大豆拟茎点种腐病菌Phomopsis longicolla荧光素酶标记菌株的制备及应用  

Generation and Preliminary Application of A Luciferase-labeled Strain for Soybean Phomopsis Seed Decay Pathogen Phomopsis longicolla

作　　者：张晨 陈汉 王志昂 董莎萌[1] 王源超[1] 叶文武[1] ZHANG Chen;CHEN Han;WANG Zhi-ang;DONG Suo-meng;WANG Yuan-chao;YE Wen-wu(College of Plant Protection,Nanjing Agricultural University/Key Laboratory of Soybean Disease and Pest Control of Ministry of Agriculture and Rural Affairs,Nanjing 210095,China)

机构地区：[1]南京农业大学植物保护学院/农业农村部大豆病虫害防控重点实验室,江苏南京210095

出　　处：《大豆科学》2023年第3期344-351,共8页Soybean Science

基　　金：国家自然科学基金(32172374);财政部和农业农村部:国家现代农业产业技术体系(CARS-004)。

摘　　要：大豆拟茎点种腐病菌(Phomopsis/Diaporthe longicolla)可侵染大豆引起种腐病和茎枯病等多种病害。本研究根据该病菌基因组的密码子偏好性,优化了外源的荧光素酶基因序列,通过酵母同源重组构建了荧光素酶表达载体,利用PEG介导的原生质体转化法获得了稳定表达荧光素酶的大豆拟茎点种腐病菌菌株PlYC2-1-Luc。结果显示:荧光素酶基因的引入不影响大豆拟茎点种腐病菌的生长速率和致病力。以PlYC2-1-Luc菌株进行示踪可直观发现:健康大豆中分离的3株内生镰孢菌能够显著抑制PlYC2-1-Luc生长,具有生防潜力;含苯醚甲环唑、精甲霜灵·咯菌腈和咯菌腈·噻霉酮的种衣剂均可抑制PlYC2-1-Luc向大豆种子扩展,表明这些药剂或其成分可用于病害防控;PlYC2-1-Luc对大豆品种Williams的致病力强于齐黄34,表明齐黄34更加抗病。该荧光素酶标记方法具有较好的有效性和稳定性,该研究为进一步开展大豆拟茎点种腐病菌的致病机理及病害防控技术研究提供了一种可视化、高效的菌株材料。Phomopsis longicolla(syn.Diaporthe longicolla)causes a variety of soybean diseases,including seed decay and pod and stem blight.We optimized the sequence of an exogenous luciferase gene according to the codon preference of P.longicolla genome,constructed the expression vector using yeast-homologous recombination technology,and finally obtained the luciferase-labeled P.longicolla strain PlYC2-1-Luc by PEG-mediated protoplast transformation system.The introduction of luciferase gene did not affect the growth and virulence of P.longicolla.By tracing PlYC2-1-Luc,we obviously found that:the growth rate of PlYC2-1-Luc could be inhibited by three endophytic Fusarium strains isolated from healthy soybean,revealing them as candidates of biocontrol strain;the growth of PlYC2-1-Luc toward soybean seeds treated with three seed coating products(difenoconazole,mefenoxam+fludioxonil,and fludioxonil+benziothiazolinone)was inhibited,reveling these fungicide products or effective ingredients are useful of disease control;and the disease level of PlYC2-1-Luc against the soybean cultivar Williams was stronger than that against Qihuang 34,revealing that Qihuang 34 is more resistant to disease.These results indicate that the labeling method is effective and stable,and this study provides a visual and highly effective strain material for further research on the pathogenic mechanism and disease control technology of P.longicolla.

关 键 词：大豆拟茎点种腐病菌 Phomopsis longicolla 荧光素酶 密码子偏好性 病原菌示踪 

分 类 号：S435.651[农业科学—农业昆虫与害虫防治]