2009年, 第39卷, 第5期　刊出日期：2009-10-10

  

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病原学
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  进境苹果果实中梨火疫病菌的套式PCR检测

  贾平乔, 周国梁, 吴杏霞, 张卫东, 杨晓君, 徐殿胜, 易建平

  植物病理学报. 2009, 39(5): 449-457.

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  针对进境商用苹果果实携带梨火疫病菌Erwinia amylovora数量有限的特点,选取源于病菌pEA29质粒的2对引物P29A/P29B和PEANT1/PEANT2配对组合成套式PCR,其检测灵敏度可达0.15 pg菌体DNA,检测灵敏度高于EPPO推荐的单管套式PCR方法和常规PCR方法。分别利用这3种PCR检测方法对美国、新西兰、日本和智利等国进境的166批苹果样品进行检测,3种检测方法的样品阳性率分别为53.6%、38.0%和8.4%,试验结果表明此套式PCR检测方法可用于进境商用苹果的梨火疫病菌快速检测。进境样品的检测结果证实了进境商用苹果果实中存在梨火疫病菌的可能性。
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  葡萄卷叶伴随病毒2号和3号辽宁分离物部分基因组的序列分析

  王萌, 费菲, 周涛, 程玉琴, 范在丰

  植物病理学报. 2009, 39(5): 458-465.

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  将采自辽宁兴城地区在生长期间具典型卷叶病症状的金星无核(Venus Seedless)葡萄品种休眠枝条,用RT-PCR检测4种葡萄卷叶伴随病毒(Grapevine leafroll-associated viruses,GLRaVs),扩增得到了葡萄卷叶伴随病毒2号(GLRaV-2)和葡萄卷叶伴随病毒3号(GLRaV-3)两种病毒的主要外壳蛋白(major coat protein,CP)基因的完整序列(GenBank登录号分别为FJ786017和FJ786016)。这表明该葡萄植株受到了GLRaV-2和GLRaV-3辽宁分离物(GLRaV-2-LN和GLRaV-3-LN)的复合侵染。根据检测结果,克隆了GLRaV-2-LN基因组3'端CPm (minor capsid protein)、p19(19-kDa protein)和p24(24-kDa protein)基因(GenBank登录号分别为FJ786018、FJ786019和FJ786018)。序列分析表明,GLRaV-3-LN的CP基因全长942 nt,与已报道的国内外其它分离物CP基因全序列相比,核苷酸序列同源性为89.8%~91.8%,由此推导的氨基酸序列同源性为94.9%~97.4%。GLRaV-2-LN的CP、CPm、p19和p24基因全长分别为597 nt、672 nt、486 nt和618 nt。与国外报道的几个分离物的相应蛋白基因全序列相比,核苷酸序列同源性分别为88.3%~100.0%、78.7%~99.9%、75.1%~99.4%和87.5%~99.5%;由此推导的氨基酸序列同源性分别为92.9%~100.0%、89.2%~100.0%、73.9%~99.4%和89.3%~99.0%。
细胞生物学、生理学、生物化学、分子生物学
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  基因枪法导入GUS基因获得小麦条锈菌突变体

  卢丽丽, 张如佳, 王美南, 王阳, 井金学, 李振岐

  植物病理学报. 2009, 39(5): 466-475.

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  采用基因枪转化法用带有β-葡糖醛酸酶(GUS)报告基因的质粒pGUS6L20转化小麦条锈菌,建立了优化的转化体系;在转化当代的条锈菌中得到了GUS基因瞬时表达的菌株;连续继代转接培养后,在转化后第1代(T1)和第2代(T2)条锈菌中检测到了GUS基因稳定表达的菌株;利用GUS报告基因和毒性标记相结合的方法,经过3代筛选鉴定,在小麦条锈菌中国鉴别寄主尤皮Ⅱ号和抗引655上,各获得1个稳定的毒性突变体,经PCR及PCR-southern blot证实外源片段已整合到毒性突变菌株的基因组中。该研究表明基因枪转化法是获得条锈菌毒性突变体的有效方法。
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  转录激活因子FleQxoo原核表达及其与鞭毛基体基因fliExoo启动子的结合

  傅本重, 吴茂森, 陈华民, 何晨阳

  植物病理学报. 2009, 39(5): 476-481.

