2010年, 第40卷, 第6期　刊出日期：2010-12-10

  

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病原学
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  长时间低温处理有助于加强黄瓜花叶病毒CMV-BF 2b基因的多态性

  侯焱, 张兴桃, 庄木, 李艳红

  植物病理学报. 2010, 40(6): 561-567.

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  将被黄瓜花叶病毒CMV-BF株系侵染的三生烟(Nicotiana tabacum var.Samsun NN)的不同单株分成两组分别置于高温(30±5℃)和低温(10±5℃)条件下, 观察和分析CMV-BF侵染30、80 d时植株的症状表现及CMV2b基因的序列变异规律。结果表明:侵染30d时, 在高温环境中的植株表现出畸形叶片较多、病毒症状较严重的现象, 而低温培养的植株细弱, 部分叶片大面积白化;2b基因DNA序列的多态性也以高温处理组略高于低温组, 但差异未达到显著水平。侵染80 d时, 高温组植株的病毒症状有所恢复, 畸形叶片减少, 而低温组植株除白化现象更严重外, 畸形叶片增多;2b基因的多态性以低温处理组明显高于高温组, 且达到显著水平(P<0.05)。因此, 长时间低温处理有助于加强CMV-BF2b基因的多态性, 而且2b基因的多态性可能与植株的病毒症状表现相关。
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  樱桃病毒A北京分离物外壳蛋白的原核表达及抗血清制备

  郭颂, 张福兴, 怀晓, 周颖, 张瑞, 范在丰, 周涛

  植物病理学报. 2010, 40(6): 568-573.

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  根据前期研究获得的樱桃病毒A北京分离物(Cherry virus A isolate Beijing, CVA-BJ)外壳蛋白基因序列设计引物, 将此基因克隆到原核表达载体pET-28a后转化大肠杆菌BL21(DE3), 以终浓度为1 mmol/L的IPTG进行诱导表达。Ni珠吸附法纯化表达产物后免疫家兔制备抗血清。间接ELISA测得抗血清效价为1:2048。以纯化后的蛋白和樱桃叶片总蛋白为检测材料, Western blot分析表明抗血清具有高度特异性。间接ELISA方法检测樱桃病叶, 结果呈阳性, 与RT-PCR检测结果一致。表明制备的抗血清可用于感病樱桃样品中CVA的检测。
细胞生物学、生理学、生物化学、分子生物学
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  水稻瘤矮病毒S11基因沉默抑制子的原核表达及抗血清制备

  赵芹, 沈文锦, 张翼翔, 刘福秀, 李华平

  植物病理学报. 2010, 40(6): 574-578.

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  以采自广东省水稻瘤矮病的典型病株为材料, 通过RT-PCR法扩增和克隆了水稻瘤矮病毒(Rice gall dwarf virus, RGDV)S11组分核苷酸序列全长。序列分析结果表明, RGDV广东分离物S11(登录号EF532326)核苷酸序列全长1168bp, 含有一个1068bp的开放阅读框, 编码一条355个氨基酸的多肽, 推测分子量约40kD, 与泰国分离物基因结构基本一致, 其核苷酸与氨基酸序列相似性分别为94.3%和98.8%。将广东分离物S11基因克隆至pBV221温敏表达载体上, 在大肠杆菌DH5α中实现了高效表达。以诱导蛋白为抗原免疫家兔获得了抗血清。利用ELISA法测定抗血清的效价为1:4096, Western blot分析结果表明, 抗血清能与S11蛋白发生特异血清学反应。这可为进一步研究S11蛋白的结构和功能, 揭示其抑制基因沉默的作用机制提供参考。
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  水稻条斑病菌hrpB1基因决定寄主致病性和非寄主过敏反应的功能研究

  刘之洋, 邹丽芳, 邹华松, 陈功友

  植物病理学报. 2010, 40(6): 579-586.

