Investigation On The Method Of Rapid Screening Tobacco Mutants Resistant Against Alternaria Alternata

Tobacco is an important economic crop. The occurrence of tobacco brown spot seriously affects the yield and quality of tobacco. Planting resistant cultivar has been considered to be the most cost-effective way. In order to screen anti-brown spot mutants from mutant library with high number, a rapid and high-throughput method is of significance. hi this paper, six bacterial brown spot pathogens strained from tobacco were isolated and identified, and a strong strain-Al was screened. We designed and compared three resistant identifying methods, and got the rapid and high-throughput one. At the same time, we selected, identified and studied parts of mutant materials.The findings are as follows:(1) The seeds of the tested materials were disinfected and placed in the medium with brown bacteria toxins at a dilution ratio of1:8. Tobacco seedling root length was investigated after25days and the level of anti-sense tobacco brown bacteria was determined. To verify the feasibility, indoor and field disease indexes of Jingyehuang, Beinhart1000-1, NC89, G140, K326, G28, Zhongyan100and other conventional anti-sense control varieties were compared and analyzed. The results showed a significant positive correlation (r=0.8823). It is consistent between the degree of anti-sense resistance indoor and outdoor by comparing the inhibition of toxin on tobacco seedling roots to determine material on the brown spot disease, which can be a representative of the field disease. The method provides foundation for rapid high-throughput screening to identify anti-tobacco brown spot diseases.(2) Anti-brown spot mutants were screened from1229mutants in Zhongyan100M2mutant library by EMS mutagenesis. Six resistant mutants were obtained by comparing mutant materials’root length which significantly longer than control wild-type tobacco root after25days. Microscopic observation on the tested material roots shows that:root tips of mutation were smooth, straight and with sufficient root hairs; root tips browning the original moisten or necrosis, root hairs dehydrate bendy. The seeds of Zhongyan100EMS M3mutation that resistant brown spot disease were obtained by fruit bagging and analyzed using primer J9. The result shows that the fingerprints of five mutants were in consistent with resistant variety, the match rate were83.3%.The innovation of this paper is firstly established a rapid and high-throughput method to screen tobacco mutants by the toxins medium combined according to root length measurement. Six anti-brown spot mutant materials were screened successfully through the method. Our results would not only provide foundation for screening tobacco mutants, but also show materials and support for tobacco brown spot disease gene screening, cloning and functional identification, and anti-tobacco brown spot disease mechanism.