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  为了阐明转录激活因子FleQxoo对水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae,Xoo)鞭毛基体基因fliExoo的转录调控机理,本研究用PCR方法从Xoo野生型菌株PXO99^A中克隆了fleQxoo基因,构建了原核表达载体pET21-fleQxoo,在大肠杆菌BL21(DE3) pLysS中进行了诱导表达,通过Ni-NTA亲和层析纯化得到FleQxoo蛋白;EMSA检测到FleQ-xoo与鞭毛基体基因启动子fliExoo-p片段的特异结合作用。这些结果为阐明FleQxoo直接调控鞭毛基体基因fliExoo的转录提供了分子证据。
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  心叶烟NPR1基因全长cDNA序列的克隆及表达分析

  张良, 张颖, 石静, 张露, 郭兴启

  植物病理学报. 2009, 39(5): 482-490.

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  NPR1(non-expressor of pathogenesis-related gene 1)基因在拟南芥系统获得抗性中起着关键作用,可调控拟南芥植株广谱抗性的发生。本文报道了从心叶烟中克隆NPR1同源基因(NgNPR1)及其表达特性的研究结果。NgNPR1 cDNA全长2253 bp,编码588个氨基酸。将NgNPR1基因组全长与cDNA序列进行比对发现,NgNPR1基因组DNA含有4个外显子和3个内含子。Southern杂交分析表明,在心叶烟基因组中NgNPR1为单拷贝基因。采用绿色荧光蛋白在洋葱表皮瞬时表达的试验,证明了NgNPR1蛋白在水杨酸诱导时会从细胞质转运到细胞核中。Northern杂交分析发现,NgNPR1基因可以被与植物抗病相关的信号分子如水杨酸、茉莉酸甲酯、过氧化氢和乙烯所诱导。进一步研究发现,植物病原物如赤星病菌、青枯病菌和烟草花叶病毒对心叶烟植株的侵染也会使NgNPR1表达量增加。这些结果表明,NgNPR1基因在心叶烟植株抵御病原物侵染过程中可能起着重要作用。
致病性与抗病性遗传
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  氮胁迫条件下稻瘟病菌分泌蛋白致病性分析

  周晓罡, 苏源, 李成云, 丁玉梅, 张绍松, 孙茂林, 李进斌

  植物病理学报. 2009, 39(5): 491-500.

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  以CH-63和TH-162个致病性差异的稻瘟病菌株为材料,在缺氮培养条件下进行液体培养。经培养2 d后,采用铵盐沉淀及透析纯化处理获得稻瘟病菌培养滤液中的分泌蛋白。分别采用致伤接种及浸泡处理对该蛋白作用水稻植株后的病理反应进行系统研究,结果表明经致伤接种的水稻叶片及茎杆接种斑处出现明显的坏死病斑,其病斑直径是对照的2~4倍。浸泡处理的水稻幼根出现严重褐化,植株生长矮小,株高仅为对照的1/2。对上述稻瘟病菌株分泌蛋白进行酶解处理后病症消失或减弱,进一步确证其为引起上述病理反应的主要因子。将该分泌蛋白接种24个水稻单基因系,接种后72 h根据病斑长度将24个IRRI水稻单基因系按抗、中、感病性状分组。对稻瘟病菌菌株CH-63和TH-16的缺氮分泌蛋白致病力强弱差异与菌丝块接种结果进行比较,结果表明CH-63致病力强于TH-16。
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  甜瓜细菌性果斑病菌致病性突变体筛选与hrcR基因的克隆

  任争光, 侯磊, 宋治国, 张力群

  植物病理学报. 2009, 39(5): 501-506.

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  细菌性果斑病(Bacterial Fruit Blotch,BFB)是西瓜和甜瓜的重要病害。本实验从发病甜瓜果实上分离得到致病菌株MH21,经鉴定为燕麦食酸菌西瓜亚种(Acidovorax avenae subsp. citrulli)。利用转座子Mini-Tn5构建MH21菌株的突变体库,Southern印迹杂交结果显示Mini-Tn5在菌株MH21染色体上可随机、单拷贝插入。通过浸种处理甜瓜种子进行突变体的致病性检查,筛选得到1株致病性完全丧失的突变体M543。对M543中转座子插入基因的克隆和测序表明其突变基因为燕麦食酸菌Ⅲ型分泌系统(TTSS)中的保守基因hrcR。推测菌株MH21中hrcR基因编码的蛋白定位于细菌内膜,是分泌通道主要成分之一。hrcR基因互补菌株的致病性检查结果显示其致病力恢复到野生型的84%。研究同时发现hrcR基因突变后MH21菌株丧失了在烟草上诱导过敏性坏死反应的能力。本研究从遗传学角度说明TTSS是西甜瓜果斑病菌致病性的重要因子。
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  1个小麦NBS类抗病基因同源cDNA序列的克隆与鉴定

