关键词: 链格孢菌, BOX-PCR, 抗硫酸铜突变体, 枣缩果病, 枣树

Abstract: [Objective] In order to determine the suitable agent and method for labeling Alternaria alternata, the pathogen of jujube shrunken-fruit disease, and to explore its primary infection period, the labeling system with copper sulfate in A. alternata was established, which will facilitate the study of infection mechanisms and pathogenicity of the pathogen, and effective management of the disease in the field.[Method] Copper sulfate was selected for the reagent of resistance-labeling in A. alternata wild type strain CN193 from the four fungicidal chemicals, i.e. copper sulfate, cobalt chloride, silver nitrate and cycloheximide by using inhibition zone method on PDA plate. With the inhibition method of spore germination, the mixture of conidial spore suspension with either 1.0 mg·mL^-1, 1.5 mg·mL^-1 or 2.0 mg·mL^-1 concentration of the copper sulfate in a ratio of 1:1 was dropped into concave slides. The germinated spores were scored after a 12 h incubation, and the initial concentration of copper sulfate was determined. A copper sulfate-resistant mutant strain CN193Cu^r was obtained by culturing the fungus on PDA medium containing copper sulfate from 1.5 mg·mL^-1 up to 4.5 mg·mL^-1. The stability of the copper sulfate resistance of mutant CN193Cu^r was verified in the laboratory, and its infectivity was determined by pathogenicity test in a jujube plantation with inoculation of spore by spraying to the jujube flowers and stab-wounding on the surface of unripe jujube fruits, together with the analysis of BOX-PCR finger printing patterns.[Result] Copper sulfate had highly inhibitory effect on A. alternata while cobalt chloride and silver nitrate were less inhibitive, and cycloheximide was not inhibitive. The initial concentration of copper sulfate was set at 1.5 mg·mL^-1, which produced a mutation rate of 12%, and the final concentration of copper sulfate in this study was 4.5 mg·mL^-1. Inoculation tests showed that the mutant CN193Cu^r had the same pathogenicity as did its wild type strain CN193. The mutant CN193Cu^rp, isolated from diseased fruit through inoculation of spore spraying onto the flowers, could grow normally on PDA plate with high concentration (4.5 mg·mL^-1) of copper sulfate, while the wild type strain CN193 could not. The CN193Cu^rp grew faster than the wild strain on PDA plates without copper sulfate. The fruits could be infected after inoculation with conidia suspension of CN193Cu^r at the stage of blossom, and the mutant strain CN193Cu^rp was able to be isolated again from the diseased fruits, demonstrating that the mutant strain was able to infect jujube flowers.[Conclusion] Copper sulfate could be a suitable label to A. alternata, which may provide a technique for labeling other pathogenic fungi and bacteria. The method of increasing concentrations of antibiotic or chemicals can obtain stable mutants, which will facilitate the study of infection mechanisms in plant pathogenic fungi or bacteria. BOX-PCR fingerprint techniques can be used to quickly detect the fungal diversity, which may provide more effective intraspecific information of different strains. The mutant CN193Cu^r has stable resistance to copper sulfate and strong pathogenicity to jujube fruits, and can be applied to study the infection mechanism and pathogenesis of the pathogen causing jujube shrinkage fruits.

Key words: Alternaria alternata, BOX-PCR, copper sulfate resistant mutant, jujube shrunken-fruit disease, Zizyphus jujuba