噻枯唑防治水稻白叶枯病和柑橘溃疡病的作用机制研究

发布时间：2017-12-28 22:14

本文关键词：噻枯唑防治水稻白叶枯病和柑橘溃疡病的作用机制研究　出处：《南京农业大学》2016年博士论文　论文类型：学位论文

【摘要】：噻枯唑是我国目前用来防治黄单胞菌属致病细菌引起的水稻白叶枯病(Xanthomonas oryzae pv. oryzaae),水稻细菌性条斑病(Xanthomonas oryzae pv.oryzicola)和柑橘溃疡病(Xanthomonas citri pv. citri)的常用杀细菌剂。水稻白叶枯病是由水稻黄单胞菌引起的一种在全世界水稻种植地区十分常见并且会造成严重经济损失的的细菌性病害。除了利用品种抗性作为控制水稻白叶枯病流行危害的关键措施以外,在病害发生初期采用杀菌剂进行化学防治是重要的应急措施。由于在水稻上长期频繁使用,已经在田间检测到抗药性菌株。噻枯唑抗型菌株表现活体抗药性,而在离体条件下不表现抗药性。1.噻枯唑敏感性菌株ZJ173与抗性菌株2-1-1转录组分析本文以噻枯唑敏感性菌株ZJ173和在药剂处理条件的水稻上筛选到的ZJ173抗药性突变体2-1-1为研究对象,首先研究了这两个菌株的生物学表型差异,包括致病力,生长能力,胞外多糖产量,生物膜形成,游动性以及碳源利用率。其次对这两个菌株进行高通量转录组测序,筛选出表达量有显著性差异的基因并进行功能分析。结果表明,ZJ173与2-1-1的致病力与生长能力没有显著性差异,但是2-1-1的胞外多糖产量显著高于ZJ173,生物膜产量高于ZJ173,细胞游动性弱于ZJ173。与这一结果对应的是,在转录组得到的差异基因中,也包含了与细胞游动与趋化性相关的基因。这说明噻枯唑抗性菌株可能具有更强的细胞聚集能力,形成生物膜结构来抵抗外界化学药剂的伤害。同时,在分析对碳源利用率后发现,ZJ173需要从外界环境中吸收的碳水化合物种类与数量均显著多于2-1-1,转录差异表达基因中也包含了与三羧酸循环以及琥珀酸合成相关的基因,这说明2-1-1菌体内的碳水化合物代谢活力高于ZJ173。以上结果表明,噻枯唑抗性机制可能与菌体的游动聚集能力以及碳水化合物代谢有关。2.硫氨素合成途径相关基因thiG对Xoo的致病性与药敏性的影响thiG基因是硫氨素合成途径中的重要基因,主要负责硫氨素的噻唑环的合成。在上一章的研究中发现在转录组测序得到的差异表达基因中有硫氨素合成途径的相关基因。Xoo的thiG基因缺失突变体表现致病力下降,其生长速率在不含硫氨素的培养基中下降,在含有硫氨素的培养基中和水稻叶片组织中生长速率恢复野生菌株的水平。在离体条件下,thiG缺失突变体对噻枯唑药敏性增强。这些结果说明thiG突变体需要从外界环境中吸收硫氨素来维持自身正常生长,我们推测thiG缺失突变体可能同时吸收与硫氨素结构相似的噻枯唑,从而导致低浓度药剂对突变体的抑菌效果优于野生型菌株。同时,与野生型菌株ZJ173相比,thiG基因的缺失突变体细胞的聚集能力会增强,包括生物膜产量增加,而细胞游动性和迁移能力降低,这些结果说明,可能由于thiG缺失突变体在水稻上的侵染和扩散能力下降,所以表现致病力下降。双组份调控系统rpfC和rpfG编码的蛋白质可以通过第二信使cyclic di-GMP调控细胞的聚集,研究结果显示rpfC和rpfG基因的表达量在thiG缺失突变体中表现下调。突变体的胞外多糖产量虽然没有发生明显变化,但是与胞外多糖合成与分泌的相关基因,像gumD,gumE,gumH和gumM,均出现了上调表达,而这四个gum基因同时也逆向调控生物膜的形成,进一步证明突变体的细胞聚集能力增强与胞外多糖生物合成相关。这些结果说明硫氨素合成途径中的噻唑环合成基因thiG对Xoo的致病力和细胞聚集能力有至关重要的作用。3.Xoo的胞外多糖合成相关gum基因家族对噻枯唑药敏性影响水稻白叶枯病菌(Xoo)的胞外多糖作为一种重要的致病因子,与病原菌的侵染,吸附以及聚集都密切相关,并且可以保护菌体抵抗外界不良环境的干扰。本文研究发现,噻枯唑敏感性菌株ZJ173和抗药性菌株2-1-1的胞外多糖产量存在很大差异。通过构建ZJ173与2-1-1各个胞外多糖合成基因簇gum基因家族的缺失突变体,在水稻上分别测定各个突变体对噻枯唑的药敏性。结果表明,在水稻上ZJ173的突变体由于致病力下降,在噻枯唑未处理与处理后病斑长度没有显著性差异,因此不能判断噻枯唑是否对突变体发挥抑菌效果。然而,2-1 -1的gum突变体2-1-1△gumC和2-1-1△gumI在活体条件下的致病力没有降低,同时对噻枯唑表现出抗性水平下降,说明gumC和gumI基因的缺失会提高抗性菌株2-1-1的药敏性。综上所述,Xoo缺失gum基因后的噻枯唑药敏性发生不同变化,由于gum基因簇在胞外多糖合成与分泌中负责的功能不同,gum基因缺失不同程度地改变了 Xoo的胞外多糖产量和结构,并直接影响对噻枯唑的药敏性。4.噻枯唑抑制柑橘溃疡病菌的生长以及诱导柑橘防卫基因的表达柑橘溃疡病是由Xanthomonas citri pv. citri (Xcc)引起的一种在柑橘种植地区广泛存在的并造成严重经济损失的细菌性病害。在本研究中,我们证实了噻枯唑通过抑制Xcc的生长和诱导寄主的抗病反应,可以有效的控制柑橘溃疡病。噻枯唑处理柑橘会诱导致病相关基因(PR1, PR2, CHI和RpRd1)以及NPR基因(NPR1,NPR3和NPR4)的上调表达,尤其是在处理初期。另外,我们发现噻枯唑会诱导类黄酮合成途径中的标志性基因CitCHS和CitCHI以及水杨酸合成途径中的关键基因PAL的表达。而且,噻枯唑还可以诱导防卫反应的启动效应的诱导基因AZI1的上调表达。综上所述,噻枯唑处理柑橘后诱导的防卫反应可能与SA信号传导途径和启动效应有关,同时这一研究为以后在柑橘溃疡病的化学防治方面提供了一种新的选择。
[Abstract]:Saikuzuo in China is currently used in the treatment of Xanthomonas leaf blight of rice caused by pathogenic bacteria (Xanthomonas oryzae pv. oryzaae), rice bacterial leaf streak (Xanthomonas oryzae pv.oryzicola) and Citrus Canker (Xanthomonas citri pv. citri) commonly used bactericide. Rice bacterial leaf blight is a bacterial disease caused by Xanthomonas Oryza, which is very common in rice growing areas all over the world and will cause serious economic losses. In addition to using variety resistance as a key measure to control the epidemic of rice bacterial blight, it is an important emergency measure to use fungicides for chemical control in the early stage of disease. Drug resistant strains have been detected in the field because of the long-term use of rice on the rice. Saikuzuo resistant strains showed in vivo resistance, and in vitro expression of resistance. 