondary metabolites in T.cuspidata through similar pathways compared to exogenous application of IAA.By analyzing the synthesis pathway of paclitaxel in T.cuspidata treated with rhizosphere soil promoting fungi,we found significant changes in multiple key enzyme genes in the synthesis pathway.This includes a significant upregulation of the 3’-N debenzoyl-2’-deoxypacilitaxel N-benzoyltransferase,which is involved in the key step of converting the precursor compound of paclitaxel,"3’-N debenzoyl-2’-deoxytaxol,into 3’-N debenzoyl-2’-deoxytaxol.And cytochrome P450 enzyme CYP720B23 was significantly up-regulated,participating in the catalytic conversion of paclitaxel precursor compound 10-deacetyl-10-hydroxybacardienone(10-DAB III)to paclitaxel precursor compound 7-neneneba acetoxyneneneea 10-hydroxybacardienone(10-DAB II).The cytochrome P450 enzyme CYP750C28 was significantly upregulated,which was mainly involved in the conversion and synthesis of key intermediates in the taxol synthesis pathway.In the second part of this study,we isolated,purified,and screened fungi from the rhizosphere soil of Taxus cuspidata.We had obtained excellent rhizosphere growth promoting fungus,which could lay a foundation for further development and utilization of rhizosphere growth promoting fungi.We also conducted in vivo growth promoting experiments on rhizosphere growth promoting fungi in seedlings of Taxus cuspidata,and studied its impact on the content of target metabolites,physiological indicators,key genes and transcription factors of secondary metabolic pathways of Taxus cuspidata.This will provide important data reference and theoretical basis for the subsequent targeted incremental cultivation of Taxus cuspidata,the analysis of taxol biosynthesis pathways and metabolic regulation mechanisms,and the efficient utilization of Taxus cuspidata resources. |
PDF Full Text Request