摘要
本研究用两个马铃薯品种：春薯4号(对晚疫病高度抗病)和津引8号(对晚疫病高度感病)对采自中国马铃薯主产区(河北、云南、四川、黑龙江)的60个马铃薯晚疫病菌菌株进行了致病性测定，结果表明菌株的致病性、寄生适合度和其地域来源有一定的相关性，来源于河北的菌株致病性最强、寄生适合度最高；而来源于四川的菌株致病性最弱，寄生适合度也最低；91.30％菌株在春薯4号上的寄生适合度小于在津引8号上的寄生适合度。
对晚疫病菌DNA的提取方法进行了摸索、改进，发现SDS法更适于提取马铃薯晚疫病菌的DNA；用黑麦培养基培养的菌丝最适于提取DNA；新鲜菌丝并不适合于提取DNA，菌丝收集后在-20℃冰箱中存放数天再提取效果最好。
建立了适宜于马铃薯晚疫病菌扩增的最佳RAPD反应体系：体系为25μL，其中含10×PCR buffer(含10mmol·L~(-1)MgCL_2)2.5μL，Taq酶1.5U，dNTP10×10~(-3)μmol，模板DNA20～80ng，引物10×10~(-6)μmol。选用15个随机引物，对60个马铃薯晚疫病菌菌株进行了RAPD分析，每个引物扩增出的条带数为4-12条，共扩增出106个RAPD标记，其中多态性标记87个，占82.08％。聚类分析结果表明来源于4个省区的菌株间的亲缘关系极为复杂，菌株间亲缘关系均比较远。由聚类图还可看到，对寄主寄生适合度最大的19个菌株中有12个在相似系数为0.84时就全部聚在一起，亲缘关系相对比较近。
选用5个随机引物，对45个F_1代菌株及其亲本菌株进行了RAPD分析，每个引物扩增出的条带数为5-9条，共扩增出31个RAPD标记，其中多态性标记26个，占83.87％。通过聚类分析表明F_1代的RAPD指纹图谱和其亲本相比变异不太明显。在0.76的相似系数下，供试菌株可分为5个RAPD群，RGs1～RGs5，两个亲本菌株及大多数后代菌株(36个)都在RGs5内，只有9个后代菌株分散在RGs1～RGs4内。说明大多数后代菌株与亲本相比变异不太大，只有少数与亲本的亲缘关系比较远。
Potato late blight is an important obstacle in potato production. It is a destructive disease which causes stem death and tuber rotting on potato. Now it has been one of the most important grain crop diseases. Only in China, it causes 8 billion yuan losses every year.
Sixty isolates of Phytophthora infestans were isolated and purified from potato in four potato production areas including Hebei, Yunnan, Heilongjiang and Sichuan province. Infection frequency, lesion size, sporulation capacity and parasitic fitness of these isolates were conducted on two potato varieties(Chunshu 4, high resistance to late blight; Jinyin 8, high susceptible to late blight). The results revealed that there were significant differences in infection frequency, lesion size, sporulation capacity and parasitic fitness of the 60 isolates. There were certain correlation between parasitic fitness and geographical origin. The isolates from Hebei province had the highest parasitic fitness and the isolates from Sichuan Province had the lowest fitness. The parasitic fitness of 91.30% isolates was higher on Jinyin 8 than that on Chunshu 4; But the pathogenicity of the isolates which collected from Sichuan province and Heilongjiang province were stronger on Chunshu 4 than it on Jinyin 8.
The DNA extraction method for P. infestans were modified. The results showed that SDS method was better than CTAB method to extract DNA of P. infestans; the mycelia fom rye culture medium was the best for DNA extraction. After stored for several days or longer in -20 C freezer, the mycelia was more suitable for DNA extraction.
Based on the genomic DNA, the main factors that might affect
the results of RAPD were analyzed and the optimal RAPD protoco
1 for P. infestans was established. A 25uX solution with 2.5uL 10
XPCR buffer(10mmol L-1 MgCL2), 1.5U Tag DNA polymerase, dNTP
10x10-3p,mol, 20-80ng genomic DNA, random primer10x10-6umol was
the best for amplification reaction. Reaction mixtures were progr-
amered for 1 cycle at 94C for 4 min, and 45 cycles at 94C for 1 min, 36C for 2 min, 72 V for 2 min and 10 min at 72 C.
A total of 15 primers was employed for PCR amplification of 60 P. infestans isolates. 87 DNA markers out of 106 markers were polymorphic, the frequency of polymorphic marker was 82.1%.
The result of clustering analysis showed that the genetic relationships among the isolates from four areas were very complex.
With 5 random primer, 45 F1 progenies were used to study the relation between the parents and F1 progenies of Phythophthora infestans based on RAPD analysis. There were total of 31 RAPD markers, and 26 markers were polymorphic, the frequency of polymorphic marker was 83.87%. Results indicated that there was not significant variation between parental isolates and F1 progenies isolates. All isolates tested were classfied into 5 RAPD groups (RGsl~RGs5) at 0.76 genetic similarity. Two parental isolates and 36 F1 progenies were all in the RGs5, and only 9 F1 progenies were in RGsl~RGs4. There was not significant variation between parental isolates and most of F1 progenies isolates, only a few of progenies isolates had distant genetic relationships with parental isolates.
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