不同产区花生青枯病菌遗传多样性的分析

花生青枯病是一种重要的毁灭性病害。本研究将菌落PCR引入到花生青枯菌的鉴定及基因组研究工作中,通过菌落PCR直接对四川省内不同花生产区收集得到的花生青枯病菌进行菌株鉴定并对供试菌株的重要功能基因phcA、hrp以及hcp进行遗传多样性的比较分析。结果表明,菌落PCR能快速有效地扩增得到不同片段大小的目标片段,通过菌落PCR产物的直接测序并进行序列比对,发现来源于不同生态环境、不同花生品种的青枯病菌株其重要的功能基因在序列上不存在差异,表明这些重要的功能基因具有高度保守的特性。菌落PCR省去了菌株的再培养以及DNA的提取等繁琐过程,不仅可实现对花生青枯菌高通量的分子鉴定,还可快速地开展基因克隆及基因组学方面的研究工作,在花生青枯菌的筛选、鉴定以及植物检疫方面具有较强的应用性,在花生青枯菌的基因组研究中具有重要的应用价值。     

丛枝菌根真菌响应桉树茄科雷尔氏菌侵染进程基因的筛选与鉴定

【背景】桉树(Eucalyptus)青枯病危害严重,丛枝菌根真菌(arbuscular mycorrhizal fungi,AMF)与桉树共生影响桉树对青枯病的抗性,而AMF响应桉树青枯菌侵染的机制仍不清楚。【目的】探索AMF响应桉树茄科雷尔氏菌(Ralstonia solanacearum)的侵染机制。【方法】以非菌根化和异形根孢囊霉(Rhizophagus irregularis)菌根化巨桉(Eucalyptus grandis)分别受茄科雷尔氏菌侵染0、24、48和96 h接种后(hour post-inoculated, hpi)的根系组织为研究对象,基于转录组测序筛选和鉴定菌根化巨桉根系中异形根孢囊霉响应茄科雷尔氏菌侵染的基因信息。【结果】与对应非菌根化桉树受茄科雷尔氏菌侵染的时间点相比,菌根化桉树中异形根孢囊霉响应青枯菌侵染显著差异表达基因为3 382–5 989个,随青枯侵染时间进程的增加,异形根孢囊霉特异性响应茄科雷尔氏菌侵染差异表达基因数量逐渐增多。茄科雷尔氏菌侵染24 hpi时,异形根孢囊霉显著富集共生体生长、孢子形成和凋亡信号通路、铁载体等相关基因;茄科雷尔氏菌侵...     

香蕉花芽分化期抗枯萎病相关基因表达分析

通过对‘巴西蕉’和‘宝岛蕉’花芽分化期响应枯萎病菌4号生理小种(Fusarium oxysporum f.sp.cubense race 4,Foc4)侵染的转录组进行分析,获得了大量差异表达基因。本研究以‘巴西蕉’和‘宝岛蕉’花芽分化期根部组织为材料,采用实时荧光定量PCR(q PCR)方法测定了10个与特定抗病机制相关基因的表达变化。结果表明,10个基因在‘巴西蕉’和‘宝岛蕉’花芽分化过程中均有表达,但其表达量存在差异。与对照‘巴西蕉’相比,除laccase-4(A)基因在‘宝岛蕉’中的表达量明显下调外,laccase-4(B)、periplasmic beta-glucosidase precursor、glutathione-S-transferase Cla47、class Ⅲ peroxidase 136 precursor、peroxidase N、similar to T-phylloplanin、snakin-1、putative chitinase及cellulose synthase catalytic subunit 12等9个基因的表达量均上调,其中peroxidase N基因的表达量呈极显著上调,相对表达量约为‘巴西蕉’的1 682倍。10个差异表达基因可能参与‘宝岛蕉’花芽分化期对病原菌的防卫反应,为进一步探讨‘宝岛蕉‘复杂的抗病防御反应分子机理奠定基础。     

花生抗青枯病分子标记研究

利用抗、感青枯病的花生品种为素本配制杂交组合中花5号×远杂9102,构建重组近交系,以其F6为研究材料,分析青枯痛抗性遗传规律,结果表明,花生青枯病抗性是由两时主效基因控制的遗传,并且主效基因的遗传力较高,为84%;同时采用AFLP技术和BSA分析方法,获得两个与花生青枯病抗性连锁的分子标记,标记与抗性间的遗传距离分别为8.12cM和11.46cM.利用获得的分子标记对抗、感青枯病的花生种质进行了分子鉴定,证实了标记P3M59与膏枯病抗性的符合率为70%,标记PIM58的符合率为50%,从而为花生青枯病抗性辅助选择育种提供理论基础.     

