番茄砧木抗根结线虫特性及其根系分泌物的化感作用研究

发布时间：2018-01-06 00:33

  本文关键词：番茄砧木抗根结线虫特性及其根系分泌物的化感作用研究　出处：《沈阳农业大学》2016年博士论文　论文类型：学位论文

  更多相关文章： 番茄 根系分泌物 化感作用 南方根结线虫 基因表达

【摘要】：植物根结线虫(Meloidogyne spp.)病是一种世界性病害,且以南方根结线虫危害最为广泛。番茄是我国主栽蔬菜品种,由于多在保护地栽培,根结线虫危害逐年加重。20世纪40年代,番茄抗线虫基因Mi被发现,后来被证明是所有番茄栽培种的唯一抗源,利用该基因现已选育出许多抗根结线虫番茄栽培品种和嫁接砧木,被认为是目前防控根结线虫病最安全、有效的理想途径。近年来,根系分泌物的化感作用研究逐渐成为各国学者的研究热点。植物根系能够向根际释放大量的物质,例如氨基酸、复杂的多糖、蛋白质、挥发的亲脂性小分子等,这些物质可以对土壤生物产生直接或间接的影响,当然也包括线虫。根系分泌物可以吸引线虫向根部移动或反向排斥,或抑制线虫的运动能力,甚至使线虫死亡。然而,到目前为止根结线虫识别和导向寄主植株根部的具体机制现在仍不清楚。此外,根结线虫病属于土传病害,研究其抗性机制时需要综合植株-线虫-土壤生物学活性三者之间的关系,根系分泌物对土壤微生物数量和土壤酶活性的影响是否与植株抗根结线虫特性存在某种相关性呢?目前未见报道。本文以不同抗性番茄砧木为试材,通过南方根结线虫接种抗性鉴定,筛选出抗感差异显著的高抗、中抗和高感3个番茄砧木,通过根系分泌物GC-MS检测,分别鉴定其根系分泌物组分和含量变化,从中选择可能与抗病性有关的特异物质进行研究。通过实验室及田间试验,明确特异物质对南方根结线虫卵孵化、二龄幼虫(J2)存活和趋化性及植株抗性的影响。通过化感验证,明确特异物质对种子萌发和植株生长发育、生理生化以及土壤生物学活性的影响。最后通过差异基因分析,对特异物质进行基因功能验证。旨在通过上述问题的研究探讨为植物抗根结线虫提供理论基础及依据。具体研究结果如下：1.采用温室盆栽人工接种法,鉴定了12个番茄材料对南方根结线虫(M. incognita)的抗性程度。抗病性可涵盖高抗、中抗、抗病、感病、高感五种类型,其中高抗砧木Baliya病情指数最低,高感对照L-402病情指数最高。通过测定生理指标、根际土壤微生物数量和土壤酶活性,并与抗性进行相关分析,明确了番茄抗南方根结线虫能力和体内PAL、PPO、POD活性显著正相关,和相对电导率、丙二醛含量显著负相关,和土壤中细菌、放线菌数量、过氧化氢酶、过氧化物酶、多酚氧化酶和脲酶活性显著正相关。2.通过高抗(Baliya)、中抗(RS2)和高感(L-402)番茄根系分泌物对南方根结线虫卵孵化、二龄幼虫(J2)存活和趋化性的调查发现：(1)抗病和感病番茄根系分泌物都可抑制南方根结线虫卵孵化和J2存活,抗性番茄根系分泌物的抑制效果更为明显。(2)感病番茄根系分泌物对J2具有吸引作用,而抗病番茄根系分泌物对J2具有排斥作用。通过根系分泌物分离、纯化和GC-MS鉴定,对比分析后确定接种南方根结线虫后抗性番茄根系分泌物中酯类和酚类物质含量明显增加,其中邻苯二甲酸二甲酯、邻苯二甲酸二丁酯、L-抗坏血酸-2,6-二棕榈酸酯和2,6-二叔丁基对甲苯酚含量变化最大。最后通过根系分泌物模拟成分的室内和田间外源施加实验验证：(1)4种根系分泌物都可以抑制南方根结线虫卵的孵化和J2存活。其中L-抗坏血酸-2,6-二棕榈酸酯2 mmol·L-1浓度对南方根结线虫卵的孵化抑制作用最强,邻苯二甲酸二甲酯2 mmol·L-1浓度抑制J2存活效果最佳；(2)邻苯二甲酸二丁酯对J2具有排斥作用。(3)4种根系分泌物均可显著降低感病番茄病情指数,其中L-抗坏血酸-2,6-二棕榈酸酯2 mmol·L-1浓度抗病效果最佳。进一步证实了番茄根系分泌物在南方根结线虫抗性中的作用。3.通过种子萌发、田间外源施加实验,明确根系分泌物对番茄种子和植株的化感作用：(1)对种子的化感作用：L-抗坏血酸-2,6-二棕榈酸酯表现促进作用,邻苯二甲酸二甲酯表现明显的抑制作用,2,6-二叔丁基对甲苯酚和邻苯二甲酸二丁酯表现低浓度促进高浓度抑制。(2)对植株的化感作用：4种根系分泌物对番茄植株生长发育的影响整体表现“低促高抑”,浓度在0.5mmol·L-1左右对株高、茎粗、地上和地下鲜重表现明显的促进作用,而且促进侧根生长,使根系更加粗壮。同时该浓度对植株根系活力、叶绿素含量、PAL的促进作用也最强。而不同浓度对POD、PPO活性均表现抑制作用。4.通过模拟物质对番茄根际土壤微生物种类数量、土壤理化性质和土壤酶活性的影响研究,验证其对土壤生态环境的调控作用。外源施加模拟物质后,总体上增加的指标有：土壤pH值、速效磷含量,微生物总量、细菌和真菌数量,脲酶、过氧化氢酶和多酚氧化酶活性,降低的指标有：土壤速效钾含量、放线菌数量、过氧化物酶活性。其中2,6-二叔丁基对甲苯酚、邻苯二甲酸二丁酯和邻苯二甲酸二甲酯浓度在0.5-1.0 mmol·L-1之间土壤酶活性最高、土壤微生物结构、土壤理化性质最适宜,是植株生长的理想状态。5.采用基因表达谱测序并分析L-抗坏血酸-2,6-二棕榈酸酯处理后根部差异表达基因,结果检测到1896个差异表达基因,其中上调基因442个,下调基因1454个。经差异表达基因的功能注释和分析,明确L-抗坏血酸-2,6-二棕榈酸酯提高番茄抗性及其高浓度条件对番茄植株化感作用的分子机制。
[Abstract]:Plant root knot nematode (Meloidogyne spp.) disease is a worldwide disease, and to the southern root knot nematodes most widely. Tomato is a vegetable varieties in our country, because through protected cultivation, root knot nematodes increased year by year.20 in 40s, Mi tomato nematode resistance gene was found, was later proved is the only source of all anti tomato cultivars, the gene has bred many root knot nematode resistant tomato cultivars and rootstocks, is currently considered the prevention and control of root knot nematode disease is the safest and ideal effective way. In recent years, the Allelopathy of root exudates has gradually become the research hotspot of the plant. Roots can release large amounts of substances into the rhizosphere such as amino acids, polysaccharides, protein complex, volatile lipophilic small molecules, these substances can produce direct or indirect effects on soil organisms, including of course Nematodes. Root exudates can attract nematodes to root