链格孢菌SC-018培养工艺优化及其致病性研究

Study on Optimization of Culture Technology and Pathogenicity of Alternaria alternata SC-018

链格孢菌是一种丝状真菌,可以通过其菌丝产生的多种致病性毒素侵染植物组织,能够有效的用于防除水田杂草野慈姑。通过单因素试验对链格孢菌SC-018培养基中的碳源及氮源进行了筛选,得到其最适碳源为D-半乳糖,最适氮源为硝酸钠。通过单因素试验确定该菌培养温度及培养基初始pH值的最适范围。对D-半乳糖、硝酸钠、培养温度及初始pH值4因素进行响应面Box-Behnken实验,对链格孢菌SC-018的培养基中D-半乳糖、硝酸钠的含量及培养条件进行了优化。结果表明,优化后的培养基为马铃薯浸出液1000mL,D-半乳糖21.50g·L-1,硝酸钠3.15g·L-1,初始pH值7.81;培养温度25.94℃;通过对链格孢菌SC-018侵染水田杂草野慈姑的最适接种浓度、最佳作用时间的筛选,可得链格孢菌SC-018的孢子浓度每毫升大于1.00×105个时,便会对野慈姑植株表现出明显的致病性,且孢子浓度每毫升为2.0×106个时,作用效果最佳;接种1～2叶期的野慈姑作用效果明显优于其他时期;通过考察环境因子对链格孢菌SC-018致病性的影响可知,该菌成功侵染野慈姑并致其发病的最佳温度为25～28℃,光照条件为10h光照/14h黑暗交替,结露时间大于48h则可引起90%以上的接种野慈姑死亡。

Alternaria is a filamentous fungus,which can infect plant tissues by various pathogenic toxins produced by its mycelium,and can be effectively used to control weeds in paddy fields.In this paper,the carbon and nitrogen sources of Alternaria alternata SC-018 medium were screened by single factor experiments.The optimum carbon sources were D-galactose and the nitrogen source were sodium nitrate.The optimum range of culture temperature and initial pH of culture medium was determined by single factor experiment.Then,response surface Box-Behnken experiments were conducted on four factors including D-galactose,sodium nitrate,culture temperature and initial pH.The content and culture conditions of D-galactose and sodium nitrate in the culture medium of Alternaria alternata SC-018 were optimized.The results showed that the optimized medium was potato extract 1000mL,D-galactose 21.50g·L^-1,sodium nitrate 3.15g·L^-1,initial pH 7.81,culture temperature 25.94℃.Through screening the optimum inoculation concentration and the optimum action time of Alternaria alternata SC-018 to infect the paddy field weed Sagittaria trifolia,which can be concluded that when the spore concentration of Alternaria alternata SC-018 is greater than 1.00×10^5 per milliliter,it will show obvious pathogenicity to Sagittaria vulgaris plants,and when the spore concentration is 2.00×10^6 per milliliter,the effect wasthe best.The effect of inoculating Sagittaria trifolia in 1-2 leaf stage was obviously better than that in other periods.By investigating the influence of environmental factors on the pathogenicity of Alternaria alternata SC-018,the optimum temperature for the successful infection of Alternaria alternata SC-018 is 25-28℃,the light condition is 10 hours light/14 hours darkness alternation,and the dew time is longer than 48 hours,which can cause more than 90% of the death of the inoculated Sagittaria trifolia.