Study On Genetic Differentiation Of Phytophthora Infestans Isolates From Potato Areas Of China

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 affectthe results of RAPD were analyzed and the optimal RAPD protoco1 for P. infestans was established. A 25uX solution with 2.5uL 10XPCR buffer(10mmol L-1 MgCL2), 1.5U Tag DNA polymerase, dNTP10x10-3p,mol, 20-80ng genomic DNA, random primer10x10-6umol wasthe 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.