RAPD Analysis Of Ralstonia Solanacearum From Ginger Blast In Sichuan

Ginger has a long history in China. It is not only a useful kind of spice, but also a important kind of agricultural product. And it has large economical value. Ginger is playing a very important role in daily life and farmers' income. In rencent years, the occurrence of the ginger blast is increasingly serious, hardly affecting the output and quality. We amazedly found the output loss of the ginger could be 10-30%, even to 100%, when we investigated the pathogenic ginger in Sichuan Ya'an, Leshan, Meishan, Wenjiang, and Jianyang. Ginger blast caused ginger turning yellow thoroughly,then withered, at last made the stem rot.The identified pathogenic bacteria Ralstonia solanacearum were isolated from ginger. We studied the bacteriology of the pathogen. Results showed that different temperature and PH value had obvious effect on Ralstonia solanacearum. The suitable growth culture medium are NA, PDA, and TTC; the most suitable temperature id 32℃, and the bacteria can't growth over 40℃;the most suitable pH value is 7.5. Ralstonia solanacearum are G , and their sizes ranged from 0.5-1×1.5-4μm. We also studied physiological and biochemical characteristics of Ralstonia solanacearum and indentifir their utilizing carbon compounds such as lactose, cellbiose, isinglass etc.Pathogenicity was also tested in tomato, pepper, eggplant and ginger. Results showed different strains had different pathogencity, the strains isolated from finger can infect ginger, but nearly no pathogenicity to tomato, pepper and eggplant. Fifty-five strains Ralstonia solanacearum isolated for biotype test according to the standard established by Hayward(1964). Four biotypes including I , III, IV and V were discovered. Thirty-two strains belonged to biotype III and fifteen belonged to I , which accounted for 58.1% and 27.3%. Biotype I in ginger was first reported.Genetic diversity of 32 Ralstonia solanacearum strains were analyzed using random amplified polymorphic DNA(RAPD) molecular markers on the basis of sample collection and pure culture. Best RAPD technique system for Ralstonia solanacearum was established after filtering the best concentration of all the factors. In 25μl reaction solution, containing 10× buffer2.5μl, random primer 0.3μmol/l, template DNA30ng, dNTP50μmol/l, M_g~2+2.5μmol/l, TaqDNA polymerase2.0U, and annealing temperature 36℃. The PCR procedure is 4min at 94℃ for 1 cycle and 1min at 94℃ , 2min at 72℃ for 40 cycles, and 10min at 72℃ for extention, 4℃ for hold. We extracted the genome DNA of 32 strains using an improved method and chose 11 10-base oligonucleotides for random primers from 127 pieces. The amplified results showed some polymorphism among the different strains. The amplified fragments were almost between 0.3-3.5Kb. There were 619 amplified DNA bands for 11 primers to 32 strains including 580 polymorphism amplified DNA bands, which was 93.7% to the total . Amplified polymorphism of primers OPD07, OPD20, OPI06, OPI14, OPK19 and IPQ18 are very high even over 95%. RAPD is an effective and sensitive method to detect the polymorphism of DNA. By clustering using ntsys software, the 32 strains can be separated into 4 clusters including cluster I , II, III and IV. Cluster I came from Wenjiang, Yibing and Yaan in part isolated in 2004; cluster II from Yaan isolated in 2004and on a strain isolated from pepper in 2004; cluster III from Yaan in part isolated in 2004 and cluster IV all from Yaan isolated in 2003. The cluster results showed genetic differentiation is related to the space and time of collection. Meanwhile, the complex heredity and variation of Ralstonia solanacearum are obvious. It can be concluded that there exists high genetic diversity within the strains for test coming from Sichuan province.We try to use molecular biology(RAPD) to analyze the genetic diversity of Ralstonia solanacearum. All the factors effecting RAPD were discussed; how to establish the RAPD technique system for Ralstonia solanacearum was also discussed. Some of them may be useful for the basic research, prevention and cure of ginger blast.