move or reverse rejection, or inhibition of nematode movement ability, even the mortality of nematodes. However, the specific mechanism so far root knot nematode identification and orientation of host plant roots is still not clear. In addition, root knot nematode disease is a soil borne disease, study the resistance mechanism the relationship between comprehensive plant - soil biological activity of three nematode, root exudates on soil microbial population and soil enzyme activity and plant root knot nematode resistant characteristics have some correlations? Is not currently reported. Based on the different resistant Tomato Rootstocks as test materials, nematode inoculation by southern roots. Screening of resistant and susceptible significantly high resistance, resistant and susceptible tomato rootstock by 3, root exudates of GC-MS detection, the root exudates were identified from the changes of the component and content. In the selection of specific substances may be related to disease resistance was studied. Through laboratory and field experiments, identification of specific substances on egg hatching of Meloidogyne incognita, two instar larvae (J2) affect the survival and chemotaxis and plant resistance. Through the sense of validation, growth and development of clear material specific seed germination and plant. Physiological and biochemical effects of soil and biological activity. Finally, the difference of gene analysis, gene functional verification of the specific material. Through the above discussion aims to provide theoretical foundation and basis for plant root knot nematode resistance. The main research results are as follows: 1. by artificial inoculation in greenhouse, 12 tomato materials knot to the south the identification of the root (M. incognita). The degree of resistance resistance can cover high resistance, resistance, disease resistance, disease, high sense of five types, including high resistant rootstock Baliya disease index minimum, high sense of control L-40 2. The highest disease index by measuring the physiological indicators, rhizosphere microorganisms and soil enzyme activity, and were correlated with the resistance, the resistance of tomato to root knot nematode and the levels of PAL, PPO, a significant positive correlation between POD activity and relative conductivity, MDA content had a significant negative correlation, and soil bacteria, put the number of actinomycetes, catalase, peroxidase, polyphenol oxidase and urease activity was positively related to.2. through high resistance (Baliya), resistance (RS2) and high sensitivity (L-402) of tomato root exudates on egg hatching of Meloidogyne incognita, two instar larvae (J2) survival and chemotaxis survey found: (1) resistant and susceptible tomato root exudates could suppress Meloidogyne incognita hatching and survival of J2, the inhibitory effect of resistant tomato root exudates was more obvious. (2) susceptible tomato root exudates has attracted effect on J2, while the resistant tomato root exudates With the rejection of J2. The separation by root exudates, purification and identification of GC-MS, identified esters and phenols content in root exudates increased resistance to tomato root knot nematode inoculation, the adjacent benzene two formic acid two methyl phthalate two formic