【作者】 田丰；

【导师】 段玉玺；

【作者基本信息】 沈阳农业大学 ， 植物病理学， 2014， 博士

【摘要】 大豆起源于中国,与根瘤菌之间存在协同进化关系,而大豆胞囊线虫是大豆生产中首要的病原物,利用根瘤菌防控胞囊线虫对大豆的危害是本文重点内容。本文系统研究了费氏中华根瘤菌Sinorhizobium fredii Sneb183抑制大豆胞囊线虫病的作用机理,并揭示了根瘤菌、大豆和胞囊线虫三者之间的互作关系。通过Sneb183发酵液处理二龄幼虫,研究该菌株对线虫活性、致死率、卵孵化率、呼吸作用等生命活力的影响,明确了根瘤菌Sneb183对大豆胞囊线虫的作用方式；采用裂根法明确菌株Sneb183可以诱导大豆产生对胞囊线虫的系统抗性,通过影响胞囊线虫在大豆根系内的发育,进一步抑制线虫对大豆根系的危害；从差异蛋白质组的角度揭示了根瘤菌诱导大豆根系在线虫侵染的胁迫条件下,产生的差异蛋白质组,从而明确了三者间的关系和根瘤菌Sneb183诱导大豆抗胞囊线虫的作用机理。主要研究结果如下：1.明确了根瘤菌Sneb183对大豆胞囊线虫二龄幼虫的作用方式。利用根瘤菌Sneb183发酵液处理大豆胞囊线虫二龄幼虫,发现二龄幼虫行动能力受到明显抑制,且发酵液对二龄幼虫具有触杀作用,72h的致死率达到84.3%。经发酵液处理的线虫卵的孵化率也明显降低,孵化抑制率达62.4%。同时根瘤菌的发酵液可抑制二龄幼虫呼吸,并造成体液渗漏,最终抑制线虫活性甚至死亡。2.验证了根瘤菌Sneb183对大豆胞囊线虫病的防治效果。在温室试验中,接种根瘤菌Sneb183的大豆根系对大豆胞囊线虫二龄幼虫和胞囊的抑制率分别达到40.13%和37.8%。在大田试验中,接种根瘤菌Sneb183后,大豆根系对胞囊的抑制率为46.2%,同时根瘤菌Sneb183也表现出对大豆苗期生长的促生作用。在大豆成熟期,根瘤菌Sneb183处理的大豆株系,单株荚数和单株豆粒数与对照相比均有显著提高,分别为对照的1.34和1.37倍。3.验证了根瘤菌Sneb183诱导大豆产生抑制胞囊线虫的系统抗性。通过裂根法证明接种根瘤菌Sneb183可以诱导大豆产生系统抗性,从而抑制线虫二龄幼虫的侵染,抑制率为38.8%;通过冰冻切片技术观察植物根系的组织变化,表明根瘤菌Sneb183可以诱导大豆根部表皮增厚,并使中柱鞘细胞加厚,结构更为致密,从而抵御线虫侵染并限制发育；通过测定大豆根系防御酶的酶活变化,表明菌株Sneb183可以激发大豆根系抗性相关酶活性增加,进一步确定根瘤菌Sneb183可以通过诱导大豆产生抗性从而抑制胞囊线虫病害。4.证明了根瘤菌Sneb183可以抑制线虫在大豆根系内的发育。接种根瘤菌Sneb183的大豆根系中,不同龄期线虫均表现出发育迟缓；接种根瘤菌Sneb183后的大豆根系分泌物对大豆胞囊线虫二龄幼虫具有一定的毒杀作用,说明根瘤菌Sneb183的接种使大豆的根系分泌物成分发生了改变,产生对J2有毒杀作用的成分,从而表现出对J2的致死作用；通过测定距根瘤不同距离的根系分泌物对J2的作用,以及二龄幼虫在根系不同部位的侵染数量,没有出现显著性差异,从而证明根瘤菌Sneb183接种的大豆根系产生对线虫的抑制作用是一种系统抗性。5.测定了在线虫侵染的胁迫条件下,接种根瘤菌Sneb183产生的差异蛋白质组。从蛋白质组学的角度揭示了根瘤菌Sneb183在诱导大豆抵御线虫中的作用。共鉴定出456个差异蛋白质点,其中上调的有244个蛋白,下调的有212个蛋白。这些蛋白充分参与了植物的生物过程、分子功能和细胞组分等。6.获得了差异蛋白质组参与的代谢通路。在鉴定出差异蛋白质组的基础上,利用BLAST2G0进行蛋白质的功能注释并分类,用KEGG来对鉴定到的蛋白质在生物通路(pathway)层面的信息进行注释,并得到生物通路图。鉴定出的差异蛋白共参与了118种代谢途径,其中钙信号途径、类黄酮合成途径、磷酸戊糖途径和谷胱甘肽代谢途径等与植物能量代谢和抗胁迫相关,从而诱导大豆抵御线虫侵染,提高植物抗性。更多还原