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  水稻条斑病菌(Xanthomonas oryzae pv.oryzicola, Xoc)的hrp基因决定了病原菌在非寄主植物上的过敏反应(hypersensitive response, HR)和在寄主植物上的致病性(pathogenicity), 基因产物形成Ⅲ型分泌系统(type-Ⅲ secretion system, T3SS)将致病性效应分子注入寄主细胞从而引起水稻产生抗病性或者感病性反应。以位于hrpB操纵单元的首个hrpB1基因为对象, 通过基因敲除方式对其进行了突变, 发现hrpB1突变体丧失了在水稻上的致病性和在烟草上激发HR的能力, 并且在水稻组织中的生长能力显著降低。RT-PCR测定结果表明, hrpB1的转录表达受HrpG和HrpX的正调控。免疫杂交结果显示, HrpB1蛋白可通过T3SS进行分泌。这些结果不仅明确了hrpB1基因在病原菌致病性中的功能, 而且提示了hrp结构基因不仅仅局限于形成Ⅲ型分泌系统, 部分hrp基因产物本身也通过Ⅲ型系统分泌到胞外, 并且可能起到效应分子的功能。
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  小麦蓝矮植原体免疫膜蛋白(Imp)的跨膜区影响其在大肠杆菌中的表达

  孙润红, 于祥泉, 陶烨, 杨洋, 吴云锋, 张银武, 李艳

  植物病理学报. 2010, 40(6): 587-592.

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  根据跨膜区预测结果, 设计ImpF1/ImpR1和ImpF2/ImpR2两对特异性引物。以ImpF1/ImpR1, ImpF1/ImpR2, Imp-F2/ImpR1和ImpF2/ImpR2共4个组合经PCR扩增得到4个目标片段, 即:免疫膜蛋白Imp基因的全长(Imp-B);切除C-端跨膜区的Imp基因的基因序列(Imp-N);切除N-端跨膜区的Imp基因的基因序列(Imp-C);切除N-和C-端跨膜区的Imp基因的基因序列(Imp-S)。经酶切连接到原核表达载体Pmal-c2x, 并转入E.coliBL21(DE3)PlysS菌株中。SDS-PAGE电泳验证, 只有切除N-、C-端跨膜区的Imp基因的基因序列(Imp-S)得到大量表达, 结果表明:小麦蓝矮植原体免疫膜蛋白的跨膜区影响其在大肠杆菌中的表达。根据跨膜区预测结果, 设计ImpF1/ImpR1和ImpF2/ImpR2两对特异性引物。以ImpF1/ImpR1, ImpF1/ImpR2, Imp-F2/ImpR1和ImpF2/ImpR2共4个组合经PCR扩增得到4个目标片段, 即:免疫膜蛋白Imp基因的全长(Imp-B);切除C-端跨膜区的Imp基因的基因序列(Imp-N);切除N-端跨膜区的Imp基因的基因序列(Imp-C);切除N-和C-端跨膜区的Imp基因的基因序列(Imp-S)。经酶切连接到原核表达载体Pmal-c2x, 并转入E.coli BL21(DE3)PlysS菌株中。SDS-PAGE电泳验证, 只有切除N-、C-端跨膜区的Imp基因的基因序列(Imp-S)得到大量表达, 结果表明:小麦蓝矮植原体免疫膜蛋白的跨膜区影响其在大肠杆菌中的表达。
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  大麦条纹花叶病毒中国株编码βC蛋白可以自身相互作用

  孙现超, 赵文军, 薛杨

  植物病理学报. 2010, 40(6): 593-600.

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  以分别含有BSMV-CH基因组3个组分ds-cDNA全长的pMD-α、pMD-β和pMD-γ质粒为模板, PCR扩增BSMV-CH编码各蛋白基因, 克隆至酵母双杂交载体, 检测各蛋白是否存在自身激活特性, 用酵母双杂交分析BSMV-CH编码蛋白之间的相互作用情况, 测定β-半乳糖苷酶活性确认蛋白在酵母体内的作用, Western-blot分析蛋白的体外相互作用。结果表明成功克隆到BSMV-CH编码蛋白基因, 并正确克隆至酵母双杂交载体, 酵母双杂交检测未发现BSMV-CH编码蛋白之间的相互作用, 但确定BSMV-CH编码的βC蛋白可以自身相互作用, 体外可以单体或二聚体形式存在。
致病性与抗病性遗传
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  云南景洪疣粒野生稻抗白叶枯病相关基因表达分析

  付坚, 吕广磊, 程在全

  植物病理学报. 2010, 40(6): 601-608.

