| Auricularia polytricha, an edible fungi with important nutrition value and health care function, is cultivated widely in Sichuan, Fujian and other places of China. Scytalidium lignicola, one pathogen of ascomycetes fungi, affects the mycelia growth of Auricularia polytricha, then leads to slippery scar disease. What is more, S. lignicola was host-specific to A. polytricha, which was found in our preliminary studies. To make the pathogenic mechanism clear, the pathogenicity of S.lignicola on different culture substrates against the mycelia of A.polytricha, the exploration of pathogenic protein factors, the production conditions of pathogenic toxins and the basic characteristics of toxins were analyzed in this study and different methods of how to extract and purify toxins were explored. The results were as follows:1. The symptoms of slippery scar disease in different types of substrates were analyzed. The mycelia of A. polytricha and S. lignicola were inoculated in two opening ends of bags, respectively. The mycelia of A. polytricha were infected by that of S. lignicola, which generated the spots of slippery scar disease. Nevertheless, in sawdust plate, the obverse and reverse of the plate did not form the disease scar after both of mycelia contact; yet, A. polytricha hypha would turn yellow. The parts of mycelia contact generated disease scar in double-end tube experiment. From the perspective of triangular flask culture, the mycelia adhering to the flask-cell could form the disease scar after mycelia contact, but the mycelia upside would turn yellow.2. Inhibition abilities of S. lignicola culture broth on A. polytricha and other mushroom mycelia were analyzed. The result showed that the inhibition ability to A. polytricha mycelia increased with an increase of concentrated culture broth in medium. A. polytricha mycelia stopped growing when the portion of the fermentation broth was 50%. It had a strong inhibition ability to Pleurotus ostreatus, Lentinula edodes, A. auricular-judae and P. geesteranus, yet had a weak inhibition ability to other mushrooms. Conversely, it could promote the growth of Hericium erinaceus.3. Analysis of pathogenic proteins of pathogen. It showed that water extract of scab and S. lignicola gained from infected A. polytricha cultivation bags could inhibit the mycelia growth of A. polyticha. Moreover, both them were thermal instability.4. Analysis of pathogenic toxins of pathogen. The most suitable culture conditions for toxin production were analyzed. The optimal culture was CYM, and 8 days was the optimal cultural time, as well as continuous vibration was required, yet p H4~9 did not influence the production. p H2~10, protease K, UV and heat could not influence the activity of pathogenic factor. Moreover, its molecular weight was less than 1000 and the polarity was medium. The pathogenic factor was inferred to be toxin.Tried to use sephadex column, anion exchange resin and cation exchange resin to purify the toxin, but the results were not desirable. Chloroform extract from culture filtrate was separated by pre-TLC, and then the active components were separated by pre-TLC again. Among the isolates, components in Rf=0 and Rf=0.08 had the highest inhibition ratio of 69.8% and 63.5%. Meanwhile, the composition of Rf=0 was simple which was observed by the spectrum of HPLC and the highest absorption peak was at 1.602 min. The composition of Rf=0 had a strong absorption leak in 220~250 nm. So the toxin was speculated to be acid compounds containing two unsaturated conjugated bond.To sum up, the pathogenic factors of S. lignicola causing disease of A. polytricha were enzymes and toxins. The toxins were speculated to be acid compounds. This study laid an foundation on making clear the molecular basis of host specificity of pathogen and comprehensive control. |
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