| Endophytic fungi with the rich biodiversity and secondary metabolites diversity,in recent years,has become an important source of biologically active compounds,and has developed a new hot spot of research in the field of agriculture,health care and so on.Medicinal plants pigeon pea[Cajanuscajan(L.)Millsp.],mainly distributed in tropical and subtropical countries,has the functions of relieving heat,detoxification,antibacterial,and anti-inflammatory activities.All these presented strong potential to found new endophytic fungi.In this study,endophytic fungi were isolated from pigeon pea,after anti-pathogenic activity screening and LC-MS/MS detection,three flavipin-producing endophytic fungi were identified.The isolate ELP-8-9 with high flavipin amount was chosen to make an optimization of fermented conditions.Furthermore,separation and purifying flavipin from endophytic fungus ELP-8-9,the inhibition activity of fungal flavipin against Fusarium oxysporum and soybean root rot were studied,the results were as follows:1.For the first time,three endophytic fungi producing:flavipin were isolated from pigeon pea and systematic identified;Endophytic fungi were isolated from pigeon pea roots,stems,leaves,flowers,bean-pod and seeds,a total of 156 strains endophytic fungi were obtained.After a preliminary screening,29 strains showed antifungal activity against at least one of the plant pathogenic fungi(inhibition rate of more than 50%),accounting for 18.59%of the total endophytic fungi number of tests.The anti-plant pathogen activity of crude extracts of active strains were rescreening,ELP-5-3?ELP-7-7?ELP-8-9?ELP-9-2?ELP-17-1?ESP-10-2?ERP-11-4 strains with good anti-plant pathogen activities were obtained and three endophytic fungi(ELP-7-7?ELP-8-9?ELP-9-2)producing flavipin were obtained by LC-MS/MS method.After charactered by morphology and molecular methods,they seemed most closely related to Chaetomium globosum.2.The best fermented technology conditions of endophytic fungus ELP-8-9 were obtained by optimization of the fermented medium and culture conditions;ELP-8-9 was chose to optimize the fermentation conditions for flavipin production effected by different factors.It is mainly refers to kinds of fermented medium,carbon source,nitrogen source,culture time,temperature,initial pH value,and so on.The results showed that the optimal fermentation medium:PDB medium,2%glucose as carbon source.Culture time,temperature,initial pH value were selected to central composite design and response surface optimization.The best fermented technology conditions:culture time:8 days,temperature:24?,pH value:5.6.The maximum production of flavipin from ELP-8-9 was 1.267 ± 0.032 mg/L.3.The purification technology of fermented EtOAc extracts flavipin was obtained by normal-phase medium pressure silica gel column chromatography;Endophytic fungus ELP-8-9 fermented extracts were separated and purified for flavipin by normal-phase medium pressure silica gel column chromatography.Purification technology conditions:300-400 mesh silica;the quantity ratio of sample/silica gel was 1:10;an gradient elution was performed with n-hexane-ethyl acetate(20:1,10:1,7:1,5:1,v/v)on the column,the flow rate was 10 mL/min.In the process of the column chromatography,the target composition containing a high content of flavipin were combined,and reduced pressure concentration to a certain volume.When there is deep yellow particle precipitation,it is the crude of flavipin.Endophytic fungus EtOAc extracts was purified to 97.35%of flavipin by normal-phase medium pressure silica gel column chromatography,crystallization and recrystallization.4.The inhibition activity of fungal flavipin against Fusarium oxysporum was studied.Then,the inhibition activity of fungal flavipin against soybean root rot was investigated by greenhouse experiments;In this study,flavipin inhibited the mycelial growth and the spore germination of F.oxysporum.When the flavipin concentration of 80 ?g/mL,the inhibition zone reached 25.8 ±0.5 mm,and the inhibition rate of spore germination reached 91.5%,with the increase of flavipin concentration,inhibition rate of spore germination was increased.The conductivity of F.oxysporum mycelia was increased after treated with flavipin,suggested that the increase of cell membrane permeability lead to damage of cell membrane structure.Determination results of malondialdehyde(MDA)also showed that a high degree of membrane lipid peroxidation,cell membrane was badly damaged.The efficacy of biological control of flavipin on soybean seedlings was investigated.The results showed that flavipin concentration was 80 and 100 ?g/mL,the control effects on soybean root rot were 81.25%and 85.14%after treating 15 days,which was slightly lower than 10 ?g/mL carbendazim(89.76%).Therefore,flavipin has potential applications in the field for the prevention and control of soybean root rot.In this study,three endophytic fungi producing flavipin were isolated from pigeon pea and identified systematic;the best fermented technology conditions of endophytic fungus ELP-8-9 with high level flavipin were obtained by optimizing the fermented medium and culture conditions;the purification technology of fermented EtOAc extracts flavipin were obtained.Addtionally,the inhibition activity of fungal flavipin against Fusarium oxysporum was studied.Then,the inhibition activity of fungal flavipin against soybean root rot was investigated by greenhouse experiments.In summary,this study has laid a solid foundation for the effective use of pigeon pea endophytic fungi and its active ingredient flavipin,also provided a new source of microbial pesticide research and development new path and method!... |
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