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  从已经构建的白叶枯病菌胁迫的景洪疣粒野生稻抑制差减杂交文库(SSH文库)中筛选一批应答基因。通过测序比对, 文库中抗逆基因占了绝大部分, 涉及抗病、耐旱、抗冻、耐盐相关基因片段以及信号转导因子和植物激素调控蛋白等。其中NBS-LRR和STK类抗病基因各1个, ME207基因与泛素结合酶相似, 与过敏性反应信号传导相关。ME022基因与金属硫蛋白MT2基因同源, 通过降低细胞内的自由金属离子浓度阻止病症的进一步扩展。综合抗逆相关基因功能的分析, 初步推断景洪疣粒野生稻高抗白叶枯病是以抗病基因、信号传导因子和其他抗逆相关基因共同调控的过程。
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  新疆野苹果和秦冠的抗黑星病特性

  胡小平, 张吉光, 陈婧, 周书涛, 杨家荣, 康振生

  植物病理学报. 2010, 40(6): 609-614.

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  透射电镜观察表明, 苹果叶片上表皮角质层厚度在品种间存在显著差异, 新疆野苹果和秦冠叶片的角质层厚度显著高于富士和嘎啦的;同一品种不同龄期叶片的角质层厚度随着叶龄增长而增厚, 且黑星病严重度与叶片上表皮角质层厚度间存在显著的负相关关系。苹果品种抗病性组分分析结果表明, 新疆野苹果的病害严重度最低, 约为嘎啦的1/22, 潜育期最长, 为嘎啦的2.0倍, 无(或少有)病斑出现, 不产孢。秦冠的严重度约为嘎啦的1/14, 潜育期约为嘎啦的1.5倍, 产孢量约为嘎啦的1/26。黑星病菌在新疆野苹果和秦冠叶片上的侵染概率及病斑扩展速率均显著低于富士和嘎啦。因此, 新疆野苹果和秦冠对黑星病的抗病性表现在抗侵入和抗扩展两个方面。
流行学与生态学
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  陕西省小麦禾谷镰刀菌的遗传多样性研究

  刘恒, 侯丽娟, 马红娜, 白耀博, 李强, 王保通

  植物病理学报. 2010, 40(6): 615-621.

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  为了明确陕西省小麦禾谷镰刀菌混合群(Fusarium graminearum species complex)的遗传多样性, 利用4对EcoRⅠ和MseⅠ引物对来自陕西省19个区县的162株小麦禾谷镰刀菌菌株进行了AFLP扩增。结果表明, 4对引物均能扩增出数量不等的多态性条带, 最少的6条, 最多的20条, 大部分扩增片段在100~750 bp之间。利用NTSYS-2.1软件聚类分析表明, 不同地区禾谷镰刀菌可分为两大类群, 即类群A和类群B。这两大类群的分化和地理来源有明确相关性, 类群A主要分布在关中地区, 类群B主要分布在陕南地区。初步判断可能与两个地区生态环境和小麦主栽品种差异有关。各类群内的菌系与地理位置间的关系较为复杂, 一些菌系与地理来源存在明确关系, 而个别菌系与地理来源间的关系尚不能完全明确。还需进一步研究以明确各菌株与地理来源之间的关系。
植物病害及其防治
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  应用实时荧光定量RT-PCR检测几种食用菌蛋白抗烟草花叶病毒的活性

  廖芳, 严红, 赵素芳, 李兴红

  植物病理学报. 2010, 40(6): 622-627.

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  根据烟草花叶病毒(Tobacco mosaic virus, TMV)外壳蛋白(CP)RNA的特异性序列设计TaqMan荧光探针及其引物, 利用实时荧光定量RT-PCR检测食用菌蛋白抗TMV的活性。经食用菌蛋白处理后, TMV病毒汁液中TMV的RNA浓度下降了22.02%~87.93%, 传统生物学测定的食用菌蛋白抗TMV的平均枯斑抑制率为10.88%~83.97%;相关性分析表明, 实时荧光定量RT-PCR测定的病毒RNA浓度的下降与传统生物学方法测定的平均枯斑抑制率之间呈正相关(r=0.818 8), 具有较好的一致性。利用实时荧光定量RT-PCR检测蛋白抗烟草花叶病毒活性的方法, 具有特异性好、快速、简便、重复性高的特点, 适合于蛋白抗烟草花叶病毒活性的痕量快速高效检测。
研究简报
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  进境澳大利亚油菜籽中茎基溃疡病菌的检测

  易建平, 周国梁, 印丽萍, 陈仲兵, 郑建中

  植物病理学报. 2010, 40(6): 628-631.

