大蒜抗叶枯病变异系的离体筛选及其抗性分析

【目的】利用病菌粗毒素离体筛选大蒜抗叶枯病变异系，建立抗病变异系离体筛选方法。【方法】以蒜瓣茎盘为外植体诱导愈伤组织；以大蒜叶枯病菌培养滤液制取的粗毒素为筛选剂，离体筛选大蒜抗叶枯病变异系；通过接种病菌粗毒素和病菌孢子鉴定变异株的抗病性，分析变异株的防御酶活性。【结果】大蒜叶枯病菌粗毒素对大蒜愈伤组织增殖有显著抑制作用，抑制作用随病菌粗毒素质量分数的增加而增强，不同大蒜品种愈伤组织对病菌粗毒素的抗性不同；利用不同体积分数的病菌粗毒素分步筛选分别获得大蒜品种G039变异系苗12株，G073变异系苗2株，它们对大蒜叶枯病菌粗毒素具有稳定的抗性。经病菌孢子悬浮液接种鉴定，变异系苗较其对照抗病性提高。大蒜抗叶枯病变异系苗接种病菌后叶片过氧化物酶（POD）、多酚氧化酶（PPO）和苯丙氨酸解胺酶（PAL）的活性在短时间内即达到峰值，且与抗病品种G064接近或更高，而感病品种G039的酶活性则明显低于前两者。【结论】利用不同体积分数的大蒜叶枯病菌粗毒素分步筛选可以获得大蒜抗叶枯病变异系，适宜大蒜抗叶枯病变异系分步筛选的上限粗毒素体积分数为30%；POD、PPO和PAL活性可作为大蒜抗叶枯病鉴定的生化指标。

In vitro selection of garlic mutants resistant to leaf blight and analysis of their resistance

【Objective】 The study was to select garlic resistant mutants and to set in vitro selection method by adding the pathogen crude toxin in culture medium.【Method】 The resistant mutants were selected using callus derived from clove base explants and the filtrate(crude toxin) of garlic leaf blight pathogen culture solution as the selective agent,and identified by inoculation of crude toxin and pathogen spores to the mutants,respectively.The activities of some defense enzymes in mutant garlic plantlets were measured to analyze the relation between garlic leaf blight resistance and those enzymes.【Result】 The crude toxin of pathogen inhibited the induction and multiplication of garlic callus.The inhibition increased with the increase of crude toxin concentration.The callus of different garlic cultivars showed different resistance to the pathogen crude toxin.Using step by step screen method to increase the concentration of pathogen crude toxin,12 mutant plantlets from garlic cv G039 and 2 plantlets from garlic cv G073 were selected.These mutants were proven to attain stable resistance to pathogen crude toxin.The mutants were also proven to gain improved resistance to leaf blight than their control by pathogen spore inoculation test.The activities of POD,PPO and PAL in leaves of the mutant increased sharply and reached the peak values in short time after inoculation of pathogen,which were close to or even higher than those of the resistant cv G064.However,the activities of those enzymes in disease sensitive cv G039 were much lower than those of the mutant and the resistant cv G064.Disease resistance identification by inoculation of pathogen spore solution showed that the mutant lines were more resistant to the disease than their controls.The change of POD,PPO and PAL activities in screened mutants after inoculation of the pathogen spores reflected their disease resistance.【Conclusion】 It is practicable to select garlic mutants resistant to leaf blight by increasing the concentration of pathogen crude toxin in the selective culture medium step by step.The proper maximum concentration of crude toxin for in vitro selection is 30%.The enzyme activities of POD,PPO and PAL can be taken as biochemical indexes for garlic resistant identification to leaf blight.