两种砧木对樱桃番茄青枯病抗性及根际微生物数量的影响

青枯病是危害我国华南地区番茄生产最严重的土传性病害之一。为探讨在人工接种青枯菌条件下,两种不同砧木嫁接对番茄青枯病的抗性及根际微生物数量的影响。该研究分别用砧木“番砧1号”和“茄砧21号”与樱桃番茄“粉贝贝”进行嫁接,采用稀释平板法对各嫁接组合的根际微生物进行分离。结果表明:(1)采用抗病砧木嫁接显著提高了番茄的抗病性,降低了发病率和病情指数,延缓了发病时间。(2)青枯菌在侵染植株过程中呈现动态变化,其数量从根系到地上茎部逐渐减少;砧穗嫁接植株、砧木自根嫁接植株根际基质和根系中的青枯菌数量降低,且地上茎部中的病原菌数量显著低于接穗自根嫁接植株;接种青枯菌后,各嫁接组合根际基质中的细菌、真菌和放线菌的数量随发病期的变化呈先增加后降低的趋势。(3)采用抗病砧木嫁接总体上提高了植株根际微生物总量、细菌及放线菌的数量,降低了真菌的数量,改善了嫁接植株根际微环境,对降低番茄青枯病发病率具有重要作用。     

烟草青枯雷尔氏菌噬菌体资源的发掘

由青枯雷尔氏菌(Ralstonia solanacearum)引起的烟草青枯病是烟草种植过程中一种重要的细菌性病害。为了开展利用噬菌体防控烟草青枯病的研究,本研究以烟田产区的7株烟草青枯菌为宿主,从烟田土壤中分离到了14个对烟草青枯菌具有侵染性的烈性噬菌体,说明烟田土壤中普遍存在烟草青枯菌噬菌体。选取噬菌体?PB2和?PB34,电镜观察确定其具有十二面体的头部和粗短的尾部,属于肌尾噬菌体科。噬菌体的宿主谱分析显示,筛选到的噬菌体对分别从烟草、西红柿和花生分离的青枯菌株系的侵染性有所差异。本研究分离发掘的14个对烟草青枯菌侵染的噬菌体,为利用噬菌体防控烟草青枯病的生防策略研究提供了噬菌体资源。     

青枯病病菌对茄子相关防卫信号基因表达及活性氧含量的影响

本文以茄子抗青枯病自交系和感病自交系为试材,在接种青枯病菌后,对调控抗性反应的不同信号传导基因进行表达特性分析。结果表明,在9个基因中,EDS1、PAD4、NPR1、SGTI和WRYK70等5个基因的表达均随着接种诱导时间延长而增加,而且在抗病自交系‘E-31’中的表达量要高于感病自交系‘E．32’;而JAR1、NDR1、EIN2YeaRARl等基因的表达随着接种诱导时间的增加,表达水平变化不大。初步推断EDS1、PAD4、NPRI、SGTI和WRYK70可能与调节茄子抗青枯病抗性反应有关。同时茄子在接种后活性氧的含量均增加,但是感病植株的含量高于抗病植株。     

嫁接辣椒根系特征及根际土壤酶活性与青枯病抗性的关系

以‘卫士’辣椒为砧木,‘新丰2号’为接穂嫁接,通过人工接种青枯病菌研究嫁接和自根辣椒根系特征、根际土壤微生物及酶活性的变化,探讨嫁接辣椒的抗病机理.结果显示:接种青枯病菌前,嫁接辣椒的根系重量、总长度、总体积、表面积、根尖数和分叉数均显著高于自根苗,根系活力、根际土壤放线菌数量和比例,以及根际土壤酶(多酚氧化酶、过氧化物酶和脱氢酶)活性也明显高于自根苗.接种青枯病菌后,嫁接辣椒的根系受伤程度较自根苗轻,根系重量、总长度、总体积、表面积、根尖数和分叉数的降低幅度均显著小于自根苗,根系活力、根际土壤微生物数量、放线菌比例及土壤酶活性明显大于自根苗.研究表明,嫁接辣椒根系发达,根系活力增强,根际土壤放线菌比例增加及酶活性提高是其青枯病抗性增强的重要原因.     

