發(fā)布時(shí)間：2018-01-06 00:33

  本文關(guān)鍵詞：番茄砧木抗根結(jié)線蟲(chóng)特性及其根系分泌物的化感作用研究　出處：《沈陽(yáng)農(nóng)業(yè)大學(xué)》2016年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 番茄 根系分泌物 化感作用 南方根結(jié)線蟲(chóng) 基因表達(dá)

【摘要】：植物根結(jié)線蟲(chóng)(Meloidogyne spp.)病是一種世界性病害,且以南方根結(jié)線蟲(chóng)危害最為廣泛。番茄是我國(guó)主栽蔬菜品種,由于多在保護(hù)地栽培,根結(jié)線蟲(chóng)危害逐年加重。20世紀(jì)40年代,番茄抗線蟲(chóng)基因Mi被發(fā)現(xiàn),后來(lái)被證明是所有番茄栽培種的唯一抗源,利用該基因現(xiàn)已選育出許多抗根結(jié)線蟲(chóng)番茄栽培品種和嫁接砧木,被認(rèn)為是目前防控根結(jié)線蟲(chóng)病最安全、有效的理想途徑。近年來(lái),根系分泌物的化感作用研究逐漸成為各國(guó)學(xué)者的研究熱點(diǎn)。植物根系能夠向根際釋放大量的物質(zhì),例如氨基酸、復(fù)雜的多糖、蛋白質(zhì)、揮發(fā)的親脂性小分子等,這些物質(zhì)可以對(duì)土壤生物產(chǎn)生直接或間接的影響,當(dāng)然也包括線蟲(chóng)。根系分泌物可以吸引線蟲(chóng)向根部移動(dòng)或反向排斥,或抑制線蟲(chóng)的運(yùn)動(dòng)能力,甚至使線蟲(chóng)死亡。然而,到目前為止根結(jié)線蟲(chóng)識(shí)別和導(dǎo)向寄主植株根部的具體機(jī)制現(xiàn)在仍不清楚。此外,根結(jié)線蟲(chóng)病屬于土傳病害,研究其抗性機(jī)制時(shí)需要綜合植株-線蟲(chóng)-土壤生物學(xué)活性三者之間的關(guān)系,根系分泌物對(duì)土壤微生物數(shù)量和土壤酶活性的影響是否與植株抗根結(jié)線蟲(chóng)特性存在某種相關(guān)性呢?目前未見(jiàn)報(bào)道。本文以不同抗性番茄砧木為試材,通過(guò)南方根結(jié)線蟲(chóng)接種抗性鑒定,篩選出抗感差異顯著的高抗、中抗和高感3個(gè)番茄砧木,通過(guò)根系分泌物GC-MS檢測(cè),分別鑒定其根系分泌物組分和含量變化,從中選擇可能與抗病性有關(guān)的特異物質(zhì)進(jìn)行研究。通過(guò)實(shí)驗(yàn)室及田間試驗(yàn),明確特異物質(zhì)對(duì)南方根結(jié)線蟲(chóng)卵孵化、二齡幼蟲(chóng)(J2)存活和趨化性及植株抗性的影響。通過(guò)化感驗(yàn)證,明確特異物質(zhì)對(duì)種子萌發(fā)和植株生長(zhǎng)發(fā)育、生理生化以及土壤生物學(xué)活性的影響。最后通過(guò)差異基因分析,對(duì)特異物質(zhì)進(jìn)行基因功能驗(yàn)證。旨在通過(guò)上述問(wèn)題的研究探討為植物抗根結(jié)線蟲(chóng)提供理論基礎(chǔ)及依據(jù)。具體研究結(jié)果如下：1.采用溫室盆栽人工接種法,鑒定了12個(gè)番茄材料對(duì)南方根結(jié)線蟲(chóng)(M. incognita)的抗性程度�？共⌒钥珊w高抗、中抗、抗病、感病、高感五種類(lèi)型,其中高抗砧木Baliya病情指數(shù)最低,高感對(duì)照L-402病情指數(shù)最高。通過(guò)測(cè)定生理指標(biāo)、根際土壤微生物數(shù)量和土壤酶活性,并與抗性進(jìn)行相關(guān)分析,明確了番茄抗南方根結(jié)線蟲(chóng)能力和體內(nèi)PAL、PPO、POD活性顯著正相關(guān),和相對(duì)電導(dǎo)率、丙二醛含量顯著負(fù)相關(guān),和土壤中細(xì)菌、放線菌數(shù)量、過(guò)氧化氫酶、過(guò)氧化物酶、多酚氧化酶和脲酶活性顯著正相關(guān)。2.