【作者】 张正波；

【导师】 余柳青；

【作者基本信息】 中国农业科学院 ， 农业微生物， 2007， 博士

【摘要】 化学防治杂草给农业的发展做了较大的贡献，但伴随着环境污染、杂草抗性增加、农产品安全等问题。因此开发新型环保型除草剂、减少化学除草剂的使用是未来农业发展的趋势。发展微生物除草剂，实现杂草的生物防治，可以大幅度减少化学除草剂的使用，实现农业的可持续发展。禾长蠕孢菌稗草专化型（Helminthosporium gramineum Rabenh f.sp．echinochloae，HGE）和弯孢菌（Curvularia lunata，CL）是中国水稻研究所杂草室从自然感病稗草上分离得到的两株病原真菌，它们是稗草的生防潜力菌株。HGE对稗草致病力强，但孢子产量低，而CL菌孢子产量高，但对稗草的致病力弱。本研究根据这两个菌株的特性，采用原生质体融合技术，将两者优良特性结合起来，培育出孢子产量提高、代谢产毒能力强、适合工业化发酵的优良工程菌株。并对探讨该菌株的发酵产毒素工艺、毒素微乳剂的制备技术，调查融合菌株的毒素田间防治杂草效果。获得以下主要结论：1．确定了禾长蠕孢和弯孢菌的原生质体释放最佳条件。2％溶壁酶+2％蜗牛酶、2％纤维素酶+2％蜗牛酶分别为弯孢菌（CL）和禾长蠕孢菌（HGE）最佳溶菌酶组合；CL与HGE菌丝体释放原生质体最佳培养时间分别为16h、24h。原生质体经过液体培养基再生后，获得了较高的再生频率，CL和M1的原生质体再生率分别为14.70％±1.27％、11.11％±1.43％。2．获得了较高的融合率。低浓度PEG甘氨酸溶液有利于弯孢菌与禾长蠕孢菌的原生质体融合，其融合率为2.6×10^-3和2.1×10^-4；通过对融合子生物学性状的评价，筛选出四株产毒素提高的融合子，其中融合子CM1—037毒素Ophiobolin A的合成能力较其亲本M1提高了34％；温室评价CL与M3之间的融合子，获得了两株孢子产量和生防潜力提高的融合菌株CM3—012、CM3—053，其孢子产量较其亲本M3分别提高了45.57％和17.72％、对稗草鲜重防效和干重防效分别提高了1.77％和4.89％、9.73％和12.55％。3．RAPD指纹分析融合子与亲本的亲缘关系，验证了融合子遗传物质偏亲现象和融合过程中发生了遗传重组。4．确定了融合子液体产毒素的摇瓶发酵条件为：菌株CM1—037；培养基为马铃薯200g，葡萄糖30g，麸皮20g,酵母膏1g，KH₂PO₄ 2g,MgSO₄.7H₂O 1g；菌丝体接种量10ml/L。5．确定了融合子CM1-037粗毒素的10％微乳剂剂型配方：10％粗毒素+15％助溶剂（8％9^#+7％5^#）+15％乳化剂（4.5％1^#+10.5％9^#）+2％氮酮+58％水。该制剂在大田条件下对丁香蓼、节节菜和水莎草的防治效果较好；结合低剂量化学除草剂（苄嘧磺隆+二氯喹啉酸）复合使用，该制剂对稗草和鸭舌草的防治效果达到80％以上。更多还原

【Abstract】 The chemical weed-control had made large contribution to the development of agriculture.But the environmental pollution, increase of weed type with resistance against herbicide and safety of agricultural products came with the use of the chemical herbicide. Therefore, the new type herbicide had to be developed to reduce the usage of the tradiontal chemical herbicide, which was the trend of agriculture in future. Expending microherbicide, biologically control the weeds would reduce the usage of chemical herbicide, then to realize the sustainable development of the agriculture.Helminthosporium gramineum Rabenh f. sp. echinochloae and Curvularia lunata were isolated by the China National Rice Research Institute, which were fungal pathogens of the barnyargrass from the natural infected weed plants and had potentials to develop into biological agents. HGE strains had high virulent to the barnyardgrass but low spore yield, while the CL strain had high spore yield but low virulent to the weed. The objectives of this study was to combine the advantages of the two strains into new ones by protoplast fusion, which provide higher spore yield and richer toxin, as well as to the favor of industrial production, then to study the procedures to make the microbe emulsion of the crude toxin, and to investigate its efficiency to control weeds in rice fields. The major results of the study showed as follows:1. The optimal condition for protoplasts releasing was decided as that the most effective lytic enzyme mixture for CL was 2% lywallzyme plus 2% snaiiase and that for M1 was 2% cellulase plus 2% snaiiase, that the optimum incubation time was 16 h for CL and 24 h for M1. The protoplasts were regenerated in the liquid regeneration medium and gave a high regeneration frequency, which was 14.70%±1.27% for CL and 11.11%±1.43% for M1.2. The protoplast fusion process gave a high fusion frequency, which was 2.6×10^-3 between M1 and CL、2.1×10^-4 between M3 and CL. There were four fusant strains that improved toxin production, especially for CM1-037, which gave a 1.34 times ophiobolin A production than its parent strain M1. And there were two fusant strains between M3 and CL, which gave a higher spore yield and improved biologolical control efficiency against barnyardgrass in greenhouse, and its fresh weight control efficiency and dry weight control efficiency of the fusatnt strain CM3-012 and CM3 - 053 were 1.77% and 4.89%, 9.73% and 12.55% respectively higher than their parent strain.3. RAPD analysis on the relationship of fusant strains and the parent strains showed that genetic recombination occurred during protoplast fusion.4. The condition for toxin production was that CM1-037 as strain, potato 200 g/L, glucose 30 g/L, wheat bran 20 g/L, yeast extract 1g/L, KH₂PO₄ 2g/L, MgSO₄.7H₂O 1g/L as medium, and mycelia 10 ml/L as inoculums.5. The 10% crude toxin microbial emulsion were that 10% crude toxin + 8% solvent 9^# + 7% solvent 5^#+4.5% emulsifying agent 1^# + 10.5% emulsifying agent 9^#+ 2% lanrocapran+58% water, which had a high efficiency control weeds such as false loosestrife, indian rotata, Sedge. When combination with the low usage of chemical herbicide （ bensulfuron methyl + quinclorac） , it improved the efficiency against barnyardgrass and Monochoria above 80%, against false pimpernel above 75%.更多还原