| Tobacco is one of the important economic crops of Shandong Province, and its high quality and stable yield were restrained by diseases, especially tubers diseases in succession planting fields for many years. Tobacco root diseases such as black shank, bacterial wilt, sore shin, black death, black root rot occurred frequently and serious in Shandong Province. Various research institute and production sector have been committed to black shank disease, nematode control and breeding for disease resistance in the past few years , so these sustainable management of the diseases has reached remarkable achievement. But the evolution of microorganism in rhizosphere, followed by soil-borne diseases increasing caused secondary disease such as root rot becoming more and more serious for succession planting .Diagnoses and controlling tobacco black root rot were difficult because it often occurred with other tobacco roots diseases and some other factors in fields. Molecular detection and chemical fungicide screening for Thielaviopsis basicola will facilitate the development of effective diagnoses and control strategies for tobacco black root rot, and will be important for hold stable quality and sustainable development. The result showed:1. Construct a rapid and efficient PCR method to detect or identify T. basicola. Total DNA extraction of T. basicola strains obtained from Shandong province, and PCR were carried out as described previously. Universal primers used for amplifying rDNA-ITS designed according to fungal pathogens sequences from GeneBank. The purified PCR products were ligated into pMD18-T according to the manufacturer's instructions, followed by transformation into Escherichia coli DH5α. Positive clones were sequenced and the result were submitted to GeneBank(EU794001). Specific primers (TB-5/TB-3) used for amplifying rDNA-ITS of Thielaviopsis basicola , designed according to the sequences difference between Thielaviopsis strains (including EU794001) and other tobacco fungal pathogens from GeneBank. The result showed that 400bp fragment was acquired by PCR only from T. basicola but other fungi strains wasn's detected separated from tobacco. T. basicola was also specifically detected in tobacco tissues and soil with TB-5/TB-3, and the sensitivity of detection was 100 fg genomic DNA per 25μL PCR reaction volume. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring, as well as guiding plant disease management.2. Screening chemical fungicides of T. basicola. Seven fungicides for controlling tobacco black root rot were selected for testing under laboratory experiment. The results showed that the inhibition of spores germination of carbendazim in 12h and 24h were 56.0% and 58.6%, and the other six fungicides reached 100%. Thiophanate-methyl, Carbendazim ,Dithane M-45 and Sandofan M8 had good and stable control effects to this hypha of pathogen.3. Evaluate toxicity of the four chemical fungicides to T. basicola. On the screening basis, the toxicity of four fungicides to Thielaviopsis basicola were tested by the mycelium growth rate methods. The results showed that the EC50 of Carbendazim, Thiophanate methyl , Dithane M-45 and Sandofan M8 were 0.4251,5.3134,30.2686 and 37.2234mg/L, and the EC90 were 0.8170,11.4312,110.1398 and 160.5393 mg/L separately. Considering the compartion of active ingredients of fungicides and their working mechanisms,we find that the best way of applying fungicides is to apply Carbendazim and Dithane M-45 alternatively. |
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