Study On The Anti-TMV Activity Polysaccharides Of Two Endophytic Fungus From Ficus Microcarpa

As the world’s oldest virus,TMV（Tobacco Mosaic Virus）annually causes economic losses to global agricultural production of more than one hundred million dollars.Isolating endophytic fungi from medicinal plants and studying their biologically active metabolites has been a hot topic in natural product pesticides research in recent years.Ficus microcarpa is a traditional Chinese herb from southern China.Very little research has been done on its endophytic fungi and their metabolites anti-TMV.In this study,the endophytic fungi of Ficus microcarpa and their metabolites were systematically investigated for their anti-TMV activity.The specific results are as follows:（1）A total of 132 endophytic fungi were isolated from Ficus microcarpa.Two active strains,designated FA109 and FA114,were screened for anti-TMV activity and secondary metabolite production.（2）Identification using morphological and molecular biological classification methods.Construction of a 5-gene joint maximum likelihood tree for CAL,EF1-α,ITS,TUB2 and HIS3,the endophytic fungus FA109 was identified as Diaporthe yumnanensis（Registry Number:CGMCC 3.18289T）,The endophytic fungus FA114 was identified as Diaporthe acutispor（Registry Number:CGMCC 3.18285T、LC 6142、LC 6160）.（3）Isolation of two anti-TMV active polysaccharides SH-3-1 and DH-3-1 from the metabolites of endophytic fungi FA109 and FA114.Their IC₅₀values were 15.87 mg/L and 15.47 mg/L and both provided more than 50%protection against tobacco.Molecular mass of polysaccharides SH-3-1 and DH-3-1 of 53740 Da and 46826 Da,respectively,determined by HPLC.Determination of polysaccharide composition using ion chromatography.The polysaccharide SH-3-1 was determined to composed of rhamnose,arabinose,glucosamine hydrochloride,galactose,glucose and mannose in the molar ratio2.50:1.10:0.30:79.20:3.60:13.40 and DH-3-1 was determined to be composed of arabinose,galactose,glucose and mannose in a molar ratio of 1.70:73.60:10.70:14.00.Both polysaccharides were determined by infrared spectroscopy to be furanose.（4）Optimization of medium fermentation using response surface methodology for 2 endophytic fungi strains.The optimum medium ratio for FA109 was 25.61 g/L lactose,3.14%sodium nitrate mass fraction and1.11 g/L inorganic salt Co Cl₂·6H₂O,producing a maximum polysaccharide concentration of 225.43 mg/L.The optimal medium ratio for FA114 was 20.86 g/L of maltose,4.47%mass fraction of sodium nitrate,and 0.98 g/L mass of inorganic salt Co Cl₂·6H₂O,yielding a maximum polysaccharide concentration of 285.13 mg/L.（5）By analysing the expression levels of disease-stage related genes and defence enzyme activities in Nicotiana benthamiana after polysaccharide treatment,it was clear that the two polysaccharides induced disease resistance,induced up-regulation of PR1,PR3 and PR5gene expression levels and increased defence enzyme POD,SOD and PPO activities in Nicotiana benthamiana.Conclusion:The first large-scale isolation and purification of an endophytic fungus from Ficus microcarpa,with good anti-TMV activity of its metabolites.Two polysaccharides extracted from the metabolites of two endophytic fungal strains with good protective effects on tobacco.They induce up-regulation of the Nicotiana benthamiana disease resistance genes PR1,PR3 and PR5 and increase the activity of defence enzymes such as POD,SOD and PPO.The results indicate that the metabolites of the endophytic fungus of Ficus microcarpa have good biological activity against plant viruses and provide new ideas and approaches for the development of new anti-phytovirus agents.