【作者】 叶德友；

【导师】 陈劲枫；

【作者基本信息】 南京农业大学 ， 蔬菜学， 2011， 博士

【摘要】 南方根结线虫(Meloidogyne incognita)是危害黄瓜(Cucumis sativus L.,2n=2x=14)生产的重要病原。栽培黄瓜种内南方根结线虫病抗源匮乏,致使黄瓜南方根结线虫病抗性育种滞后。通过种间杂交将野生种中的抗线虫性状引入栽培黄瓜是黄瓜抗线虫育种取得重要突破的有效途径。甜瓜属野生种酸黄瓜(Cucumis hystrix Chakr.,2n=2x=24)与栽培黄瓜已成功实现杂交,对其杂种染色体加倍后获得双二倍体新种(Cucumis hytivus Chen and Kirkbride,2n=4x=38),该种间杂种是进行黄瓜遗传改良的重要基因资源。全文从发掘和利用黄瓜南方根结线虫病抗性基因资源出发,以酸黄瓜、栽培黄瓜以及酸黄瓜与栽培黄瓜种间杂交后代为研究材料,研究酸黄瓜的南方根结线虫病抗性特点及其遗传转移,探讨其抗性形成的内在机制,在种间杂交基础上,筛选抗南方根结线虫病的黄瓜-酸黄瓜渐渗系,分析其在抗性基因定位与黄瓜遗传改良中的利用价值。主要研究内容及结果如下：1.黄瓜根结线虫病原鉴定与抗病种质资源筛选对供试的3个根结线虫样本进行了形态学观测和同工酶分析,结果表明其雌虫会阴花纹形态与南方根结线虫(Meloidogyne incognita)和爪哇根结线虫(M. javanica)相似,苹果酸脱氢酶(MDH)和酯酶(EST)表型与南方根结线虫相同,确定供试线虫样本为南方根结线虫。根结线虫适宜寄主筛选结果表明,粉果番茄品种‘苏粉3号’繁殖南方根结线虫效果优于红果番茄‘农大508’,夏秋季繁殖效果好于冬春季,培养时间以45d～55d为宜,4℃离体保存选择在4d以内。采用温室盆栽苗期人工接种鉴定技术,对14份甜瓜属(Cucumis)种质资源进行了南方根结线虫抗病性鉴定。通过聚类和方差分析,将供试材料分为高感、中感、感病和高抗4类,2份野生种质对南方根结线虫表现高度抗性,甜瓜属其它栽培种为感病类型。酸黄瓜与栽培黄瓜种间杂交后代材料均为抗病类型,表明酸黄瓜的南方根结线虫病抗性已通过种间杂交部分转移至后代中。2.酸黄瓜南方根结线虫病抗性的形成机制为探明酸黄瓜南方根结线虫病抗性的解剖学及其细胞学机理,对线虫侵入与幼虫发育、根的解剖学及线虫诱导形成的取食位点的细胞学结构进行了研究。结果表明,抗病材料酸黄瓜根系中线虫侵入率极显著低于感病材料北京截头(P<0.01),酸黄瓜能够有效抵抗南方根结线虫的侵入；抗、感材料根系中雌雄虫个体比例分别为1：12和1：5(P<0.01),抗性反应能够抑制线虫取食和幼虫发育；抗病反应中细胞发生过敏性坏死,感病反应中无此类现象发生。抗侵入、抑制取食和坏死反应是抗病材料酸黄瓜抗性反应的主要特征。采用高效液相色谱(HPLC)分析法,研究了南方根结线虫病抗性与葫芦素B含量的关系。供试材料根系中的葫芦素B含量以酸黄瓜的最高(1.31μg/g),非洲角次之(0.85μg/g),北京截头最低(0.35μg/g),表明酸黄瓜中葫芦素B的本体含量较高。酸黄瓜、非洲角、北京截头受线虫侵染后根系中的葫芦素B含量较相应未接种对照分别升高了198%、214%和40%,表明酸黄瓜经线虫诱导后葫芦素B含量上升较大。酸黄瓜受线虫危害低于非洲角并显著低于北京截头,说明酸黄瓜的抗线虫能力较强。供试材料受害程度与葫芦素B含量相关性分析表明,作物的抗线虫能力与根系中的葫芦素B含量呈正相关关系,其含量越高,抗性越强,酸黄瓜的高葫芦素B含量是其南方根结线虫病抗性的化学证据。以抗、感甜瓜属种质为试材,研究了线虫侵染对根系和叶片活性氧(ROS)代谢、防御酶活性及相关抗性指标的影响。结果表明,抗病材料酸黄瓜中超氧阴离子(O2·-)和过氧化氢(H2O2)含量受线虫侵染增加的幅度显著小于感病材料北京截头,超氧化物歧化酶(SOD)、过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)活性变化率与其病情指数分别呈显著正相关(r=0.8278*)、极显著负相关(r=-0.9679**)和显著负相关(r=-0.8499*),线虫侵染后酸黄瓜根系中增加了3条POD同工酶谱带(Rf=0.126,0.188,0.944),而北京截头只增加了1条弱带(Rf=0.396),线虫侵染导致电导率(EL)上升和丙二醛(MDA)含量增加幅度北京截头高于酸黄瓜,根系活力下降幅度酸黄瓜显著小于北京截头。酸黄瓜受线虫侵染氧化胁迫较小,根系活力较高,从而使酸黄瓜对线虫侵染表现出了较好的耐性。为解析酸黄瓜南方根结线虫病抗性的光合响应机制,研究了线虫侵染对黄瓜叶片光合作用、叶绿素荧光参数及相关生理指标的影响。线虫侵染使酸黄瓜叶片叶绿素(Chl)含量降低幅度显著小于‘北京截头’,酸黄瓜Chl随氮(N)素含量降低较‘北京截头’平缓；线虫侵染导致二者净光合速率(Pn)、气孔导度(Gs)下降,酸黄瓜胞间CO2浓度(Ci)下降,而北京截头接种14d后Ci急剧上升,酸黄瓜Gs对叶片相对含水量(RWC)下降的反应较北京截头敏感；实际光化学效率(φPSⅡ)、光化学猝灭系数(qP)降低幅度酸黄瓜小于北京截头,非光化学猝灭系数(qN)升高幅度酸黄瓜显著高于北京截头；线虫侵染对酸黄瓜Pn/Ci初始斜率影响不大,而使北京截头急剧降低。研究结果说明,酸黄瓜具有相对较高的养分及水分利用率,叶绿素含量下降较小,能够较多地将光能用于光化学反应,热耗散能力较强,从而保持了较高的净光合速率,使得线虫侵染对酸黄瓜植株生长造成的影响不大。3.抗南方根结线虫病黄瓜-酸黄瓜渐渗系的筛选鉴定通过对黄瓜-酸黄瓜种间杂交后代群体进行抗线虫鉴定、形态学观测和分子生物学鉴定,筛选出抗南方根结线虫病黄瓜-酸黄瓜渐渗系ILs-10-1。渐渗系ILs-10-1抗南方根结线虫,其抗性表现稳定；田间农艺性状和形态学观测发现,其具有小叶、短果、棕色瘤刺、多分枝的特点,且与栽培黄瓜杂交亲和；利用SSR分子标记分析发现,SSR18648能够在渐渗系ILs-10-1中稳定重复地扩增出野生亲本酸黄瓜的特异DNA片段,从分子水平上证实了渐渗系ILs-10-1为酸黄瓜与栽培黄瓜发生渐渗杂交的种间杂交后代。更多还原

【Abstract】 The root-knot nematode Meloidogyne incognita is an important pathogen and causes significant yield losses to cucumber production throughout the world. Breeding for resistance to M.incognita in cucumber was lagged behind other crops resulting from the poverty of resistance in cultivated cucumber. Introducing resistance in wild into cultivated cucumber based on hybridization is an effective way to make breakthrough in M.incognita-nematode breeding of cucumber. A successful cross has been made between Sour cucumber and cultivated cucumber and Sour cucumber is the important genetic resources for cucumber improvement through interspecific hybridization. In order to exploiting cucumber gene resources resistant to M.incognita, Sour cucumber, cultivated cucumber and interspecific progenies derived from the cross between these two species were used as primary research materials in this paper. Resistant to M.incognita and resistance transfer to its progenies in Sour cucumber were studied, and the internal mechanisms of resistance in Sour cucumber were also investigated. Breeding of cucumber-Sour cucumber introgression lines resistant to M. incognita based on