【作者】 贾双双；

【导师】 徐坤；

【作者基本信息】 山东农业大学 ， 蔬菜学， 2012， 博士

【摘要】 番茄（Lycopersicon esculentum Mill.）是我国设施栽培面积最大的蔬菜之一，因其复种指数高、连作重茬栽培较为普遍，致使番茄根结线虫病高发，生产实践中，采用抗性品种和利用抗性砧木嫁接栽培已成为防控根结线虫病的有效方法。因此，鉴定筛选高抗番茄根结线虫病的砧木品种，并确定其抗性机制具有重要的理论意义和应用价值。为此，本研究在鉴定评价番茄砧木对南方根结线虫抗性水平基础上，系统研究了不同抗性番茄砧木幼苗相关生理代谢及特异蛋白表达差异，以期揭示番茄砧木抗南方根结线虫的生理机制，为选育高抗根结线虫番茄砧木品种提供理论依据。主要研究结果如下：1.通过测定16个供试番茄砧木幼苗接种南方根结线虫50d时的相对生长量及相关病理指标，结果表明，南方根结线虫侵染对不同番茄砧木幼苗各器官相对生长量、相关病理指标及其变异系数的影响显著不同，其中生长量的变异系数以相对根鲜质量较大，为20.20%，相对茎粗较小，仅6.22%。不同番茄砧木幼苗的根结指数（GI）、卵粒指数（EI）、繁殖系数（RF）和病情指数（DI）也存在显著差异，且其变化也不一致，表明根据单一指标对幼苗抗病性进行分类的结果会存在一定差异。2.研究表明，采用多指标综合聚类分析较单一指标聚类分析更能准确划分番茄砧木抗南方根结线虫的类群，而根据综合指标进行隶属函数运算，则可确定供试番茄砧木对南方根结线虫抗性的顺序，由此建立了以系统聚类分析和隶属函数运算相结合的番茄砧木对南方根结线虫抗性综合鉴定评价方法。通过聚类分析和隶属函数相结合的方法，确定Baliya为免疫材料，BESUPA、坂砧2号、砧木606、砧木002、砧木001、TMS-150、Support、MIKADO、砧木401为抗病材料，Anka-T为耐病材料，坂砧1号、北京1号为感病材料，128、BF兴津101、Ls-89为高感材料。3.研究了高感品种Ls-89与高抗品种坂砧2号幼苗接种南方根结线虫前后活性氧代谢的差异。结果表明，未接种南方根结线虫的番茄砧木幼苗，其根系与叶片活性氧水平及SOD、POD、CAT等相关酶活性未因品种抗性不同而表现出显著差异。接种南方根结线虫后，两品种幼苗根系与叶片O2·-生成速率和H2O2含量均表现出周期性升高，且坂砧2号显著高于Ls-89，但MDA含量则以Ls-89显著高于坂砧2号，表明高抗品种番茄砧木幼苗膜脂抗氧化能力较强；两品种幼苗根系与叶片SOD活性均在侵染早期降低，且以坂砧2号降幅较大；但POD、CAT活性在侵染早期变化不大，至侵染中后期则显著升高，表明番茄砧木幼苗SOD活性对南方根结线虫侵染较为敏感。4.不同抗性番茄砧木幼苗苯丙烷类代谢活性显著不同，即使未接种南方根结线虫，高抗品种坂砧2号幼苗根系与叶片苯丙烷类代谢相关酶活性及酚类物质含量显著高于高感品种Ls-89；接种南方根结线虫后，两品种根系与叶片PAL、TAL、PPO等3种酶活性与总酚、类黄酮等物质含量变化规律基本一致，均呈周期性升高，但坂砧2号升幅显著高于Ls-89；而根系与叶片木质素含量逐渐升高，并且坂砧2号较Ls-89积累速率快且升幅大。5.南方根结线虫侵染显著影响高感砧木Ls-89根系活力及叶片色素含量，而对高抗砧木坂砧2号影响不大。未接种南方根结线虫的番茄砧木幼苗，其根系与叶片可溶性糖、可溶性蛋白、脯氨酸、细胞壁羟脯氨酸含量及几丁质酶与β-1,3-葡聚糖酶活性变化并未因品种抗性不同而有显著差异；接种南方根结线虫后，相关物质含量及酶活性变化规律基本一致，均呈现周期性升高，且坂砧2号升幅显著高于Ls-89。6.不同抗性番茄砧木幼苗根系分泌物对卵孵化的影响不同，同一品种不同时期的根系分泌物对卵孵化的影响也不同。未接种南方根结线虫的Ls-89根系分泌物刺激卵孵化，而坂砧2号与Baliya的根系分泌物则显著抑制卵孵化。接种南方根结线虫后，Ls-89幼苗根系分泌物刺激卵孵化的效果显著高于其未接种幼苗；而坂砧2号在接种南方根结线虫后7d、14d与35d的根系分泌物对卵孵化具有显著的抑制效果，而接种后第21d与28d抑制效果不显著，且与其未接种对照差异不显著；Baliya根系分泌物均显著抑制线虫卵的孵化，且与其未接种对照差异也不显著。7.通过对高感砧木Ls-89、高抗砧木坂砧2号及免疫砧木Baliya幼苗接种南方根结线虫21d前后根系分泌物的成分分析表明，不同抗性品种根系分泌物成分有所不同，同一品种接种南方根结线虫前后根系分泌物成分也有所不同。通过对比分析初步确定，不同抗性番茄砧木幼苗接种线虫21d根系分泌物中对根结线虫侵染可能起作用的物质为：邻苯二甲酸二异辛酯、2,2′-亚甲基双(4-甲基-6-叔丁基)苯酚、邻苯二甲酸二丁酯、L-抗坏血酸-2,6-二棕榈酸酯、2-苯并噻唑酮、环莠隆、邻苯二甲酸,6-乙基-3-辛基-2-乙基己酯、异硫氰酸环己酯、8-硬脂酸甲酯、顺-9-十八碳烯醛、顺-9,10-环氧硬脂酸、9,10-二羟基硬脂酸、3,7-二甲基-2,6-辛二烯醛、黄葵内酯、硬脂酸,2-(2-羟基乙氧基)乙酯、磷酸,三(2-乙基己基)酯。8.通过对不同抗性番茄砧木幼苗根内南方根结线虫发育进程及根际基质卵及二龄幼虫数量的研究发现，接种南方根结线虫导致Ls-89与坂砧2号幼苗根内线虫数量增加，但Ls-89根内线虫数量显著高于坂砧2号，而免疫砧木Baliya根内未发现二龄幼虫。二龄幼虫侵入坂砧2号根系的时间不仅迟于Ls-89，数量也显著低于Ls-89，且侵入根系的二龄幼虫发育为三龄及四龄幼虫数量也显著低于Ls-89。Ls-89与坂砧2号幼苗根际基质内二龄幼虫数量因二次侵染均增加，但以Ls-89显著高于坂砧2号，而Baliya根际二龄幼虫数量持续下降；根际卵数量变化较大，侵染后期Ls-89显著高于坂砧2号，而Baliya幼苗根际未发现线虫的卵。9.通过对接种南方根结线虫的高感砧木Ls-89、高抗砧木坂砧2号与免疫砧木Baliya幼苗叶片全蛋白鉴定分析，共检测到682个蛋白，其中32个蛋白点差异表达显著，经质谱分析和NCBI数据库搜索比对后，共鉴定了17个蛋白，分别是：peroxidase precursor（过氧化物酶前体），glutathione S1-transferase, class-phi（谷胱甘肽S1-转移酶），ATPsynthase CF1alpha subunit（叶绿体ATP合酶α亚基），vacuolar H+-ATPase A1subunitisoform（液泡H+-ATP酶A1亚基异构体），ATP synthase beta chain（ATP合酶β亚基），chlorophyll a/b binding protein（叶绿素a/b结合蛋白），photosystem I light-harvestingchlorophyll a/b-binding protein （光系统Ⅰ捕光叶绿素a/b结合蛋白），ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit（1,5-二磷酸核酮糖羧化酶/加氧酶大亚基），50S ribosomal protein L12, chloroplastic（叶绿体核糖体蛋白），heat shock70protein（热激蛋白），HSP68=68kda heat-stress DnaK homolog（热胁迫蛋白），Threoninedeaminas（e苏氨酸脱氨酶），leucine aminopeptidas（e亮氨酸氨基肽酶），hypothetical proteinSynRCC307-1195（假定蛋白）。更多还原

