Studies On Agricultural Bioactivity Components Of Endophytic Fungus In Celastrus Angulatus

Endophytic fungus of strain 2B isolated from Celastrus angulatus has been studied on the taxonomy,agricultural bioactivities, isolation and identification of active components, breeding of high yield strains,and optimization of fermentation conditions in this paper. The main results are as follows:1 By means of observation of aerial mycelium, macroconidia, microconidia and colony morphologycharacteristics of strain 2B using optics microscope, comparised with related classify system, strain 2B wasidentified as Fusarium proliferatum.2 The agricultural bioactivities of the strain 2B fermentation products were tested. The results showedthat the methanol extracts of mycelium had evident fungicidal activity. The extracts was tested againstAlternaria solani, Alternaria longipes, Bipolarissorokiniana shoem, Exserohilum turcicum, the resultsindicated that the inhibiting rates against mycelium growth and spore germination were both more than80% at the concentration of 1000 μg·mL-1. The results of pot test indicated that the methanol extracts ofmycelium exhibited more than 70% for both protective efficacy and therapeutic efficacy againstPseudoperonospora cubensis disease at the same concentration.3 By means of open column chromatography on silica gel and preparative HPLC, four compounds,2B-1, 2B-2, 2B-3, 2B-4, were isolated from the methanol extracts of fermentation products of strain 2B.On the basis of spectral technology (MS, FT-IR, H NMR, 1 13C NMR, COSY), chemical methods andcomparison with related reports of literatures, their structures were identified as enniatinB, enniatinB1,enniatinA1 and ergosterin, respectively. At the concentration of 100 μg·mL-1, the inhibiting rates ofcompound 2B-1, 2B-2, 2B-3 against the mycelium growth of Exserohilum turcicum were 68.18%, 87.49%,90.91%, respectively.4 The analytical method of HPLC for enniatins was set up. The operating conditions were chromatogramcolumn Hypersil ODS (C18, 300mm × 9mm i.d.,10 μm), inject volume 5μL at room temperature,methanol-water (88:12, V/V) as mobile phase with a flow rate of 1.0mL·min-1and UV detection at 229nm.The relative standard deviation (RSD) was less than 1%, and the average recovery was over 98%.5 Using Endophytic fungus of strain 2B as starting strain, mutagenized with ultraviolet (UV) , EMS andultrasonic+UV + EMS in sequence, at the same time ,based on the relationship between the variation ofmodality and the capcity of the mutants' yield, the mutant strains which had great variation of modalitywere selected. Through three times mutagenized and the shake flask fermentation test, a mutant UE-152high-producing enniatins was obtained, which had much higher productive qualitied than its original strain.Its enniatins yield reached 59.72 mg·100mL-1 as 2.9 times high as that of original strain, and the hereditycharacter of the high productivity was stable.6 The optimum composition of culture medium and fermentation conditions of the strain UE152 weredemonstrated by the test of uni-factor and orthogonal design, respectively. The optimum culture mediumconsisted of glucose 2%, potato 20%, peptone 0.5% , NH4Cl 0.3%, MgS04 0.1%, CaCO3 0.1% and originalpH6.0-7.0. Inoculum volume was 10 entries per 50mL medium. A 250mL flask containing 50mL mediumwas cultivated at 26℃ for 120h and the rotatory velocity was 160 rpm. Under the best culture conditions,the general yield of Enniatins was 88.68 mg·100 mL-1. The results of orthogonal test showed if increasedthe aeration level, the yield could be higher.