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  41 fungal isolates with similar morphological characteristics to Leptosphaeria maculans were obtained by the deep-freezing filter paper method from 2100 seeds of Brassica napus imported from Australia.The isolate 8129-5 showed a slower growth on PDA at 20℃with growth rate of 2.8 mm/day.The colonies on PDA at 20℃ had an irregular or regular margin with white or grayish white compact aerial mycelium.No diffusible pigment was produced on PDA at 31℃ or in liquid Czapek-Dox media at 20℃.PCR detection showed that the isolate 8129-5 could be amplified by L.maculans-specific primers LmacF/LmacR and got expected product of 331 bp.The sequence analysis revealed that the ITS sequence of isolate 8129-5 had 99.8% identity with L.maculans.Pathogenicity of the isolate 8129-5 was confirmed on cotyledons of rape seed by artificial inoculation compared with typical symptom of L.maculans.Based on the morphological characteristics, PCR detection and the result of pathogenicity test, the isolate 8129-5 was identified as L.maculans.
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  应用细菌磁颗粒实时荧光RT-PCR检测南瓜花叶病毒

  孙宁, 邓丛良, 么磊, 陈继峰

  植物病理学报. 2010, 40(6): 632-635.

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  A novel real-time RT-PCR method, BMPs based real-time RT-PCR which integrated with magne-tic separation technique of bacterial magnetic particles(BMPs), was set up for detection of Squash mosaic virus(SqMV).After SqMV particles in crude sap were concentrated by BMPs, viral RNAs were released and detected by real time RT-PCR.The results indicated that BMPs based real-time RT-PCR was efficient, and the detection sensibility was equivalent to that of the Trizol based real-time RT-PCR, of which Trizol reagent was used for viral RNAs extration.Comparing to Trizol-based method, the BMPs-based method had advantages of simplicity on operation, time saving for RNA extraction and without using noxious organic chemicals.
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  3种瓜类枯萎病菌Fusarium oxysporum的分子检测

  张淑梅, 赵晓宇, 张先成, 孟利强, 李晶, 张云湖, 王玉霞

  植物病理学报. 2010, 40(6): 636-641.

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  Fusarium oxysporum is one of the most important phytopathogens and cause Fusarium wilt disease in cucumber, watermelon and melon, etc.In this study, a pair of species-specific primers Fc-1 and Fc-2 was synthesized based on differences in internal transcribed spacer sequences of Fusarium genus.With the primers, a specific 315 bp PCR product was amplified from five F.oxysporum isolates isolated from cucumber, watermelon and melon, infected cucumber and watermelon tissues, while no product was obtained from other fourteen fungi, healthy cucumber and watermelon tissues.The detection sensitivity is 100 fg for genomic DNA of F.oxysporum and 1 000 spores/g soil for the soil pathogens.In contrast, the nested PCR with two pairs of primers(ITS1/ITS4 and Fc-1/Fc-2) increased the sensitivity by 100-fold.In addition, one-step PCR could also detect F.oxysporum in symptomless cucumber root of 7 dpi(days post inoculation) and in infected cucumber and watermelon tissues at the early stage of disease development.Therefore, the developed PCR-based method enabled rapid, sensitive and reliable detection of F.oxysporum.It also provides the detection method for early monitoring and diagnosis of the pathogen as well as the plant disease management guidance.
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  安徽桑黄花型萎缩病植原体16S rDNA序列分析及分子检测

  卢全有

  植物病理学报. 2010, 40(6): 642-646.