通過(guò)高抗(Baliya)、中抗(RS2)和高感(L-402)番茄根系分泌物對(duì)南方根結(jié)線蟲(chóng)卵孵化、二齡幼蟲(chóng)(J2)存活和趨化性的調(diào)查發(fā)現(xiàn)：(1)抗病和感病番茄根系分泌物都可抑制南方根結(jié)線蟲(chóng)卵孵化和J2存活,抗性番茄根系分泌物的抑制效果更為明顯。(2)感病番茄根系分泌物對(duì)J2具有吸引作用,而抗病番茄根系分泌物對(duì)J2具有排斥作用。通過(guò)根系分泌物分離、純化和GC-MS鑒定,對(duì)比分析后確定接種南方根結(jié)線蟲(chóng)后抗性番茄根系分泌物中酯類(lèi)和酚類(lèi)物質(zhì)含量明顯增加,其中鄰苯二甲酸二甲酯、鄰苯二甲酸二丁酯、L-抗壞血酸-2,6-二棕櫚酸酯和2,6-二叔丁基對(duì)甲苯酚含量變化最大。最后通過(guò)根系分泌物模擬成分的室內(nèi)和田間外源施加實(shí)驗(yàn)驗(yàn)證：(1)4種根系分泌物都可以抑制南方根結(jié)線蟲(chóng)卵的孵化和J2存活。其中L-抗壞血酸-2,6-二棕櫚酸酯2 mmol·L-1濃度對(duì)南方根結(jié)線蟲(chóng)卵的孵化抑制作用最強(qiáng),鄰苯二甲酸二甲酯2 mmol·L-1濃度抑制J2存活效果最佳；(2)鄰苯二甲酸二丁酯對(duì)J2具有排斥作用。(3)4種根系分泌物均可顯著降低感病番茄病情指數(shù),其中L-抗壞血酸-2,6-二棕櫚酸酯2 mmol·L-1濃度抗病效果最佳。進(jìn)一步證實(shí)了番茄根系分泌物在南方根結(jié)線蟲(chóng)抗性中的作用。3.通過(guò)種子萌發(fā)、田間外源施加實(shí)驗(yàn),明確根系分泌物對(duì)番茄種子和植株的化感作用：(1)對(duì)種子的化感作用：L-抗壞血酸-2,6-二棕櫚酸酯表現(xiàn)促進(jìn)作用,鄰苯二甲酸二甲酯表現(xiàn)明顯的抑制作用,2,6-二叔丁基對(duì)甲苯酚和鄰苯二甲酸二丁酯表現(xiàn)低濃度促進(jìn)高濃度抑制。(2)對(duì)植株的化感作用：4種根系分泌物對(duì)番茄植株生長(zhǎng)發(fā)育的影響整體表現(xiàn)“低促高抑”,濃度在0.5mmol·L-1左右對(duì)株高、莖粗、地上和地下鮮重表現(xiàn)明顯的促進(jìn)作用,而且促進(jìn)側(cè)根生長(zhǎng),使根系更加粗壯。同時(shí)該濃度對(duì)植株根系活力、葉綠素含量、PAL的促進(jìn)作用也最強(qiáng)。而不同濃度對(duì)POD、PPO活性均表現(xiàn)抑制作用。4.通過(guò)模擬物質(zhì)對(duì)番茄根際土壤微生物種類(lèi)數(shù)量、土壤理化性質(zhì)和土壤酶活性的影響研究,驗(yàn)證其對(duì)土壤生態(tài)環(huán)境的調(diào)控作用。外源施加模擬物質(zhì)后,總體上增加的指標(biāo)有：土壤pH值、速效磷含量,微生物總量、細(xì)菌和真菌數(shù)量,脲酶、過(guò)氧化氫酶和多酚氧化酶活性,降低的指標(biāo)有：土壤速效鉀含量、放線菌數(shù)量、過(guò)氧化物酶活性。其中2,6-二叔丁基對(duì)甲苯酚、鄰苯二甲酸二丁酯和鄰苯二甲酸二甲酯濃度在0.5-1.0 mmol·L-1之間土壤酶活性最高、土壤微生物結(jié)構(gòu)、土壤理化性質(zhì)最適宜,是植株生長(zhǎng)的理想狀態(tài)。5.采用基因表達(dá)譜測(cè)序并分析L-抗壞血酸-2,6-二棕櫚酸酯處理后根部差異表達(dá)基因,結(jié)果檢測(cè)到1896個(gè)差異表達(dá)基因,其中上調(diào)基因442個(gè),下調(diào)基因1454個(gè)。經(jīng)差異表達(dá)基因的功能注釋和分析,明確L-抗壞血酸-2,6-二棕櫚酸酯提高番茄抗性及其高濃度條件對(duì)番茄植株化感作用的分子機(jī)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.

【學(xué)位授予單位】：沈陽(yáng)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S436.412
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本文編號(hào)：1385568