根癌农杆菌介导火龙果溃疡病菌遗传转化体系的构建及转化子筛选

Establishment of Agrobacterium tumefaciens-mediated transformation system for Neoscytalidium dimidiatum and Screening of Transformants

为了探究火龙果溃疡病病原菌的致病机理,笔者利用农杆菌介导的遗传转化技术,分析了根癌农杆菌浓度、分生孢子萌发时间以及共培养温度等3个主要因素对遗传转化效率的影响,建立火龙果溃疡病菌的遗传转化技术体系,并对转化子进行筛选。结果表明,当农杆菌浓度OD_(600)=0.5、分生孢子萌发时间24 h、共培养温度25℃时遗传转化效率最高(833个转化子/10^(6)分生孢子);随机挑选200个转化子进行筛选,获得3株菌落形态与野生型菌株差异较大的转化子,4株产孢量减少的转化子,5株产孢量增加的转化子,6株致病力下降的转化子。

Pitaya is a nutrient-rich fruit with high economic value and great development potential in tropical and subtropical region.Pitaya stem canker caused by Neoscytalidium dimidiatum is one of the most serious fungal diseases infecting pitaya,which has become a main factor restricting the development of pitaya.In order to control the disease effectively,it is necessary to study the pathogenic mechanism of the pathogen causing pitaya stem canker.Agrobacterium tumefaciens-mediated genetic transformation was hence used to establish a genetic transformation system for N.dimidiatum.The effects of three main factors,concentration of A.tumefaciens,germination time of conidia,and co-culture temperature,on the transformation efficiency were analyzed,and transformants were screened.The results showed that the genetic transformation efficiency of N.dimidiatum was the highest,as high as 833 transformants/106 conidia,when the OD600 of A.tumefaciens was 0.5,the germination time of conidia was 24 h,and the co-culture temperature was 25℃.Two hundred transformants were randomly selected from the genetic transformation for screening,of which 3 transformants with different colony morphology,4 with decreased spore production,5 with increased spore production,and 6 with decreased pathogenicity were generated.