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  Mulberry yellow dwarf(MYD)disease is an quarantine disease and the causal agent is a phytoplasma.Two pairs of published universal primer, P1/P7 and Rm16F2/Rm16R1, based on the 16S-23S rDNA sequence of phytoplasma and total DNA extracted from infected mulberry tissues were employed for PCR and nested-PCR detection.The results revealed that a phytoplasma-specific 1 830 bp fragment with a G+C content of 46.01% was sequenced(GenBank accession No.GQ249410).The sequence shared 99.7% and 99.8% identity with aster yellows, the representatiive phytoplasma in 16SrI group, and mulberry dwarf phytoplasma classified into subgroup B in 16SrI group and named as the MYD phytoplasma strain Anhui(MYD-Anh).A phylogenetic tree based on 16S rDNA sequences was constructed and showed that MYD-Anh was clustered into 16SrI group.Identity of 16S rDNA sequence between MYD-Anh and mulberry yellow dwarf phytoplasma strain Zhenjiang(MD-zj) was nearly 100%, and they might belong to the same strain.Nested-PCR was used to detect the pathogenic phytoplasma from the differential tissues of mulberry infected with MYD-Anh.The results showed that a phytoplasma-specific 1.4 kb fragment was amplified with total DNA extracted from bark and vein.Nested-PCR was more sensitive than PCR for detecting MYD phytoplasma.
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  花生斑驳病毒青岛分离物cp基因序列克隆与分析

  刘媛媛, 侯珊珊, 常文程, 阴筱, 贾金磊, 李向东, 迟玉成

  植物病理学报. 2010, 40(6): 647-650.

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  Peanut mottle virus(PeMoV) was detected via RT-PCR from two peanut samples(QD5 and QD6) with mottle symptom collected from Qingdao, Shandong Province.The 3'-terminal 892 bp fragments of their genome were cloned and sequenced.The cp genes of QD5 and QD6 were 837 bp in length and encoded 278 amino acids(aa), with DAA at aa sites regulating aphid transmission.QD5 and QD6 shared nucleotide identities of 95.3%-99.4% and aa identities of 93.5%-99.6% in cp genes with other PeMoV isolates available in the GenBank.The phylogenetic results showed that PeMoV were clustered to three groups, America, Asia and Australia, which were consistent with their geographical origins.This is the first molecular evidence on the incidence of PeMoV in China.
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  云南甘蔗杆状病毒的分子检测及其对甘蔗品质和产量的影响

  李文凤, 黄应昆, 姜冬梅, 张志想, 张本利, 李世访

  植物病理学报. 2010, 40(6): 651-654.

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  Sugarcane bacilliform virus(SCBV) was detected by PCR from sugarcane showing chlorosis and mottle symptom from Kaiyuan, Yunnan Province.Part sequence of replicase gene of the isolate SCBV-Kaiyuan was determined.Sequence analysis indicated that the 589 bp of SCBV-Kaiyuan shared identities of 73.2%-74.0% and 83.1%-84.1% at nucleotide and amino acid levels with SCBV-Australia respectively, 66.7%-68.4% and 65.6%-67.7% with SCBV-Morocco.The quality and yield of the sugarcane infected with SCBV-Kaiyuan was also investigated.The juice extraction, sucrose content, gravity purity and average stalk weight were decreased 1.55%, 1.24%, 2.22% and 0.26 kg in plants infected with SCBV-Kaiyuan, but reducing sugar was increased by 0.21% in infected plants.
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  分子标记辅助选择小麦抗白粉病兼抗赤霉病聚合体

  宋伟, 张敏, 杨继芝, 罗培高, 龚国淑, 涂坦

  植物病理学报. 2010, 40(6): 655-658.

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  Sumai 3, a wheat variety resistant to Fusarium head blight(FHB), was crossed with Neimai 9, a commercial wheat cultivar with the resistance to powdery mildew.The SCAR(sequence characterized amplified region) markers of powdery mildew resistance gene Pm21 and four SSR(simple sequence repeats)markers flanking the major FHB resistance QTL(Qfhs.ndsu-3BS) in Sumai 3 were used to detect the resistance loci by marker assisted selection(MAS) in the plants of the F₂ population.Identification of resistance to both powdery mildew and FHB in field showed that 12 plants resistant to both diseases were obtained.In addition, the agronomic traits of these plants were better than those of Sumai 3, and are perhaps the excellent parental materials for wheat breeding.