Isolation And Identification Of Rhizoctonia Leguminicol A And Extraction Of Swainsonine From Mycelium And Fermentation Broth Of Rhizoctonia Leguminicola

0bjective The purpose of study is to establish a simple and effective method of isolatingand identifying Rhizoctonia leguminicola to meet the need of the strains required for studyingRhizoctonia leguminicola. Improve the method of extracting swainsonine from mycelium andfermentation broth of Rhizoctonia leguminicola in order to simplify the extractionstep,increase extraction yield,reduce the cost of producing and offer foundation for studyingand producing swainsonine.Methods1Rhizoctonia leguminicola was isolated from the root part of legume.By thescreening based on morphological characteristics of Rhizoctonia leguminicola,theidentification of molecular biology,detecting swainsonine of mycelium using thin layerchromatography and gas chromatography.2Rhizoctonia leguminicola was inoculated in Czapek medium and cultured for14d in25℃.Mycelium and fermentation broth was collected.Mycelium was extracted by methanolSoxhlet extraction.Acidic saturated n-butanol extracted organic impurities from acidicsolution,alkaline saturated n-butanol extracted swainsonine from alkali solution,acidic waterextracted swainsonine from acidic saturated n-butanol.Acidic water solution was madealkaline,and mixed with same volume SiO₂in order to fully evaporated water,then theammonia chloroform extracted repeatedly swainsonie from the dry matter.Vacuumsublimation purified product.The product was analyzed by thin layer chromatography and gaschromatography.3The fermentation broth collected was Pretreated by acidulating,heating,flocculationand aggregation,centrifugation,activating carbon adsorption.The fermentation broth wasevaporated to dryness into powder and extracted by methanol Soxhlet extraction.Acidicsaturated n-butanol extracted organic impurities from acidic solution,alkaline saturatedn-butanol extracted swainsonine from alkali solution,acidic water extracted swainsonine fromacidic saturated n-butanol.Acidic water solution was made alkaline,and mixed with samevolume SiO₂in order to fully evaporated water.Then The ammonia chloroform extractedrepeatedly swainsonie from the dry matter,separated swainsonie by Silica gel chromatography and purified product by vacuum sublimation.The product was analyzed by thin layerchromatography and gas chromatography.Results Rhizoctonia leguminicola screened met the morphological characteristics of Rhizoctonialeguminicola.we concluded that they were Rhizoctonia fungi by analyzing the5.8S rDNA-ITS,Swainsonine was found in mycelium by thin layer chromatography and gas chromatography; whiteCrystalline substance was extracted from mycelium of Rhizoctonia leguminicola, we determined that it isswainsonine by thin layer chromatography and gas chromatography,white powder substances was extractedfrom fermentation broth of Rhizoctonia leguminicola,we determined that it contained swainsonine by thinlayer chromatography and gas chromatography.Conclusion Four producing-swainsonine Rhizoctonia leguminicola was isolated andidentified by modified way.5mg crystal was isolated from50g dried mycelium of theRhizoctonia leguminicola.The white crystal was swainsonine by thin layer chromatographyand gas chromatography.30mg white powder was isolated from250ml fermentation broth ofthe Rhizoctonia leguminicola by modified way.we confirmed white powder was swainsonineby thin layer chromatography and gas chromatography.