【作者】 沈怀君；

【导师】 孔祥清；

【作者基本信息】 黑龙江八一农垦大学 ， 植物病理学， 2009， 硕士

【摘要】 在对由黑龙江八一农垦大学农药研究室分离获得的链格孢菌（Alternaria）菌株LD2形态特征、生物学特性、流行病学及毒性的致病机理等一系列研究后,认为该菌可作为小蓟（Cephalanoplos segetum）生防潜力菌使用并具有进一步的研究潜质。但由于野生菌种本身生防能力不强,难以得到理想的防治效果,本研究通过物理（紫外线）和化学（亚硝基胍）复合诱变的方法对野生菌种进行改造,然后对改造后菌种生长的培养基配方进行优化以及发酵条件等进行研究,最后采取20 L发酵罐对其进行实验中试即放大培养。研究结果如下:1.采用紫外线诱变链格孢菌LD2的孢子悬浮液（30min）,筛选到突变株LD2-13。该菌株较原始菌株孢子产量提高了1.33×10⁷个/mL,感病率提高了11.1%,死亡率提高了11.2%。通过传代稳定性验证试验处理,结果表明紫外线诱变得到的菌株LD2-13传代稳定性较好。采用NTG诱变出发菌株LD2-13的孢子悬浮液（75min）,筛选到突变株LD2-13-4,该菌株较野生菌株LD2孢子产量提高了2.73×10⁷个/mL,感病率提高了24.9%,死亡率提高了16.1%。将亚硝基胍诱变得到的菌株LD2-13-4进行传代稳定性验证试验处理以及对玉米、大豆的安全性试验,结果表明该菌株传代稳定性较好,而且对大豆、玉米均安全,无感病现象。2.采用单因素实验和正交实验优化出的液体摇瓶发酵培养基为:蛋白胨5.0 g/L、蔗糖20.0 g/L、麸皮50.0 g/L、KH₂PO₄ 3.0 g/L、MgSO₄·7H₂O 1.0 g/L、FeSO₄·7H₂O 0.1 g/L、无水CaCl₂ 0.1 g/L。在上述培养基条件下采用单次单因素实验筛选出的液体摇瓶发酵培养条件为:250mL三角瓶装液量100 mL,接种量4%,接种种龄3 d,初始pH7.8,摇床转数180 r/ min,30℃振荡培养15d。在上述发酵工艺条件分生孢子产量可达到5.3×10⁷个/mL,较该菌在固体（PDA）培养基上培养,分生孢子产量有所增加。3.在20 L发酵罐中,发酵放罐的最佳时间为68 h,分生孢子产量达7.2×10⁷个/mL,最终得发酵液总体积11.4 L,干重产量为76.3 g。更多还原

【Abstract】 Heilongjiang Agricultrue University pesticide laboratory separated Alternaria that named fungi-LD2.After reserched its morphological characteristics, biology,epidemiology and the pathogenic mechanism of toxicity,the results showed that this fungi can be used to biocontrol Cephalanoplos segetum and further to reserch potential. However,Due to wild species Biocontrol ability itself is not strong,it is difficult to obtain the desired effect,Therefore,This study used physical （ultraviolet） and chemical （nitrosoguanidine） composite mutagenesis method to transform the wild strains.Then reserch growth medium and the fermentation conditions of modified fungi. Finally,cultured strains in 20L fermentor as second test to enlarge production.The results are as follows:1. Through the use of spore suspension of ultraviolet mutagenesis LD2 Alternaria alternata （30min）,screening mutant LD2-13.The strain of spore production increased 1.33×10⁷ months / mL,infection rate inceased 11.1% and mortality increased 11.2% than the original strain. Through testing to verify genetic stability, the results showed that the UV-induced strain LD2-13 genetic stability was better. Through the use of spore suspension of NTG mutagenesis LD2-13 Alternaria alternata （75min）,screening mutant LD2-13-4, The strain of spore production increased 2.73×10⁷ months / mL,infection rate inceased 24.9% and mortality increased 16.1% than the original strain. The verification test of genetic stability and the security test of corn, soybean to mutant LD2-13-4, The results showed that the genetic stability of the strain was well, but also for soybean and corn were safe, non-flu patients phenomenon.2. Single factor experiments and orthogonal experiment to optimize the liquid fermentation medium are as follows:peptone 5.0g/L、sucrose 20.0g/L.bran 50.0g/L、KH₂PO₄ 3.0g/L、MgSO₄·7H₂O 1.0g/L、FeSO₄·7H₂O 0.1g/L、CaCl2 0.1g/L.In the medium under the conditions of the use of a single factor experiment screened liquid fermentation culture conditions are as follows:250mltriangle bottle capacity 100mL,inoculation quantity4%,Inoculation days3d,original pH7.8,shaker revolution180r/ min,30℃surge culture 15d. Fermentation process in the above-mentioned conditions for spore production could reach 5.3×10⁷ months / mL,more fungi in the solid（PDA）culture medium,spore production increased。3. In 20L fermentor,the best time to fermentation tank68h,spores output 7.2×10⁷个/mL,The end,the total volume of fermentation 11.4 L,dry weight output 76.3g。更多还原