【Abstract】 Tomato (Lycopersicon esculentum Mill.) is one of the vegetables cultivation area inChina, because of the higher multiple cropping index and the more common continuouscropping cultivation, resulting in the higher incidence of tomato root-knot nematode disease.In the production practice, it will become the most potential effective method for preventingand control the crop root-knot nematode disease by taking advantage of the resistant varietiesand grafting cultivation of the resistant rootstocks. So, identification and screening of the highresistance tomato rootstocks to Meoidogyne incognita, and determine its resistancemechanism had important theoretical significance and application value. Therefore, theresearch was on the base of screening and evaluation of tomato rootstocks to M. incognitaresistance level, systematically studied related physiological mechanism and specific proteinexpression differences on different resistant tomato rootstocks seedlings. In order to reveal thephysiological mechanisms to tomato rootstocks resistance to M. incognita, and it providedtheoretical basis for breeding highly resistant to M. incognita of tomato rootstocks. The mainresults were as bellow:1. The relative growth rate and relevant disease resistant index of16tomato rootstockseedlings inoculated with M. incognita were measured after50days. The results showed thatthe different relative growth rate of different organs, different disease resistant indexes of16tomato rootstock seedlings and their different coefficient variances resulted from the infectionof M. incognita were different, in which the coefficient variances of relative root fresh massof five relative growth rate was highest20.20%, but that of relative stem diameter was only6.22%. There were also significant differences about gall index (GI), egg granule index (EI),reprodutive frequency (RF) and disease index (DI) of different tomato rootstock seedlings,and the changes were also inconsistent, which indicated that the results about using of singleindicator to classfiy for seedling resistance would be some differences.2. The result of cluster analysis with multiple indexes total indexes was more accuratethan other single index for the resistant class of tomato rootstocks to M. incognita. According to multiple indexes to operate by membership function, we can sure the order of the testedtomato rootstocks resistance to M. incognita. Thereby we established the identification andevaluation method to tomato rootstocks resistant to M. incognita of the combinationsystematic cluster analysis and membership function operation. By the method of clusteringanalysis and subordinate function, we sure Baliya was immune material; BESUPA, BanzhenNo.2, Rootstock606, Rootstock002, Rootstock001, TMS-150, SUPPORT, MIKADO,Rootstock401were resistant materials; ATM052was disease tolerant material; BanzhenNo.1, Beijing No.1were sensitive materials;128, BF xingjin101, Ls-89were highly sensitivematerials.3. Differences in reactive oxygen metabolism before and after inoculating with M.incognita of highly susceptible species Ls-89and highly resistant species Banzhen