【Abstract】 The action mechanisms of Sinorhizobium fredii Sneb183against soybean cyst nematode were researched in this paper systematically, and the interaction relationship was revealed among Snebl83, soybean cyst nematodes (SCN) and soybeans. Through the effect of Sneb183on nematode activity, mortality rate, hatching rate, respiration and so on, the action mode of Sneb183on nematode was understood. The ability of Sneb183to systemically repress SCN was tested, and study revealed Sneb去83induced systemic resistance in soybean and also could inhibit nematode development in soybean root. The differential proteome was used to reveal the change of proteome in the soybean root under nematode infection condition when inoculated with Sneb183; through the differential proteome we could understand the relationship among these three and the mechanism of induced systemic resistance in soybean by Sneb183. The main results were as follows:1. To make clear the effect mode of Snebl83on soybean cyst nematode juvenile two (J2). When nematode were treated with Sneb183fermentation liquid, action capability was inhibited significantly, and72h mortality rate was84.3%. The hatching rate reduced62.4%when the egg treated with fermentation liquid. Meanwhile Sneb183could inhibit J2breathing and cause fluid leakage, and ultimately suppress nematode activity or even death.2. Verified the Sneb183inhibit soybean cyst nematode on the soybean root through greenhouse and field experiments. In greenhouse experiment, the inhibition rate of J2and cyst in soybean root treated with Sneb183was40.13%and37.8%respectively. In field experiment, the inhibition rate of cyst was46.2%after inoculation of Sneb183. Meanwhile, Sneb183also showed growth-promoting effect on soybean seedling growth. In soybean maturity period, the numbers of pods and beans of soybeans treated with Sneb183were both increased significantly, which were1.34and1.34times respectively compared with control treatment.3. Verify the induced systemic resistance of soybean by Sneb183. Through split-root method, we proved Sneb183could induce systemic resistance of soybean, thereby inhibit J2infection, the inhibit rate was38.8%. Frozen technique was used to observe changes of plant root organization, and observed that Sneb183could induce epidermal thickening, and increase pericycle dencity. The structure of root changed more compact and thus resisted nematode infestation and limited development. By measuring the disease related enzymatic activity, showed that Sneb183could stimulate the resistance related enzyme activity, which also determined Sneb183could induce systemic resistance.4. Proved Sneb183could inhibit nematode development in soybean root, and made nematode juvenile growing slowly. The root exudates of soybean inoculated with Sneb183was toxic to nematode, this improved that Snebl83could change the root exudates of soybean, and had lethal effect on J2. By measuring the root exudates effects of different distances from the root nodule to nematode, and the number of J2infestation in different parts of the root system, there was no significant difference between the Sneb183treatment and control, thus improved Sneb183induced nematode suppression effect of soybean is a system resistance.5. iTRAQ technology was used to test the differential proteome in soybean root when inoculated with Sneb183. The Sneb183treatment and control treatment were both under stress of nematode inoculation. By this way, revealed the effect of Sneb183induced soybean against nematode at proteomic perspective. A total of456differential proteome were identified, in which244proteins were increased and212proteins were reduced. These proteins are involved in the biological process, molecular function, cellular component and so on.6. On the basis of differential proteome result, BLAST2GO was use to note protein function, and then used KEGG bioinformatics method to identify the pathway which these proteins involved in, and obtain the biological map. In these differential proteome,118kinds of metabolic pathways was identified, including calcium signaling pathway, flavonoid biosynthesis, pentose phosphate pathway, glutathione metabolism and so on. These pathways were all involved in energy metabolism and systemic resistance of plant, so the soybean resistance to nematode was increased by Sneb183.更多还原