【作者】 曹慧英；

【导师】 王晓鸣；

【作者基本信息】 中国农业科学院 ， 植物病理学， 2010， 博士

【摘要】 玉米是我国重要的农作物,近年来生产一直处于上升趋势。在玉米生产中,病虫害是高产稳产的重要限制因子,而一些新的或已往仅在极少区域发生的病虫害,对我国玉米生产带来了新的威胁。2006年7月,在新疆和甘肃玉米制种区的制种亲本PS056上发生了一种不同于已往报道的茎腐病,植株发病率80%-100%,对杂交种种子的生产造成了明显的影响。病害症状主要为植株矮小,发病部位组织表现为褐色干腐。实验表明,从PS056茎秆上病组织中获得的细菌分离物具有致病性,该病与已往报道的以软腐为特征的玉米细菌茎腐病不同。因此,确诊该种细菌茎腐病是玉米上的一种新记录病害,命名为玉米细菌干茎腐病(Bacterial Dry Stalk Rot)。本文对该病害进行了详细的研究,主要结果如下：1.从2006到2009年,干茎腐病在新疆、甘肃玉米制种地父本PS056茎秆上连续发生。病害的典型症状为植株矮小,茎部叶鞘表面出现不规则的黑褐色病斑,茎节发病处表皮消失,茎髓组织变褐并形成不规则的缺刻,褐变组织表现为干腐。病株茎秆扭曲、发脆,遇风极易倒折。根据对发病组织的显微镜检查、病原菌分离和形态鉴别、致病性测定、生理生化测定、16S rRNA序列分析及全细胞脂肪酸鉴定等结果,将引起新疆和甘肃干茎腐病的病原菌鉴定为成团泛菌Pantoea agglomerans。目前,国内外尚无报道由P. agglomerans引起的玉米细菌干茎腐病的病害。2.测定了P. agglomerans 16S rDNA和16S-23S rDNA (ITS)序列,设计并筛选到一对特异性引物Pa1/Pa2(正向引物Pal:5’GCTACAATGGCGCATACAA3’,反向引物Pa2：5’CGTCCTGCTCGTTACTTTT3’),能够在P. agglomerans中特异性扩增出375 bp的条带。构建了P. agglomerans分子检测技术,利用引物Pa1/Pa2能够进行玉米种子带菌和土壤带菌的检测。3.种子带菌检测结果表明,父本PS056和F1金玉9856种子均为外部不带菌,内部带菌；母本OSL190种子的内、外部均不带菌；特异性引物PCR检测表明,F1金玉9856种子中分离菌Pag1和父本PS056种子中分离菌Pag2都可以扩增出相同的P. agglomerans特异性目标条带(375bp)。温室和田间实验表明,引起干茎腐病的病菌P. agglomerans可通过种子传播,十壤带菌也是病害的主要侵染源之一,而花粉带菌可能是病害从父本向杂交种种子传播的主要方式。4.利用转座子技术,构建了致病性菌株P. agglomerans XJ1突变体库。通过对突变菌株的接种筛选,获得11个致病力丧失的突变体。经对Tn5插入位点的侧翼扩增,确认突变体PA121和PA71是由于yhfK基因和flhD基因的插入突变而失去致病力,Tn5在突变体PA121和PA71中为单拷贝插入。yhfK基因通过调控成团泛菌中hrpA基因的表达而导致病菌对玉米致病性丧失；flhD基因的突变导致了成团泛菌鞭毛发育受阻、细菌泳动性丧失并进而引起致病力丧失。5.利用病原菌P. agglomerans XJ1菌株对OSL190(抗病亲本)×PS056(感病亲本)的F2分离群体及其相关的亲本材料进行抗性鉴定,分析了玉米对P. agglomerans的抗性遗传规律。抗性鉴定结果表明,父本PS056为100%发病,母本OSL190均抗病,正交和反交的后代F1均表现为抗病,F2群体的抗、感分离比均符合3：1的比例,表明母本OSL190含有显性抗病单基因,父本PS056含有隐性感病基因。与自交系PS056有亲缘关系的其他自交系78599-1、沈137、齐319、E28和X178表现抗病,丹341和丹340则严重感病,推断PS056的感病性可能来源于丹341。更多还原