No.2werestudied. The results showed that, reactive oxygen species generation rate and related enzymeactivities in the roots and leaves of tomato rootstock seedlings, not inoculated with M.incognita, did not show significant differences because of different resistant varieties. Afterinoculating, O2·-generation rate and H2O2content in roots and leaves of two rootstokseedlings had showed a cyclical rise, and Banzhen No.2was significantly higher than Ls-89,however, MDA content in Ls-89was significantly higher than Banzhen No.2. It was showedthat lipid antioxidant ability of resistant rootstok seedlings was stronger. SOD activities werelower in roots and leaves of two varieties seedlings in the early stage of infection, and a largerdecline in Banzhen No.2. But POD and CAT activities changed little in the early stage, to themiddle and late stage significantly increased, which indicated that SOD activities of tomatorootstock seedlings were more sensitive to the infection by M. incognita.4. Phenylpropanes metabolism activities of different resistant tomato rootstock seedlingswere significantly different. The related enzyme activities and phenolics contents ofphenylpropanes metabolism in the roots and leaves of highly resistant species Banzhen No.2seedlings, not inoculated with M. incognita, showed significantly higher than highlysusceptible species Ls-89. After inoculating, the variation of PAL, TAL, PPO activities andtotal phenolis contents, flavonoid in roots and leaves of two rootstok seedlings werebasically the same, and showed a cyclical rise, and Banzhen No.2was significantly higherthan Ls-89, but, the lignin content in roots and leaves increased gradually, and Banzhen No.2 was significantly higher than Ls-89on accumulation and increasing.5. M. incognita infection was significantly influence on root activity and leaf pigmentcontents to Ls-89, but little effect on Banzhen No.2. The soluble sugar, soluble protein,proline, cell wall hydroxyproline contents and chitinase, β-1,3-glucanase activities in the rootsand leaves of tomato rootstock seedlings, not inoculated with M. incognita, did not showsignificant differences because of different resistant varieties. After inoculating, the variationof the related substances contents and enzyme activities were basically the same, showed acyclical rise, and Banzhen No.2was significantly higher than Ls-89.6. The effect of the root exudates of different resistant tomato rootstock seedlings on egghatch was different, and the effect of the root exudates of different periods of the same specieson egg hatch was different, too. Not inoculated with M. incognita, the root exudates of Ls-89stimulated the egg hatch, but Banzhen No.2and Baliya significantly inhibited egg hatch. Afterinoculating, the root exudates of Ls-89were significantly stimulating the eggs hatch, andwere significantly higher than the control of non-inoculated. After inoculating7days,14daysand35days, the root exudates of Banzhen No.2were signifcantly inhibiting the egg hatch,but the21days and28days inhibitory effects were not significant, which were not significantdifferences to the non-inoculated control. The root exudates of Baliya