The Selection Of Anti-black Shank Mutants In High Potassium Efficiency Of Flue-cured Tobacco Lines

Tobacco black shank is a worldwide and serious rhizome disease,it influences the yield and quality of tobacco leaves seriously.In order to improve the disease resistance to tobacco black shank of high potassium of flue-cured tobacco lines with good comprehensive characters in field,and realize the promotion value of high potassium of flue-cured tobacco lines.This study based on the pathogenic characteristics of pathogenic bacteria in tobacco black shank,firstly chose five kinds of conventional medium which are suitable to the growth of phytophthora parasitica,combined with the inducing effect of root exudates from tobacco,screened out the the highly pathogenic fermentation liquid of phytophthora parasitica;Then purified the fermentation liquid of phytophthora parasitica,so that it can obtain the pathogenic factors component with better scavenging activity?the active ingredients from sterile fermentation liquid of phytophthora parasitica?;Finally utilizing the pathogenic factors as selection pressure,chose 5 kinds of potassium efficient flue-cured tobacco which have good performance in field to go for anther culture in vitro,then selected the cell mutant lines which are resistance to black shank.The main research results were as follows:1.Screening of culture medium for inducing pathogenic bacteria of black shank.The phytophthora parasitica was inoculatede into 5 kinds of medium?10%V8 juice?carrot?oats?tobacco leaf juice?potato?for the dark culture.Using the method of leaf injection in vitro,Observing the changes of leaf injection area where was injectived by 5 kinds of sterile fermentation liquids of phytophthora parasitica.The results show:potato medium?PDA?is the most obvious in injection effect,it's leaf injection area appear the hypersensitive necrosis reaction?hypersensitive resistance,HR?after 7 days of injection,and the best treatment time is 7 days for the black shank bacteria to cultivate to be fermentation liquid,so choose the potato medium?PDA?for the most suitable medium to induce strong pathogenicity of black shank bacteria.Using the method of leaf injection in vitro,judging the correlation between the root exudates of tobacco and the pathogenicity of phytophthora parasitica.The results show:the mean lesion area of A?0.56cm²??injection reaction of sterile fermentation liquids from culturing phytophthora parasitica without the inducement of root exudates?is less than B?0.80cm²??injection reaction of sterile fermentation liquids of phytophthora parasitica treated by root exudates?,the HR reaction of B?injection reaction of sterile fermentation liquids of phytophthora parasitica treated by root exudates?is more strong,so it indicated that the root exudates may promote the pathogenicity induction of sterile fermentation liquids of phytophthora parasitica.2.The preparation and crude separation of fermentation broth of black shank fungus.In this paper,silica gel column has been used to separate the sterile fermentation liquids of phytophthora parasitica,then through the preparation of liquid chromatography for purify,using the method of leaf injection in vitro,judging the pathogenicity of black shank bacteria fermenting liquid?ultrafiltrate?resin solvent?95%ethanol elution liquid.The results show:HR reaction of leaf injection area where was injectived by 95%ethanol elution liquid is the most obvious,it indicated that the pathogenicity of 95%ethanol elution liquid is stronger,while the HR reaction of leaf injection area where was injectived by resin solvent is not obvious,then it can be rejected.Evaporated and condensed the 95%ethanol eluent into dry powder,then use the ddH₂O to dissolve and save it for later use.3.The determination of selection pressure and the screening of mutant which resistanted to black shank..Using the 95%ethanol solution as the selection pressure of pathogenic factor,choosing 5 kinds of potassium efficient tobaccos?GK2?GK8?HKDN-2?HKDN-8?HKDN-5?to go for anther culture in vitro.The results show:the germination rate of five kinds of potassium efficient flue-cured tobaccos is different,all the germination rate is generally low,and the germination rate from high to low are GK2?38.0%?>HKDN-5?21.3%?>GK8?19.3%?>HKDN-8?15.3%?>HKDN-2?13.3%?in turn;With the increase of the concentration of the sterile fermentation liquid,the germination rate of the five kinds of potassium efficient flue-cured tobaccos is decrease,the mostsuitable selection pressure of these tobacco varieties?GK2?GK8?HKDN-2?HKDN-8 and HKDN-5?is respectively 3%?2%?3%?4%?3%.After the ploidy identification and double treatment of the tobacco plants which were from anther culture,they were induced to be normal tobacco plants,including235 strains,while among them,there were 44 strains of GK2?61 strains of GK8?57 strains of HKDN-2?39 strains of HKDN-8 and 34 strains of HKDN-5.Through the method of leaf injection in vitro,identified the resistance to black shank of tobacco plants which were screened out.The results indicted:2 strains?GK8?with high resistance were screened,and there are 26 strains which were moderate resistance to black shank,among them,there were 6strains of GK2?10strains of GK8?6 strains of HKDN-2?3 strains of HKDN-8 and 1 strains of HKDN-5.