Sun Zhan-Bin, Sun Man-Hong, Zhou Mo, Li Shi-Dong
Institute of Plant Protection, Chinese Academy of Agricultural Sciences, No. 2 West Yuanmingyuan Road, Haidian District, Beijing, 100193, China.
AMB Express. 2017 Dec;7(1):1. doi: 10.1186/s13568-016-0313-x. Epub 2017 Jan 3.
Clonostachys rosea is a promising biocontrol fungus active against various plant fungal pathogens. In this study, the endochitinase-encoding gene Chi67-1, the expression of which is sharply upregulated in C. rosea 67-1 when induced by sclerotia, was transformed into the original isolate by protoplast transformation, and transformants were screened against Sclerotinia rot of soybean. The transformation efficiency was approximately 50 transformants per 1 × 10 protoplasts, and 68 stably heritable recombinants were assayed. The parasitic rates of 32.4% of the tested strains increased by more than 50% compared to 43.3% of the wild type strain in 16 h, and the Rc4-4 transformant showed a parasitic rate of 100% in 16 h. The control efficiencies of the selected efficient transformants to soybean Sclerotinia stem rot were evaluated in pots in the greenhouse, and the results revealed that Rc4-4 achieved the highest efficiency of 81.4%, which was 31.7% and 28.7% higher than the control achieved by the wide type and the pesticide carbendazim, respectively. Furthermore, the expression level of Chi67-1 was 107-fold higher in Rc4-4 than in the wild type, and accordingly, the chitinase activity of the recombinant increased by 140%. The results lay a foundation for the development of efficient genetically engineered strains of C. rosea.
粉红粘帚霉是一种有前景的生防真菌，对多种植物真菌病原体具有活性。在本研究中，通过原生质体转化将在菌核诱导下在粉红粘帚霉67-1中表达急剧上调的内切几丁质酶编码基因Chi67-1转化到原始分离株中，并针对大豆菌核腐烂筛选转化体。转化效率约为每1×10个原生质体产生50个转化体，共检测了68个稳定遗传的重组体。在16小时内，32.4%的受试菌株寄生率比野生型菌株的43.3%提高了50%以上，Rc4-4转化体在16小时内寄生率达到100%。在温室盆栽中评估了所选高效转化体对大豆菌核茎腐病的防治效果，结果表明Rc4-4的防治效果最高，达到81.4%，分别比野生型对照和农药多菌灵高出31.7%和28.7%。此外，Rc4-4中Chi67-1的表达水平比野生型高107倍，相应地，重组体的几丁质酶活性提高了140%。这些结果为开发高效的粉红粘帚霉基因工程菌株奠定了基础。