''陕麦139''受条锈菌抑制表达相关基因的分析

以‘条中32’接种前后的小麦抗条锈病种质‘陕麦139’幼苗叶片为材料,利用抑制消减杂交技术,构建了条锈菌接种48 h小麦抗病品种叶片的抑制表达SSH-cDNA文库,通过测序以了解条锈菌侵染后被抑制表达的相关基因。从构建的文库中随机选取48个阳性克隆,进行测序,经过序列拼接等,获得高质量EST序列34条(Gen-Bank序列号为EL930132-EL930165),序列比对分析表明其中31条EST序列与已知功能的基因序列同源性较高,主要涉及植物的能量代谢途径、膜转运、信号传导及核酸加工等方面的基因,包括光合系统Ⅱ抗性蛋白D1、碳酸酵素、线粒体磷酸盐转运子、苹果酸脱氢酶、过敏性诱导反应蛋白和rRNA核酸内切酶等相关基因。此外,获得未知功能的EST序列3条。研究表明,小麦在条锈病菌侵染初期,许多基因的正常表达调控受到影响,如能量代谢和寄主细胞结构木质化,阻止小麦抗病过敏性反应等。     

粳稻子预44抗LP11稻瘟病菌基因Pizy6(t)的定位

稻瘟病是世界范围内严重威胁水稻(Oryza sativa)生产可持续发展的主要病害之一,每年造成10%–30%的水稻产量损失。抗瘟水稻品种的培育和育种利用是解决稻瘟病危害最经济有效的方法。对新的致病性菌株进行分离和筛选是定位与克隆抗病新基因及抗病育种的基础。选择分离自不同稻瘟病发生重灾区的单孢菌株,对广谱抗瘟水稻子预44和感病水稻江南香糯进行致病性鉴定,筛选出两材料间致病性差异明显的5个菌株;进一步利用子预44、湘资3150、9311、日本晴、丽江新团黑谷、中花11、TP309和江南香糯8个抗瘟性不同的水稻材料,对筛选的菌株进行致病性鉴定。结果显示,LP11能使广谱抗瘟籼稻湘资3150严重发病,推测其很可能是新进化出来的强致病菌株。利用子预44和江南香糯杂交构建的F2群体进行抗性遗传分析,结果表明子预44对LP11菌株的抗性是由单显性基因控制。利用SSR分子标记和图位克隆方法在子预44中定位了1个抗稻瘟病基因Pizy6(t)。研究结果不仅为抗病相关研究提供了有价值的新菌株,而且为子预44中抗稻瘟病基因Pizy6(t)的克隆奠定了基础。     

Characterization of the interaction between the bacterial wilt pathogen Ralstonia solanacearum and the model legume plant Medicago truncatula

The soilborne pathogen Ralstonia solanacearum is the causal agent of bacterial wilt and attacks more than 200 plant species, including some legumes and the model legume plant Medicago truncatula. We have demonstrated that M. truncatula accessions Jemalong A17 and F83005.5 are susceptible to R. solanacearum and, by screening 28 R. solanacearum strains on the two M. truncatula lines, differential interactions were identified. R. solanacearum GMI1000 infected Jemalong A17 line, and disease symptoms were dependent upon functional hrp genes. An in vitro root inoculation method was employed to demonstrate that R. solanacearum colonized M. truncatula via the xylem and intercellular spaces. R. solanacearum multiplication was restricted by a factor greater than 1 x 10(5) in the resistant line F83005.5 compared with susceptible Jemalong A17. Genetic analysis of recombinant inbred lines from a cross between Jemalong A17 and F83005.5 revealed the presence of major quantitative trait loci for bacterial wilt resistance located on chromosome 5. The results indicate that the root pathosystem for M. truncatula will provide useful traits for molecular analyses of disease and resistance in this model plant species.     

