The Genetic Diversity Of Phytophthora Nicotianae In Chongqing

In China, Tobacco black shank (Phytophthora nicotianae) is widely spread and extremely destructive, which can infect Flue-cured tobacco, curde tobacco, sun-cured tobacco, burley tobacco, fragrant tobaccos and all the cultivated tobacco. Chongqing is an important tobacco planting area of China where tobacco black shank desease occurred seriously and caused a huge economic loss. Due to the raising cost of control, it is more and more difficult to prevent. Phytophthora parasitica var. nicotianae has abundant genetic diversity, such as mating type, drug-fast variation, and physiological race, which cause the tobacco lose the resistance. In this paper, the technology of RAPD wa used to analyse the genetic diversity of Phytophthora nicotianae with different genetic background. The aim is to provide the principle basis for the prevention of tobacco black shank disease.1. Strain preservationFor research the longest store life of tobacco black shank disease, we tried to store Nicotianae isolates through four methods:oatmeal agar in 25℃and 13℃, sterile distilled water or liquid paraffin in 25℃. Which stored by oatmeal agar in 25℃,could preserve 9 months or so, but have to transplanted every 4 months. The way stored by oatmeal agar in 13℃could last only 6 months. The isolates stored in sterile distilled water is best for the conserving,19 months latter the percentage of survival isolates is 100%, and the percentaqe of the isolates stored in sterile liquid paraffin is 75%.2. The collection isolation and identification of the strainThe samples were collected from Youyang, Shizhu, Fuling in Chongqing in 2009, and 25 isolates had been isolated. And 10 isolates had been isolated from Fengjie, Youyang, Nanchuan in 2010. With the 56 isolates which were provided by Institute of Plant Ecology &Pathology, Southwest University, there are 91 isolates. All these isolates were collected from Youyang, Shizhu, Fengjie, Nanchuan, Pengshui, Wanzhou, Fuling, Wushan in Chongqing.and acquisition time is from 2005 to 2010.3. The determination of mating typeDetermined the mating type of 45 isolates from Chongqing, by the method of breeding on the flat. The year of the samples from Chongqing is 2006,2007 and 2009. The results showed that there is type A2 or type A0 among the samples of 2006 and 2007, but it is only type A2 in 2009. It's different from before.4. The measurement of the resistance of metalaxy and induction of resistant strainsThirty two isolates had been determined, and the average EC50 of the isolates collected from Youyang is 0.8181μg/mL, the isolates collected from Shizhu is 0.5476μg/mL. The resistant level of the isolates collected from Youyang is higher than the average level, and the isolates collected from Shizhu is lower. The resistance of the isolates from different area had big otherness.Twenty metalaxyl-resistant isolates were obtained, the highest one is RSL1-7-1 (EC50=530.5789μg/mL), its about 941 times more than original isolates. Its inducing metalaxyl-concentration is 5μg/mL, the resistance of all the mutants could be maintained stabily subculturing on OA plates. The mutants have lower fitness than original isolates, the mutants with higher resistance level have the lowest fitness. All the fitness of mutants maintained at a high level.5. Identification of physiological raceSix isolates were inoculated on the oat medium with 2,3,5-triphenyl tetrazolium chloride (TTZ), and the color showed that the isolates were race 0 or 1. According identification hosts, only the isolate SL1-7 is race 0, others were race 1.6. Analysis of genntic diversity with RAPDTwelve Phytophthora parasitica var. nicotianae isolates had been analysed the genetic diversity with RAPD, a total of 39 DNA bands were found, and showed rich genetic diversity. Taking the similarity coefficient 0.86 for threshold,12 isolates were divided into 4 genetic clustering groups (Ⅰ,Ⅱ,Ⅲ,Ⅳ). The highest resistant isolate, RSL1-7-1 was divided into a single genetic clustering group, on the similarity coefficient 0.74 for threshold. There was big difference between them and its original isolate, these may showed that the gene of RSL1-7-1 had mutanted, but the mutant RYBX2-1-3, whose resistance level was 118 times of its original isote, can not been divided. In a word, there were no obvious relativity among genetic clustering group and ources, mating type, resistance level, physiological race.