interspecific hybridization were conducted and its values in resistance gene mapping and cucumber improvement were analyzed. The main contents and results are as follows:1. Pathogen identification and screening of germplasm for resistance to the root-knot nematode in cucumberThe morphological observation and isozyme phenotypes analysis of the female were conducted for three Meloidogyne spp. samples collected from infected cucumber roots. The results showed that perineal pattern of the female was similar to Meloidogyne incognita and M.javanica. Malate dehydrogenase (MDH) and esterase (EST) phenotypes of the female were the same as M.incognita, thus the collection of Meloidogyne spp. was identified as M. incognita. Screening for suitable host of M. incognita showed that its reproduction on powder fruit tomato varieties was better than red fruit tomato, in summer and autumn better than in winter and spring. The appropriate culture and conservation time of M. incognita was respectively from 45 days to 55 days and within 4 days in vitro at 4℃after inoculation.Fourteen Cucumis germplasms were selected to evaluate their resistance to M.incognita. Cluster and variance analysis showed that they could be clustered 4 groups, i.e. high susceptibility, moderate susceptibility, susceptibility and high resistance. Among of these Cucumis species, two wild germplasms were high resistance, interspecific progenies were resistance, and other species studied were all susceptible to M.incognita. The high resistance to M.incognita in Sour cucumber was transmitted to its progenies through interspecific hybridization.2. Resistant mechanism of sour cucumber for its resistance to the root-knot nematode Meloidogyne incognitaThe differences in invasion and development of the nematode, the anatomy of root and cytology of feeding site were compared by using resistant and susceptible cucumber varieties. The results showed that the percentage of nematode invasion in Sour cucumber was significantly lower than that in Beijingjietou (P<0.01). The individual ratio of female and male nematodes was respectively 1:12 and 1:5 in resistant and susceptible roots (P<0.01). The resistant reaction observed in Sour cucumber could inhibit nematode from feeding and juvenile development. The hypersensitive necrosis of cells occured in resistant reaction, no necrosis could be detected in susceptible one. Limited invasion, inhibited feeding of nematodes and necrosis response of feeding site cells were the main features occured in Sour cucumber displaying resistance to M.incognita.High performance liquid chromatography was used to study the relationships between resistance and content of Cucurbitacin B in cucumber. The results showed that the highest content of Cucurbitacin B in roots before inoculation is in Sour cucumber (1.31μg/g), followed by the Africa Horned cucumber (0.85μg/g) and the minimum Beijingjietou (0.35μg/g), indicating that the ontology content of Cucurbitacin B in Sour cucumber were higher. The content of Cucurbitacin B in these three Cucumis species were increased by 198%,214% and 40% respectively than the corresponding controls after inoculation, indicating the increased content of Cucurbitacin B in Sour cucumber is greater. The infected degree of Sour cucumber is lower indicating its resistance is higher. The correlation analysis showed that there was positively correlation between nematode-resistant abilities of crops and the content of Cucurbitacin B in roots, the higher its content, the stronger the resistance. The resistance in Sour cucumber for its chemical aspects results from its higher levels of Cucurbitacin B in roots.Cucumis genotypes with different resistance to M.incognita were used to study the effects of nematode infection on reactive oxygen metabolism, defense