1. Saikuzuo sensitive strain ZJ173 and strain 2-1-1 transcriptome analysis based on sensitivity of Saikuzuo strain ZJ173 and treatment conditions in the pharmaceutical rice screened ZJ173 resistant mutants of 2-1-1 as the research object, firstly study the phenotype differences among the two strains, including pathogenicity, growth ability, exopolysaccharide yield, biological film forming, motility and utilization ratio of carbon source. Secondly, the two strains were sequenced by high flux transcriptome, and the genes with significant differences in expression were screened and functional analysis was carried out. The results showed that there was no significant difference in pathogenicity and growth ability between ZJ173 and 2-1-1, but the yield of exo polysaccharide in 2-1-1 was significantly higher than that in ZJ173, and the yield of biofilm was higher than that of ZJ173, and the motility of cells was weaker than that of ZJ173. In correspondence with this result, in the differential genes of the transcriptional group, it also contains genes associated with cellular movement and chemotaxis. This shows that Saikuzuo resistant strains may have stronger ability of cell aggregation, formation of biofilm structure to resist external chemical damage. At the same time, in the analysis of the utilization ratio of carbon source, ZJ173 need carbohydrates type and quantity of absorption from the external environment were significantly higher than 2-1-1. The gene also contains three carboxylic acid cycle and succinic acid synthesis related gene expression transcription differences, indicating that the carbohydrate metabolism activity of 2-1-1 in ZJ173 cell. The above results show that the resistance of Saikuzuo and possible mechanisms of cell aggregation ability and carbohydrate metabolism on swimming. 2., the influence of thiG gene on the pathogenicity and drug sensitivity of Xoo is thiG, which is an important gene in the thiosynthesis pathway. It is mainly responsible for the synthesis of thiazoles thiazole ring. In the previous chapter, we found that there are genes associated with thiin synthesis pathway in the differentially expressed genes that are sequenced in the transcriptional group. The thiG gene deletion mutant of Xoo showed a decrease in pathogenicity, and its growth rate decreased in the medium without sulfur ammonia. The growth rate of the wild strain was restored in the medium containing thionine and the tissue of rice leaves. In vitro, thiG deletion mutant of Saikuzuo drug sensitivity enhancement. These results indicate that thiG mutants need to absorb sulfur ammonia from the environment always maintain their normal growth, we hypothesized