Long Term Preservation By Using Mineral Oil Of Phytophthora Infestans And Development Of Specific Scar Makers

Potato is the fourth grain crop in the world, which is behind rice、wheat and corn."International Year of the Potato (IYP)" was declared by the United Nations in2008, and it has high-lighted the important position of potato in the global food system. China is ranked one in planting area and yield of potato. But the potato late blight caused by the oomycete Phytophthora infestans (Mont.) de Bary has been threatening the development of China’s potato industry.Saprophytic ability of P. infestans was weak, it is easy to die in the dry, high or low temperature conditions. Therefore, it is difficult to use long term preservation of other pathogenic fungi to preserve P. infestans. Seventy three isolates of P. infestans were used to investigate viability at different preservation period by using mineral oil.These isolates were collected in the provinces of Heilongjiang and Jilin from2006to2008. The results suggested that94.6%isolates of P. infestans were viable among all P. infestans isolates preserved in mineral oil. The viability rates of the isolates stored in mineral oil for4,5,6,10,12,13and15months were100%. However, the viability rates of isolates stored in mineral oil for7months were80%,90.5%for those stored for8months,91.7%for those stored for9months,88.9%for isolates preserved in mineral oil for11month and75%for isolates preserved in mineral oil for14months. This experiment determined pathogenicity of P. infestans isolates in different preservation periods by using in vitro inoculation, and calculated the composite parasitic fitness of P. infestans isolates after preservation of mineral oil. The results showed that the mean value of lesion area at preservation periods was8.68cm2, and the mean value of infection frequency was1.0, and the mean value of sporulation capacity was5,031sporangia per cm2and the mean value of composite fitness index was40,926. The pathogenicity of P. infestans isolates stored in mineral oil accounted for14806-414474sporangia per cm2. All data suggest that mineral oil preservation for P. infestans can be an easy and efficient preservation method for long term storage of P. infestans.P. infestans lurking in potato tubers is the main primary infection source of prevalence of potato late blight next year. Identification of potato late blight in the plants is very difficulty. This study designed a pair of specific primers LB-3F and LB-3R according to gene sequence of P. infestans have been reported, and amplified genomic DNA extracted from P. infestans, P. sojae, Alternaria, Pythium, Fusarium, Botrytis cinerea and Rhizoctonia by using primer LB-3F and LB-3R. The amplification results showed that about150bp DNA fragment was amplified in genomic DNA of P.infestans, while target fragment was not amplified in genomic DNA of other pathogens. The specific fragment was cloned and sequenced, and compared with gene sequence of P. infestans isolates linked with A1mating type. The results showed that inserted specific DNA fragment of P. infestans in this experiment was156bp. The similarity between gene sequence of inserted DNA and middle of the gene sequences (259～414bp) of specific DNA fragment linked with A1mating type of P. infestans was99%. This experiment synthetise probe by using genomic DNA recovered from the specific fragment, and carried out Southern blot with genomic DNA extracted from P. infestans and other pathogenic fungi by using the probe. The result showed that hybridization band was only produced in P. infestans isolates, but no produced from toher pathogenic fungi. This result of Southern bloting was consistent with PCR result.To detect presence of P. infestans in different incubation periods, this experiment amplified genomic DNA extracted from potato leaves, stems and potato tubers inoculated at different periods (0、4、8、12、24、48、72、96、120h) by using specific primer LB-3F and LB-3R.The results showed that target DNA fragment was amplified in genomic DNA extracted from potato leaves inoculated after4hours, extracted from potato stems after inoculation48h, and extracted from potato tubers after inoculation24h. Above results suggest that even though there were no obvious symptoms on the plant surface, but P.infestans may be lurking in various tissues of the plant. This experiment carried out PCR-Southern hybridization with PCR amplified product. The result was consistent with PCR amplified results. Above results suggested that this SCAR primer provide a scientific basis for the infection process of P. infestans and rapid detection of P. infestans.