acid and two L- -2,6- two butyl ester, ascorbic acid and palmitate 2,6- the biggest change of methyl tert butyl phenol content. Finally applied is verified by the root exudates of indoor and field simulation of exogenous: (1) 4 kinds of root exudates can suppress Meloidogyne incognita eggs hatching and survival of J2. The L- -2,6- two mmol 2 ascorbic acid palmitate incubation L-1 concentration on the southern root knot nematode eggs the strongest inhibition, adjacent benzene two formic acid two methyl 2 mmol concentration of L-1 inhibited J2 survival effect; (2) with the rejection of J2. Dibutyl phthalate (3) of 4 kinds of root exudates Could significantly decrease the susceptible tomato disease index, the L- 2 mmol two -2,6- ascorbic acid palmitate concentration of L-1 resistant effect is the best. Further confirmed the tomato root exudates in southern root knot nematode resistance in.3. by seed germination, field exogenous experiments, clear root exudates of tomato seed and plant allelopathy: (1) on Seed allelopathy: L- ascorbic acid two -2,6- palmitate showed promotion effect, the adjacent benzene two formic acid two methyl ester showed obvious inhibition of 2,6- tert butyl p-cresol and phthalic acid esters showed two two D low concentration promoted inhibition at high concentration. (2) effect on plants: 4 the root exudates on growth and development of tomato plants to the overall performance of low concentration and high suppression ", concentration of 0.5mmol in L-1 of plant height, stem diameter, ground and underground fresh weight showed obvious role in promoting, and To promote the growth of lateral roots, the roots more stout. At the same time the concentration on the root activity, chlorophyll content, promote the strongest effect of PAL. And the different concentration of POD, the activity of PPO showed inhibition of.4. by simulating material on Tomato rhizosphere soil microbial species, effects of soil physical and chemical properties and soil enzyme activity. Verify the regulation effect on soil ecological environment. Simulation of exogenous substances, the overall increase in the index are: soil pH, available phosphorus content, total number of microorganisms, bacteria and fungi, urease, catalase and polyphenol oxidase activity decreased, the indicators are: soil available potassium content, actinomycetes, peroxide enzyme activity. 2,6- Di tert butyl 4 methylphenol, dibutyl phthalate and phthalic acid methyl ester concentration between two two 0.5-1.0 mmol L-1 soil enzyme activity was highest, soil microbial community structure The most suitable, soil physical and chemical properties,.5. is an ideal state of plant growth and root of the difference of gene expression profile sequencing expression of L- -2,6- two after treatment with ascorbic acid palmitate by gene analysis, detection results of 1896 differentially expressed genes, including 442 up-regulated genes and 1454 down regulated genes. The expression and analysis of gene function annotation. Clear L- -2,6- two ascorbic acid palmitate increase the molecular mechanism of tomato resistance and high concentration of tomato plant allelopathy of Zhuzhou Chemical.

【学位授予单位】：沈阳农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S436.412
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本文编号：1385568