Studies On The Biosynthesis Of Nanoparticles By The Endophytic Fungus And The Antifungal Activity Of The Fungal Secondary Metabolites

1. The biosynthesis of nanoparticles by the endophytic fungusSilver/gold nanoparticles have been widely used in the medical field because oftheir unique physical and chemical properties. Chemical reduction is the conventionalsynthetic method of preparing silver/gold nanoparticles. Considering chemical agentsused in synthesis may have adverse side effects for the preparation of compounds formedical applications, it is highly desirable to develop a green avenue for the synthesisof silver/gold nanoparticles. Biological methods of synthesis have proven to be bettermethods due to they minimize the factors such as toxicity and cost.Endophytic fungi are fungal microorganisms that colonize healthy plant tissueintercellularly or intracellularly without causing any apparent symptoms of disease. Inorder to maintain a stable symbiosis, endophytic fungi secrete varieties ofextracellular enzymes and small molecule compounds to resist external pressure.These bioactive metabolites offer the potential for biosynthesis, biotransformation andbiodegradation. However, endophytic fungi are relatively unexplored as a potentialresource for the synthesis of silver/gold nanoparticles, waiting to be exploited.This study collected4species of plants, isolated58strains of plant endophyticfungi, and the screening of the biosynthesis of silver nanoparticles (Ag-NPs) wascarried out by observing the color change of the cell filtrate and using a UV–visiblespectrophotometer. Only strain Q01was able to synthesize Ag-NPs under normal airpressure and temperature, and strain Q01was identified as Epicoccum nigrumaccording to morphological characteristics and molecular analysis. Thecharacterization of the silver nanoparticles was carried out by transmission electronmicroscopy (TEM), X-ray diffraction (XRD) and Fourier transform infraredspectroscopy (FTIR). The synthesized silver nanoparticle were found to bepolydispersed spherical or near to spherical particles ranging from1to22nm andexceptionally stable.The crystal structure of silver nanoparticles was face centeredcubic (fcc). The results of FTIR confirm the presence of possible proteins or othercompounds acting as reducing and stabilizing agents for silver nanoparticles. Moreover, the reduction of the silver ions was monitored and effect of four variableson the production of silver nanoparticle was optimized. In addition, conversion ofsilver ions was evaluated. The results suggest the reaction was very rapid. PDBmedium,1.0mM AgNO3concentration, pH7and33°C favored the formation of silvernanoparticles. Under the optimized conditions, the conversion of silver ions got to69.2%by Epicoccum nigrum.In this study, the antifungal activity of the silver nanoparticles was assessed. Thebiosynthesized silver nanoparticles showed considerable activity against thepathogenic fungi (minimum inhibitory concentration,0.125-1μg/mL). The combinedeffect of silver nanoparticles with common antifungal drugs had proven to be additive,there was no antagonism phenomenon.In addition to synthesize Ag-NPs, the endophytic fungus Epicoccum nigrum alsocan biosynthesize gold nanoparticles. The synthesized gold nanoparticles appeared indifferent shapes (spherical, near to spherical or triangular). The particles were foundto be polydispersed with a modal diameter of1-30nm. The crystal structure of goldnanoparticles was face centered cubic (fcc).To the best of our knowledge, the endophytic fungus Epicoccum nigrum hasnever been reported to biosynthesize silver/gold nanoparticles and the current researchopens a new path for the green synthesis of silver/gold nanoparticles.2. The antifungal activity of the fungal secondary metabolitesEndophytes would be a source of new natural medicines. Recently, fungalinfections have become more prevalent, but the availability of antifungal drugs islimited. New organisms and many novel natural products from endophytic fungiinhibit or kill fungi. Therefore, endophytes are an interesting niche waiting to beexploited.In this study, the antifungal activity of the ethyl acetate extract of thefermentation broth of the endophytic fungus was investigated. In the screening of theantifungal activity, the strains Q41showed marked antifungal activity. The strain wasidentified as Fusarium oxysporum through morphological and molecular biologymethods. We scaled up the fermentation and isolated the antifungal chemicalconstituents of filtrate through bioassay-guided fractionation by silica gel. Theantifungal components were elucidated by gas chromatography-mass spectrometry.The results suggest the antifungal components maybe benzoic acid derivatives. This research provides a theoretical basis and technical support for the further separation ofthe monomer of the antifungal agents.