Ralstonia solanacearum extracellular polysaccharide is a specific elicitor of defense responses in wilt-resistant tomato plants

Ralstonia solanacearum, which causes bacterial wilt of diverse plants, produces copious extracellular polysaccharide (EPS), a major virulence factor. The function of EPS in wilt disease is uncertain. Leading hypotheses are that EPS physically obstructs plant water transport, or that EPS cloaks the bacterium from host plant recognition and subsequent defense. Tomato plants infected with R. solanacearum race 3 biovar 2 strain UW551 and tropical strain GMI1000 upregulated genes in both the ethylene (ET) and salicylic acid (SA) defense signal transduction pathways. The horizontally wilt-resistant tomato line Hawaii7996 activated expression of these defense genes faster and to a greater degree in response to R. solanacearum infection than did susceptible cultivar Bonny Best. However, EPS played different roles in resistant and susceptible host responses to R. solanacearum. In susceptible plants the wild-type and eps(-) mutant strains induced generally similar defense responses. But in resistant Hawaii7996 tomato plants, the wild-type pathogens induced significantly greater defense responses than the eps(-) mutants, suggesting that the resistant host recognizes R. solanacearum EPS. Consistent with this idea, purified EPS triggered significant SA pathway defense gene expression in resistant, but not in susceptible, tomato plants. In addition, the eps(-) mutant triggered noticeably less production of defense-associated reactive oxygen species in resistant tomato stems and leaves, despite attaining similar cell densities in planta. Collectively, these data suggest that bacterial wilt-resistant plants can specifically recognize EPS from R. solanacearum.     

Chromosomal locations and gonadal dependence of genes that mediate resistance to ectromelia (mousepox) virus-induced mortality.

Four genetic loci were tested for linkage with loci that control genetic resistance to lethal ectromelia virus infection in mice. Three of the loci were selected because of concordance with genotypes assigned to recombinant inbred (RI) strains of mice derived from resistant C57BL/6 and susceptible DBA/2 (BXD) mice on the basis of their responses to challenge infection. Thirty-six of 167 male (C57BL/6 x DBA/2)F1 x DBA/2 backcross (BC) mice died (22%), of which 27 (75%) were homozygous for DBA/2 alleles at Hc and H-2D. Twenty-eight percent of sham-castrated and 6% of sham-ovariectomized BC mice were susceptible to lethal mousepox, whereas 50% of gonadectomized mice were susceptible. There was no linkage evident between Hc or H-2D and loci that controlled resistance to lethal ectromelia virus infection in 44 castrated BC mice. Mortality among female mice of BXD RI strains with susceptible or intermediate male phenotypes was strongly correlated (r = 0.834) with male mortality. Gonadectomized C57BL/6 mice were as resistant as intact mice to lethal ectromelia virus infection. These results indicate that two gonad-dependent genes on chromosomes 2 and 17 and one gonad-independent gene control resistance to mousepox virus infection, that males and females share gonad-dependent genes, and that the gonad-independent gene is fully protective.     

辣椒种质资源抗青枯病的鉴定与评价

采用青枯菌FJC100301菌株对田间辣椒（Capsicum annuum）抗病品种76a和感病品种TW-1分别作了不同温度、不同接种量和不同接种方法的接种试验。结果表明,辣椒青枯病抗性的室内鉴定以接种温度28℃、浸根20 min和3×10^8cfu/mL接种浓度为宜;辣椒种质田间抗青枯病接种鉴定宜选择5月上旬进行,浸根20 min,接种浓度为3×10^8cfu/mL。采用田间抗性接种鉴定的方法,用青枯菌FJC100301菌株对106份辣椒材料进行了抗性鉴定。田间接种后每隔10 d统计病情指数,划分辣椒抗青枯病鉴定分级标准,获得了高抗材料14份、抗病材料8份、中抗材料23份、中感材料23份、感病材料20份、高感材料18份;采用离体叶片接种法对田间筛选得到的高抗和高感纯度较高品种进行抗性分析,结果与田间鉴定一致。