were significantlyinhibiting the egg hatch, were not significant to the non-inoculated control, too.7. Before and after inoculating with M. incognita21days, through analysis the rootexudates compositon of highly susceptible rootstock Ls-89, highly resistant rootstockBanzhen No.2and immune rootstock Baliya seedlings, they were showed that root exudatescompositon of different resistant varieties were different, the same species root exudatescompositon before and after inoculating with M. incognita were different, too. Afterinoculating with M. incognita21days, the substances may play a role for M. incognitainfection in different resistant tomato rootstock seedlings root exudates which were initiallyidentified by comparing analysis. They were1,2-benzenedicarboxylic acid, diisooctyl ester,2,2′-methylenebis[6-(1,1-dimethylethyl)-4-methylphenol,1,2-benzenedicarboxylic acid,dibutyl ester, L-(+)-ascorbic acid2,6-dihexadecanoate,2-benzothiazolinone, N′-cyclooctyl-N,N-dimethyl-urea, Phthalic acid,6-ethyloct-3-yl2-ethylhexyl ester, Cyclohexyl isothiocya-nate,8-octadecenoic acid, methyl ester,(Z)-9-octadecenal,3,7-dimethyl-2,6-octadienal, cis- 9,10-epoxyoctadecanoic acid,9,10-dihydroxyoctadecanoic acid, Ambrettolid, Octadecanoicacid,2-(2-hydroxyethoxy)ethyl ester, Phosphoric acid, tris(2-ethylhexyl) ester.8. M. incognita development process in different resistant tomato rootstock seedlingroots and the number of eggs and juveniles within rhizosphere matrix were found that showedthe number of juveniles in roots of Ls-89and Banzhen No.2had increased, and Ls-89weresignificantly higher than Banzhen No.2, though, juveniles were not exist in Baliya. Thejuveniles penetrated into Banzhen No.2roots not only later than Ls-89, but also the numbersignificantly lower; and only a few developed into the third instar larvae and fourth instarlarvae, the number were significantly lower than Ls-89. Because of second infection, thenumber of juveniles in Ls-89and Banzhen No.2within rhizosphere matrix increased, andthey were significantly higher in Ls-89than in Banzhen No.2, but continued to decline inBaliya. The number of eggs within rhizosphere matrix changed acutely, late in infection, theywere significantly higher in Ls-89than in Banzhen No.2, but they were non eggs in Baliya.9. Total proteins were identification and analysis from leaves in the highly susceptiblerootstock Ls-89, the highly resistant rootstock Banzhen No.2and the immune rootstockBaliya after inoculating with M. incognita.682protein spots were detected, of which32protein spots were differentially expressed significantly, through mass spectrometry andNCBI database search, only17protein spots were obtained. They were peroxidase precursor,glutathione S1-transferase, class-phi, ATP synthase CF1alpha subunit, vacuolar H+-ATPase A1subunit isoform, ATP synthase beta chain, chlorophyll a/b binding protein, photosystem Ilight-harvesting chlorophyll a/b-binding protein, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit,50S ribosomal protein L12, chloroplastic, heat shock70protein,HSP68=68kda heat-stress DnaK homolog, Threonine deaminase, leucine aminopeptidase,hypothetical protein SynRCC307-1195.更多还原