  王海燕, 刘大群, 杨文香, 李在峰, 张立荣, 张娜

  植物病理学报. 2009, 39(5): 507-513.

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  利用cDNA末端快速扩增技术对小麦抗叶锈病近等基因系TcLr35中所获得的抗病基因同源片段S2A2 5'端和3'末端序列进行扩增,并根据拼接序列设计特异性引物,进行全长基因的扩增,获得了1个通读的NBS类抗病同源基因S2A2 cDNA序列,该序列全长为3476 bp,编码866个氨基酸序列。经BLASTp比较,该片段含有NB-ARC保守结构域和多个LRR结构域,与已知植物抗病基因I2C-1、L6、RPS2等相应区域相一致。半定量RT-PCR分析表明,该基因在小麦叶片中为低丰度组成型表达。本研究在TcLr35小麦中成功获得了抗病同源基因,这为最终克隆小麦抗叶锈病基因Lr35奠定了基础。
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  水稻纹枯病寄主-病原物互作鉴别品种与菌株的筛选

  陈夕军, 王玲, 左示敏, 王子斌, 陈宗祥, 张亚芳, 鲁国东, 周而勋, 郭泽建, 黄世文, 潘学彪

  植物病理学报. 2009, 39(5): 514-520.

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  经过近十年的病原菌接种试验,从国内外数百份水稻种质中,筛选出了抗病水平不同的5个代表品种Lemont、武育粳3号、Jasmine85、C418和YSBR1,作为水稻纹枯病菌致病力的鉴别品种。从江苏、广东、广西、海南、云南、湖南、福建等省采集、分离的水稻纹枯病菌中,初步筛选出致病力有差异的30个菌株,于温室苗期接种上述5个品种,确定了不同致病力的5个代表菌株C30、GD-118、E67、YN-7和YN-3。将选出的品种和菌株在江苏扬州和浙江富阳进行大田成株期抗性和致病力分析,结果表明:品种的抗病性和菌株的致病力都存在极显著差异,分别属于5个和3个不同的显著性级别,均可暂时作为鉴定材料使用。据以上结果,初步建立了一套可供水稻纹枯病菌致病力检测和寄主抗病性鉴定的鉴别体系。本文还讨论了建立水稻纹枯病鉴别体系的必要性、可行性和需要进一步开展的工作。
植物病害及其防治
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  邻烯丙基苯酚对番茄灰霉病菌全细胞呼吸的影响

  龚双军, 刘西莉, 陈长军, 王建新, 孟昭礼, 李健强

  植物病理学报. 2009, 39(5): 521-527.

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  利用Oxygraphy液相氧电极研究了植物源杀菌剂邻烯丙基苯酚对番茄灰霉病菌(Botrytis cinerea)全细胞呼吸的影响。结果显示,邻烯丙基苯酚处理浓度为5、10 mg/L,菌丝细胞的呼吸速率较对照分别增加了19.58%和24.56%;处理浓度为150 mg/L,可显著抑制菌丝细胞的呼吸,抑制率达到77.49%,表明邻烯丙基苯酚具有低浓度刺激而高浓度抑制灰霉病菌细胞呼吸的双重影响。用药剂处理萌芽期、非萌芽期的分生孢子,测定呼吸的结果表明,药剂对孢子的呼吸作用效果与对菌丝的相似,但以萌芽期分生孢子最为敏感。以0.01 mol/L琥珀酸钠作为病菌细胞呼吸底物时,10 mg/L邻烯丙基苯酚对病菌细胞的呼吸速率具有抑制作用,而添加0.1 mol/L葡萄糖和0.01 mol/L丙酮酸钠两种底物时却表现出刺激病菌细胞呼吸的作用,表明邻烯丙基苯酚对细胞呼吸的影响和氧化底物密切相关。反应液中单独添加呼吸电子传递链特异性位点抑制剂鱼藤酮20μmol/L、嘧菌酯10 mg/L和叠氮化钠0.01 mol/L后,病菌菌丝细胞呼吸均受到显著抑制,抑制率分别为48.94%、33.08%和39.18%。随后再添加10 mg/L邻烯丙基苯酚,不改变这种抑制效果。单独添加旁路氧化酶抑制剂水杨肟酸2 mM不影响病菌菌丝的呼吸,随后再添加10 mg/L邻烯丙基苯酚后,两者协同作用则可显著抑制菌丝的呼吸。
研究简报
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  番茄叶片漆腐病病原菌鉴定

