Screening Of High-Yield Toxin Strain Of Alternaria Alternata And Mutation For Low-Yield Toxin By Restriction Enzyme Mediated Integration (REMI)

The phytotoxin of Alternaria alternata (Fr.) Keissler, isolated form Crofton weed (Eupatorium adenophorum), had the potential for developing mycoherbicide. The toxin yield and its efficacy of controlling weed were influenced by toxin-producing ability of strains. So it was necessary to screen high-yield toxin strains for industrialization. In this paper, biological traits related to toxin metabolism were compared among different strains to find out the correlation between traits and toxin production and to screen the highest-yield toxin strain. The preparation of protoplast of Alternaria alternata and then REMI transformation was conducted in order to establish optimum system and conditions of performance, and finally to induce mutant strain for low-yield toxin, so that it could be used to clone the genes related to toxin production.Strains isolated from different place had different toxin yield, which changed sometimes during the process of culturing. The highest-yield toxin strain NEW was screened from 21 owned strains by toxin liquid culture method. Some main biological characteristics of the 21 strains were studied and compared. The results indicated there was significantly positive correlation among pathogenicity, toxin yield and conidium yield of Alternaria alternata. Based upon the correlation, high-yield toxin strains would be screened out from more virulent stains.Ultraviolet was the most common method of microogranism breeding. The lethal curve of radiation time on conidium was gained, but the high-yield toxin mutation hadn't been screened. Mutations screening should be continued or substitutable methods should be found to increase toxin yield.Strain NEW with the highest-yield toxin was used for studying the effect of the mycelial age, enzyme system, digesting temperature and time and osmotic stabilizers on protoplast preparation. The optimum system and conditions for preparing protoplast were explored: the mycelia cultured in PD medium for 20 hours, enzyme mixture of 1 % Driselase, 1 % Lysing enzyme and 1 % Snailase used to digest the mycelia at 30℃for 3 hours, and 0.7 mol/L NaCl used as the osmotic stabilizer. Protoplast got by this method was satisfied with transformation both on quantity and regeneration rate aspects.REMI transformation system was studied, and the form of plasmid, the quantity of restriction enzyme and plasmid were all determined. Transformants of Alternaria alternata were successfully gained by REMI, which was certificated by PCR. Four lowyield toxin mutants, NEW001, NEW021, NEW061 and NEW073, were obtained. And the decrease of toxin yield was detected by HPLC. NEW001, which toxin yield was not more than one fiftieth of that produced by the wild type strain NEW, was a good mutant material for related gene cloning.Biological traits of four low-yield toxin mutants, such as pathogenicity, toxin yield, conidium yield, colony growth velocity and so on, were studied and compared with that of NEW. The result showed there was the positive correlation among toxin yield, conidium yield and pathogenicity too, which was a good instruction for studying pathogenicity mechanism. Four mutants, particularly NEW001, which was certificated the integration of plasmid pSH75 into the genome of pathogen, was useful for the further gene cloning.