Studies On Molecular Variation Of Tobacco Black Shank Pathogen

Tobacco black shank (Phytophthora nicotianae) is one of the most important diseases intobacco production. As a soil-born pathogen, P. nicotianae can infect a variety of plants, andcan survive in soil residues for longer time. In order to understand the pathogen’s geneticvariation and provide theoretical foundation for control of the disease, the author usedselective media to isolate the pathogen from tobacco materials sent from various parts inChina, and conduct analysis of theri molecular genetic viaration. The major results include thefollowing four aspects.The modified selective media based on carrot agar medium were tested for isolation oftobacco black shank pathogen from the diseased stems collected by the author or sent fromvarious parts in China. After washing of the diseased samples in tap water overnight, a smallpiece of pith tissue was cut at the junction between diseased and healthy parts and placed onmedium for pathogen isolation. After trials of selective media with various components ofchemicals, an effective medium was developed for isolation of tobacco black shank pathogenbased on carrot agar medium (CA). The medium was composed of Rifamycin10mg/100ml,ampicillin5mg/100ml, PCNB5mg/100ml, carbendazim5mg/100ml, streptomycin0.35mg/100ml. It is a good medium for successful isolation of the pathogen because it effectivelyinhibited the growth of other fungi and bacteria. The pathogen could be purified by growingon rye agar medium added with ampicillin after3-5days of previous culturing on sesame agarmedium.The genomic DNA of P. nicotianae was extracted by combinations of three mycelialgrinding methods and three DNA extracting reagents. The results showed that the methods ofliquid nitrogen grinding+CTAB/SDS extracting, liquid nitrogen grinding+EZ-Kitextracting, and electric drill grinding+EZ-kit extracting were better than the othercombinations. The template DNA samples extracted by these methods were clearly detectedby agarose gel electrophoresis and after EF-1α-PCR amplification. Detection of ISSR-PCRproducts with primer UBC#808also generated expected band patterns. It is concluded that thethree combinations were suitable for genomic DNA extraction of P. nicotianae. The suitablemethods of DNA extraction provide a good base for further study of the pathogen’s molecularvariation.The selected strains of P. nicotianae isolated from20cities in China were molecularlyidentified by analysis of their28s r RNA and β-tubulin gene sequences. It is found up to one or two bases were different in28s rRNA sequences of these strains. The cluster analysisindicated that these tested strains could be divided into three clades.Totally140strains of P. nicotianae isolated from the tobacco materials collected or sentin various parts of China were analyzed for genetic diversity by means of SRAP and RAPD.It is found that the majority of these strains did not show evident variation in molecular level,indicating the pathogen was genetically stable.