关键词: 玉米南方锈病, 多堆柄锈菌, 常规PCR, 巢式PCR

Abstract:

【Objective】The objective of this study is to establish a rapid detection method for Puccinia polysora during the incubation period based on the nested PCR, and to provide support for prediction and control of southern corn rust.【Method】The nested PCR primers for the specific detection of P. polysora were designed using the variant region of the ITS sequence, including outer primer NX471-F/ITS4 and inner primer NX255-F/NX255-R. For amplification, the 20 µL PCR reaction mixture contained: Ex Taq DNA polymerase (5 U·μL^-1) 0.15 μL, 10×Ex Taq Buffer (Mg²⁺ plus) 2 μL, dNTP Mixture (2.5 mmol·L^-1 each) 1.6 μL, forward and reverse primers (10 μmol·L^-1) 0.3 μL each, template DNA 1 μL, ddH₂O 14.65 μL. The PCR program was performed as follows: the outer primer NX471-F/ITS4 was used for the first amplification and denaturation at 95℃ for 7 min, 30 cycles of denaturation at 95℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 45 s, and a final extension at 72℃ for 7 min. The product was diluted 20 times and used as the template for the second run of the nested PCR, which was amplified with the inner primer NX255-F/NX255-R, and the PCR program was denaturation at 95℃ for 7 min; 30 cycles of 95℃ for 30 s, 66℃ for 30 s, and 72℃ for 26 s; 72℃ for 7 min. At the same time, the inner primer was selected for conventional PCR, in which the reaction mixture was the same as the nested PCR, and the amplification conditions were the same as the second run, except that the number of reaction cycles was 38. Under these conditions, the specificity was detected for P. polysora, Puccinia sorghi, Melampsora laricipopulina and seven other common maize pathogens, and the sensitivity of nested PCR was tested using genomic DNA of P. polysora and DNA from artificially inoculated leaves.【Result】Nested PCR could specifically detect P. polysora from all tested fungi, with a 255 bp target fragment. The lowest detection limit of the nested PCR was 10 fg·μL^-1, and the sensitivity was 500 times that of the conventional PCR. For the samples containing 500-2.5×10⁴ urediniospores per gram of artificially inoculated leaves, the detection rates of conventional PCR and nested PCR were 0 and 85.71%, respectively. Nested PCR could detect at least 1 000 urediniospores. For the samples containing 1-7 uredia per gram of artificially inoculated leaves, the detection rates of conventional PCR and nested PCR were 76.19% and 100%, respectively. Conventional PCR and nested PCR could detect at least 2 and 1 uredia, respectively. For the samples containing 1-7 infection sites per gram of artificially inoculated leaves, the detection rates of conventional PCR and nested PCR were 14.29% and 66.67%, respectively. Conventional PCR and nested PCR could detect at least 6 and 3 infection sites, respectively.【Conclusion】With NX471-F/ITS4 as the outer primer and NX255-F/NX255-R as the inner primer, a detection method for P. polysora was established based on the nested PCR, which could quickly, efficiently and accurately detect the P. polysora during the incubation period in maize leaves.

Key words: southern corn rust, Puccinia polysora, conventional PCR, nested PCR