广藿香青枯病菌无致病力突变株的构建及其生防机制研究

Pogostemon cablin(Blanco) benth is one of famous berb of Guangdong. The most devastating disease of P. cablin is bacterial wilt caused by Ralstonia solanacearum, which results in a high proportion of loss in yield and quality. Preventive measures for bacterial wilt are mainly based on crop ration and chemical control. However, all these measures have met with only limited success. And resistance of pathogenic bacteria and chemial residues and other issues have become increasingly prominent problem. Biological control reduces environmental pollution and pathogen resistance build-up. It has been considerd a viable alternative method to manage plant diseases. Avirulent strains of the microbial pathogens are adapted to the same ecological niche as the same pathogen strain and can be used to preempt or displace the pathogen from the susceptible host. They have been shown to induce resistance in treated plants against the virulent strains of the same pathogen. And also they have a broad spectrum resistance to different races of pathogenic bacteria. Hence they can be employed as biocontrol agents for providing protection against the microbial pathogens. The strong virulent strains of R. solanacearum will be used in this study was isolated from the infected P. cablin plants with typical wilting symptom. Mutants of R. solanacearum using Tn5 transposon mutagenesis will be construted and the avirulent mutants will be selected. And then, insertion site of avirulent mutants will be analysed. Biological control mechanism and effectiveness of avirulent mutants of R. solanacearum against bacterial wilt will be evaluated. This study will establish the potential of avirulent mutants of R. solanacearum as biocontrol agents against bacterial wilt.

广藿香为我国著名南药，青枯病是制约其生存和发展的毁灭性土传病害，现主要依靠合理轮作及化学农药来防治，但只能部分减轻危害。同时病原菌耐药性及农药残留等问题日益突出。生物防治具有对环境和人畜安全、不易产生抗药性等优点，是目前大力推广的无公害防治技术。病原无致力菌株的适生环境与相应致病菌基本相同，能在寄主根际与致病菌进行营养和侵染位点竞争，并能诱导植株产生抗性，对病原菌不同生理小种均具有竞争和抑制作用，是较为有效的生防因子。本研究将选择从广藿香植株上分离的高亲和性致病青枯菌菌株为材料，采用Tn5转座突变技术构建青枯菌突变体库；筛选失去致病力但仍具有较好的寄主亲和力的突变株；并对转座子插入位点进行鉴定与分析，以获得基因突变位点明确、遗传稳定的青枯菌无致病力菌株。再以无致病力突变株为材料，针对广藿香青枯病进行生防体系、防治潜能及其生防机制等的研究，为开发安全有效的防治广藿香青枯病的生物农药奠定基础。

广藿香为我国著名南药，青枯病是制约其生存和发展的毁灭性土传病害，现主要依靠合理轮作及化学农药来防治，但只能部分减轻危害。同时病原菌耐药性及农药残留等问题日益突出。生物防治具有对环境和人畜安全、不易产生抗药性等优点，是目前大力推广的无公害防治技术。病原无致病力菌株的适生环境与相应致病菌基本相同，能在寄主根际与致病菌进行营养和侵染位点竞争，并能诱导植株产生抗性，对病原菌不同生理小种具有竞争抑制作用，是较为有效的生防因子。本研究开展了广藿香青枯病病害调查、病原菌的分离、鉴定及遗传多样性研究；进行了青枯菌诱导广藿香防御相关酶活性及其同工酶研究；建立了Tn5转座子诱导广藿香青枯病菌插入突变的方法，构建了广藿香青枯病菌突变体。根据突变株Tn5转座子插入位点侧翼序列的生物信息学分析，获得了10多株Tn5转座子插入位点基因为致病相关基因的突变株。由于Tn5转座子片段的插入，可能造成插入部位的致病基因失活。经突变株泳动能力测试、致病性测定及遗传稳定性研究，得到几株致病力明显变弱且遗传稳定的突变株，为开发防治广藿香青枯病的生防菌株提供了有价值的菌株材料。相关的研究，国内外未见报道。本研究为利用基因工程手段开发防治广藿香青枯病的生防菌株奠定了基础，也为其它中药材的病害防治提供了借鉴。