摘要：
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 CN193Cur 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 CN193Cur 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 CN193Cur had the same pathogenicity as did its wild type strain CN193. The mutant CN193Curp,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 CN193Curp grew faster than the wild strain on PDA plates without copper sulfate. The fruits could be infected after inoculation with conidia suspension of CN193Cur at the stage of blossom,and the mutant strain CN193Curp 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 CN193Cur 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.%【目的】建立枣缩果病菌链格孢菌的抗硫酸铜标记，为揭示链格孢菌在枣树上的侵染规律与深入掌握枣缩果病菌的致病机制和有效控制该病奠定基础。【方法】采用平板抑菌圈法，从硫酸铜、氯化钴、硝酸银和放线菌酮4种药剂中筛选出硫酸铜可以作为标记链格孢菌 CN193菌株的药剂。应用孢子萌发抑制法，在凹玻片中按1∶1的比例放入1.0，1.5，2.0 mg·mL －1硫酸铜与链格孢菌孢子悬浮液的混合液，培养12 h后通过孢子萌发抑制率确定硫酸铜的起始浓度。将培养基中硫酸铜的浓度由1.5 mg·mL －1逐渐提高至4.5 mg·mL －1，以培养菌株对硫酸铜的抗性；选取单孢培养，得到稳定的抗硫酸铜突变体 CN193Cur。通过喷雾和刺伤接种2种方法进行 CN193Cur的田间致病性测定，并结合BOX-PCR指纹图谱分析技术，检测抗硫酸铜突变体CN193Cur菌株的稳定性及链格孢菌在枣树上的初侵染时期。【结果】硫酸铜对链格孢菌具有较好的抑制作用，氯化钴、硝酸银的抑制效果较弱，放线菌酮没有明显的抑制效果。硫酸铜初始浓度为1.5 mg·mL －1时链格孢菌的突变率为12%，试验达到最高浓度为4.5 mg·mL －1；突变体 CN193Cur与野生型菌株 CN193具有相同的致病力；经致病性测定后的 CN193Cur p 能够在含高浓度硫酸铜(4.5 mg·mL －1)的培养基上生长，野生型菌株则无法生长；突变体菌株 CN193Cur在不含硫酸铜的 PDA 培养基上表现出比野生型菌株较快的生长速度；CN193Curp与室内筛选得到的突变体 CN193Cur一样，都是链格孢菌 CN193且其突变特征稳定。枣树花期接种链格孢菌能够发病，并再次在病果中分离到突变体菌株 CN193Cur p，说明链格孢菌在花期即能侵入枣树，致使枣果发病。【结论】硫酸铜抗性可以作为链格孢菌的稳定标记，为真菌和细菌抗药性标记的建立提供了理论依据；采用抗生素或化学药剂浓度递增的方法，可以有效地获得真菌和细菌的突变体，这为其他植物病原真菌或细菌侵染机制的研究提供技术支持；BOX-PCR 指纹图谱分析技术可快速检测出真菌的多样性，能够较为有效地提供菌株的种内差异信息；CN193Cur具有稳定的突变特征及致病性，可以应用于对枣缩果病菌的侵染机制及致病机制的研究。
