Pure Culture And Toxin Determination Of The Mycelia Of Amanita Exitialis

The genus Amanita is one of the largest basidiomyceous genera with about 500 taxa described worldwide. It contains some deadly poisonous species. Most of the death cases due to eating poisonous mushroom were caused by Amanita. 22 kinds of Amanita peptide toxins have been described and mainly classified into amatoxins, phallotoxins and virotoxins. They play important roles in biological researches because a-amanitincan specifically inhibit the activity of RNA polymerase II, while phalloidin can specifically bind to F-actin. Species of Amanita are very difficult to be cultured artificially due to their biological characters of involving in ectomycorrhizal (ECM) associations. Peptide toxins products from Amanita are very expensive because all of them can only be purified from sporocarps collected in the field, and the natural resource is quite limited.Amanita exitialis Z.L.Yang & T.H.Li was described in 2000. It has caused the death of 18 persons and been proved to be a very strong poisonous mushroom. To data it has been only found in southern China. There is no report on its pure culture and its toxin determination.The biology of Amanita exitialis was studied by field observation, as well as microscopy (FM) and electron microscopy (SEM). Its nuclear behavior and the secondary wall of basidiospores were observed for the first time. The spawn was obtained by tissue isolation and identified by molecular biological method. Fairly abundant peptide toxins in the mycelia were determined by high pressure liquid chromatography (HPLC), mass spectra (MS), 1H nuclear magnetic resonance spectra (1H-NMR), 13C nuclear magnetic resonance spectra (13C-NMR) and bioactivity assaying. Amatoxins were separated from the cultured mycelia for the first time. The results showed as below: 1. Studies on biological characters1) A. exitialis has only been found in Gangdong Prov., China to date. It is associated with Castanopsis fissa (Champ, ex Benth.) Rehd. et Wils. Occurring in March and April, the basidiocarp was characterized by its white color, an apical annulus and a firm volva.2) A large number of basidia were 2-spored, while a small number of them were 1-spored. Basidiospores were (9.0)9.5 ~ 12.0(14.5)x (8.5)9.0 ~ 11.5(13.0)um. Basidiospores produced by 2-spored basidia were tetranucleate, while those produced by 1-spored basidia were octanucleate.3) The basidiospores were thin-walled when matured. However, a thicker secondary wall may be formed inside the primary wall at latter stages when necessary water supply and suitable temperature are available.2. Studies on the pure culture of mycelia1) Tissue from fruitbody and basidiospores were used to isolate the spawn of A. exitialis. The result showed only the former method was successful. Tissues from different parts of the fruitbody were usable for isolation, but the gill of young fruitbody was the best.2) Six media were used to culture the mycelia. Among them, the improved PDM was the best. The mycelium grew better on solid media then in liquid medium. The output of the mycelia approached the highest when cultured for 70 days.3) The optimal temperature for the mycelium growth was 25C, and the optimal pH is about 5.0. 20g/l000ml glucose added in PDM was the optimal consistency.4) Most of the mycelia were binucleate, few were trinucleate or tetranucleate. The mycelia at earlier stages were filamentous with hyphal cells 40-120 um x 2.5-4 but inflated to subglobose with up to 30um in diam. at latter stages.3. Molecular biological identification of the isolation1) DNA from the fruitbody of A. exitialis and the isolation from the fruitbody was extracted following the method of CTAB.The internal transcribed spacer regions were amplified by PCR using the primers ITS4 and ITS 5. The ITS sequences of the fruitbody and the isolation were the same sequence. Thus, the isolation is A. exitialis.2) The sequence similarity between A. exitialis and A. virosa reached 91%, and between A exitialis and ,4. bisporigera was 89%.4. Determination of the peptide...