【Abstract】 Maize (Zea mayz L.) is an important crop with enhanced production in recent years. In maize production, pests and diseases are the main limiting factors of high and stable yield. The occurrence of new pests and diseases or those previously occurred in limited areas have brought new threats to maize production in China. In July 2006, a new maize stalk rot in the paternal parent PS056 was observed in the hybrid seed production fields in Xinjiang and Gansu provinces, China, which was different from the previous reports. The diseased plants were scattered throughout the fields and 80% to 100% plants of the male parental line PS056 were infected, which resulted in serious yield loss because of poor pollen shed. Disease symptoms included slow growth and dwarf in diseased plants. The infected tissues showed dry rot symptoms. The symptoms of this disease were different from those of the maize bacterial stalk rot in previous reports. Thus, the new disease on maize was designated bacterial dry stalk rot. In this study, the newly found disease was discussed in details. The main results are as follows:1. From 2006 to 2009, a new bacterial disease with dry rot symptom on stalks has frequently occurred in the maize hybrid seed production fields in Xinjiang and Gansu provinces, China. The typical symptoms of the disease were the infected plants grew slower and became dwarf, and irregular dark brown spot on cortex and dry rot on stalk of maize. Sometimes, the infected parts of stalks became black and formed concavity due to browned cortex of stalk disappeared. The diseased tissues showed typical dry rot symptoms. The diseased plants were lopsided and easily broken because only one side of the stalk was infected. Based on examination results of microscope, electron microscopy, pathogenicity assay, physiological and biochemical tests,16S rDNA sequence analysis and whole-cell fatty acid identification, etc., the pathogenic bacteria isolated from the infected stalk that caused bacterial stalk rot on Xinjiang and Gansu maize was identified as Pantoea agglomerans. This is the first report of dry stalk rot disease on maize caused by P. agglomerans.2. A pair of specific primers Pa1/Pa2 (forward primer: 5’GCTACAATGGCGCATACAA 3’, reverse primer:Pa2: 5’CGTCCTGCTCGTTACTTTT 3’) were obtained by screening bacterial 16S rDNA sequences 16S-23S rDNA ITS sequences, which can specifically detect P. agglomerans in the target bacteria and reference strains. The PCR amplified production from the target bacteria was 375 bp. A molecular detection technique for P. agglomerans was established, and the specific primers Pa1/Pa2 can detect the target bacteria in the maize seeds and soil.3. Seeds infection results indicated that paternal PS056 and F, Jinyu 9856 seeds only carried bacteria internally but not externally; maternal OSL190 seeds carried bacteria neither internally nor externally. PCR amplification with specific primers showed that the same specific target band (375 bp) can be amplified from isolates Pagl isolated from paternal F1 Jinyu 9856 and isolates Pag2 isolated from PS056. The greenhouse and field experiments showed that the pathogenic bacteria can be transmitted through seeds. The P. agglomerans of soil was also one of the main sources of infection, and the pathogenic bacteria of pollen maybe the main source of infection in the transmittion from male parent to hybrid variety F1 seeds. 4. A transposon mutant library of the pathogenic strain P. agglomerans XJ1 was constructed by transposon Tn5 random insertion mutation. Eleven mutants which lost pathogenicity were obtained by injecting inoculation on maize stalks ex vivo. By amplifying the flank sequences of Tn5 insertion sites, it was confirmed that non-pathogenic mutation strains PA 121 and PA71 were due to mutation of genes yhfK and flhD. The mutation strains PA 121 and PA71 had a single-copy of Tn5 transposon. By regulating gene hrpA transcription, gene yhfK induced lost pathogenicity of P. agglomerans on maize. The mutation of gene flhD caused the loss of flagellum development and swimming motility of P. agglomerans, thus the mutation of gene flhD induced lost pathogenicity of P. agglomerans on maize.5. Using the F2 population derived from the cross between the resistant parent OSL190 and the susceptible parent PS056 and relative parental lines, the inheritance of resistance to P. agglomerans of maize was studied. Inoculation with the strain XJ 1 of P. agglomerans indicated that all the plants of the paternal parent PS056 were susceptible, but the plants of the maternal parent OSL190 were all resistant. The plants of reciprocal Fj cross were also resistant. The segregation of resistant to susceptible plants fitted the ratio of 3:1, indicating that OSL190 carries a single dominant gene for resistance and PS056 carries a single recessive gene for susceptibility. The inbred lines 78599-1, Shen137, Qi319, E28, and XI78 in the pedigree of lne PS056 were all resistant, while Dan 341 and Dan 340 were severely susceptible. This indicates that the susceptibilility was most probably derived from Dan 341.更多还原