| Tobacco root black rot is a serious soil-borne disease,which is caused by Thielaviopsis basicola.In addition,tobacco root black rot can be mixed with tobacco root rot,tobacco black shank and other diseases,which’s the symptoms of these diseases are similar,so it is difficult to identify diseases in the field,thus we often missing the appropriate control period.Because the disease is harmfu L and difficu Lt to prevent and cure,it is very important for the early warning of the disease to strengthen the monitoring of pathogenic bacteria in tobacco planting and to accurately understand the number and distribution of the pathogen in the field.In this study,a set of Real-time PCR technique was established to detect the spores of black rot pathogen of tobacco root in the field,which provided a theoretical basis for early warning of the disease.At the same time,the effects of selenium and silicon on tobacco root black rot were studied.The main resuLts of this thesis are as follows:1.Based on Real-time PCR technique,a quantitative detection system of tobacco root black rot bacteria in soil was established.Based on the difference of ITS sequence between tobacco root black rot fungus and other fungi,a pair of specific primers Tb F/ Tb R was designed,the amplified fragment was 150 bp.by optimizing the reaction system,the DNA of soil inoculated with 10 times gradient concentration of pathogen spores was used as template.The standard curve of reaction results was obtained.The linear relationship between the number of conidia in each reaction system(x)and the corresponding Ct value(y)was linear:y=-1.5373x+24.955,R2=0.9909(P<0.01)(P <0.01).The sensitivity of conidia was 1 spores per gram of soil.The reliability of the Real-time PCR standard curve of soil was verified.The coefficient of variation of the repeated technique was between 0.30-2.42%,which indicated that the method had high sensitivity and good repeatability.2.Distribution and detection of bacteria in the field.Five spot samples of five tobacco fields with tobacco root black rot in previous years were quantitatively detected.The results showed that the pathogen spores could be detected in three samples of field No.5.The results indicated that the above system could detect the content of black rot fungi of tobacco roots in the field,which indicated that the distribution of black rot fungi of tobacco roots in different fields was obviously different,and the distribution of fungi in the same field was also uneven.3.The inhibitory effects of exogenous substances and fungicides on tobacco root black rot fungi.The effects of sodium selenite and silicon on the growth of tobacco root black rot fungus mycelium were studied by laboratory viru Lence test.The resu Lts showed that sodium selenite inhibited the growth of mycelium of tobacco root black rot fungus,and its EC50 to mycelium was 1.2743μg.m L-1,while silicon had no inhibitory effect on the mycelium of pathogen.Sodium selenite and other fungicides were mixed at the ratio of 1: 1: 1: 4 and 2:3 respectively.The effects of synergistic action on mycelium were studied.It was found that the mixture of sodium selenite and carbendazim 2:3 had a certain synergistic effect on the inhibition of tobacco root black rot mycelium.4.Effects of exogenous substances on defensive enzyme activity in tobacco.The changes of defensive enzyme activity and the relative expression of PR-1,PR-2protein in tobacco were studied by filling tobacco roots with Na2 Se O3 and Si O2.The activity of PPO and ACT and the relative expression of PR-1,PR-2 protein were increased in the treated tobacco,indicating that both sodium selenite and silica might induce tobacco resistance to the disease. |
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