enzyme activities and resistance index related in roots and leaves of cucumber. The results showed that the increase rate of the content of superoxide anion radical (O2·-) and hydrogen peroxide (H2O2) were significantly lower in the resistant genotype Sour cucumber than in the susceptible genotype Beijingjietou. The correlation analysis showed that there was respectively a significant positive correlation of superoxide dismutase (SOD) (r=0.8278*), highly significant negative correlation of peroxidase (POD) (r=-0.9679**) and significant negative correlation of phenylalanine ammonialyase (PAL) (r=-0.8499*) between the rates of changes of SOD, POD and PAL activities in roots associated with the disease index for Cucumis species. POD isoenzyme electrophoresis analysis showed that there were three more enzyme bands (Rf=0.126,0.188,0.944) in Sour cucumber, but only one weak band (Rf=0.396) in Beijingjietou when compared to corresponding control. The rate of increase of EL and MDA induced by M.incognita was higher in Beijingjietou than in Sour cucumber, the root activities were significantly lower in the latter than the former. Oxidative stress infected by M.incognita was less and the root activities were higher in Sour cucumber so that it displayed resistance to M.incognita after infection.The effects of nematode infection on photosynthesis, chlorophyll fluorescence parameters and physiological indices related of cucumber were investigated. The results showed that it induced a significantly lower decrease of chlorophyll (Ch1) content in Sour cucumber leaves than in Beijingjietou. Decreased extents of its content accompany with nitrogen content reduction in Sour cucumber were less than in Beijingjietou. Net photosynthetic rate(Pn), stomatal conductance (Gs) decreased in two varieties, intercellular CO2 concentration (Ci) decreased in Sour cucumber, while its value have a sharp rise in Beijingjietou after fourteen days inoculation. Gs reduction in response to relative water content (RWC) declining in Sour cucumber was more sensitive than in Beijingjietou. The nematode infection led to a lower reduction of actual photochemical efficiency (ΦPSⅡ) and photochemical quenching (qP), while induced a significantly higher increase of non-photochemical quenching (qN) in Sour cucumber than in Beijingjietou. Nematode infection has little effect on the initial slope of Pn/Ci in Sour cucumber, leaving a sharp decrease in Beijingjietou. The results indicated that there are a relatively higher nutrients and water use efficiency, lower decrease of Chl, more light energy for photochemical reactions, stronger the heat dissipation ability, so as to maintain a higher Pn in Sour cucumber compared with nematode-susceptible ones. All these did not cause significant effect on the plant growth of resistant Sour cucumber infected by nematode.3. Breeding of cucumber-sour cucumber introgression lines with resistance to the root-knot nematode Meloidogyne incognitaInterspecific progenies from cross between cucumber and Sour cucumber were screened and an introgression lines ILs-10-1 was obtained for resistance to M. incognita through resistance evaluation. The results showed that introgression lines ILs-10-1 displayed high resistance to M. incognita and its resistance was stable. In agronomic traits and morphology, it was characterized to have small-sized leaf, short fruit, brown spine and multi-branching, and it have affinity of hybridization with the cultivated cucumber. Simple sequence repeat (SSR) analysis was carried out with the genomic DNA of ILs-10-1. A pair of primers SSR18648 screened could repeatedly amplify special DNA fragment from its wild parent Sour cucumber. Thus introgression lines ILs-10-1 was confirmed as an interspecific progenies at molecular level.更多还原