that thiG deletion mutant may also absorb and sulfur ammonia element structure similar to Saikuzuo, resulting in bacteriostasis effect is better than that of the wild type strain of low concentration of pesticides on mutants. At the same time, compared with the wild type strain ZJ173, aggregation ability of thiG gene deletion mutant cells can be enhanced, including biofilm production increased, and cell motility and migration ability decreased, these results suggest that thiG may be due to the deletion mutant in rice infection and diffusion capacity decreased, so the decline in pathogenicity. Two component regulatory system rpfC and rpfG encoded protein can regulate cell aggregation through second messenger cyclic di-GMP. The results showed that the expression of rpfC and rpfG gene was down regulated in thiG deficient mutants. Mutants of exopolysaccharide production although did not change significantly, but the genes related with extracellular polysaccharide synthesis and secretion of gumE, like gumD, gumH and gumM, were up-regulated, and four of the gum gene was also reverse regulation of biofilm formation, further evidence of mutant cell aggregation ability and exopolysaccharide biosynthesis. These results indicate that the thiazole ring synthesis gene thiG in the thiazine synthesis pathway plays a vital role in the pathogenicity and cell aggregation of Xoo. Xanthomonas oryzae pv.oryzae 3.Xoo exopolysaccharides biosynthesis gum gene family of Saikuzuo drug sensitivity (Xoo) is an important pathogenic factor for the extracellular polysaccharide, infection and pathogenic bacteria, adsorption and aggregation are closely related, and can protect the cell against the interference of the external environment is not good. This study found that the sensitivity of Saikuzuo resistant strains strains ZJ173 and 2-1-1 of the exopolysaccharide yield differences. Through the construction of ZJ173 deletion mutant and 2-1-1 various extracellular polysaccharide biosynthesis gene cluster of gum gene family, drug sensitivity of each mutant to Saikuzuo were detected in rice. The results showed that ZJ173 in rice mutants due to pathogenicity in decline, Saikuzuo untreated and treated lesion length had no significant difference, therefore cannot judge whether to play the inhibitory effect of mutant saikuzuo. However, the pathogenicity of gum mutant 2-1 -1 2-1-1 gumC and 2-1-1 Delta gumI in vivo did not decrease, at the same time, Saikuzuo exhibits resistance decreased, indicating deletion of gumC gene and gumI gene will increase the susceptibility of 2-1-1 resistant strains. In summary, Saikuzuo drug sensitivity of Xoo gene deletion of gum after the changes, because the gum gene cluster in the extracellular polysaccharide synthesis and secretion in different functions, gum gene deletion difference Xoo extracellular polysaccharide production and structure, and a direct impact
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S435.111.47;S436.66

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