  赵彦杰, 李宝聚, 石延霞, 谢学文

  植物病理学报. 2009, 39(5): 528-531.

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  A new leave disease of tomato (Lycopersicon esculentum Miller) was observed in polyethylene film-covered greenhouse in Beijing, China. This disease spreaded rapidly in the greenhouse and caused serious loss of the production of tomatoes. In the study, a fungal strain isolated from the lesion was confirmed to be pathogen for this new disease, and identified as Myrothecium roridum Tode ex Ft. based on the morphology, cultural characters on PDA plate and sequence analysis of ribosomal DNA-ITS. This is the first report of myrothecium leaf spot on tomato occurring in commercial greenhouse in China.
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  川芎茵核病的症状及病原菌鉴定

  罗玲, 黄云, 王靖, 何苗, 万英

  植物病理学报. 2009, 39(5): 532-535.

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  Symptoms of sclerotinia stem rot of Ligusticum chuanxiong were observed and the causal agent of the disease was identified. The results showed that the symptoms of this disease began with the formation of dark brown hygrophanous splotch on the basal leaves, followed by the appearance of dark brown circular spot on the basal stem. Finally, the whole plant stoped growing and died. White mycelium and/or sclerotia were formed under moisturized conditions. On PDA medium, the pathogen of this disease formed brown sclerotia with various shapes and 2-5 mm in diameter. The diameter of apothecia was 0.3-0.8 cm and the length of stipes was 2-8 cm. Asci were columniform in shape and (100-135)×9μm in size, each containing 8 ascospores sized by (6-12) μm×(3-7)μm. No asexual spore formation was observed. Based on the morphological characteristics and the sequence of ribosomal DNAITS, the pathogen was identified as Sclerotinia sclerotiorum (Lib.) de Bary.
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  苹果轮纹病菌诱导产孢方法

  冷伟锋, 李保华, 国立耘, 董娟华, 王彩霞, 李桂舫, 董向丽

  植物病理学报. 2009, 39(5): 536-539.

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  In order to get a great quantity of spores of Botryosphaeria berengeriana f. sp. piricola, several methods to promote sporulation of the pathogen were compared. The results showed that the wounded young apple fruits within 60-day old was inoculated with mycelia of B. berengeriana f. sp. piricola and induced under the 365 nm-UV-light; when the brown lesion was shown, the young apple began to form pycnidia on the lesion around the inoculation point after 3-5 days irradiation with UV-light and extruded plenty of conidia. This method induced great more conidia than that of irradiating wounded mycelia with UV-light. However, mature apple seldom produced conidia when induced with the same method.
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  来自安徽不同寄主PVY分离物的血清学鉴定及其cp基因分子进化分析

  江彤, 陈伟

  植物病理学报. 2009, 39(5): 540-543.

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  Tobacco and potato samples showing symptoms of PVY were collected from different regions in Anhui Province, and the ELISA results of partial samples were positive. The total RNA was extracted from the positive samples by TRIZOL methods. Specific primer pair was designed to amplify cp gene of PVY by RT-PCR. Sequencing results indicated that the full length of cp gene of PVY from tobacco (PVY-CP-4) and pota-to (PVY-CP-7) is 801 nts, and each of them encodes 266 amino acids. A phylogenetic tree based on alignment of cp nucleotide sequences was constructed and the sequence comparing of cp gene was conducted. The results showed that PVY-CP-4 could be grouped into one branch with PVY^O and PVY^N:O and shared the highest sequence similarity (99.4%) with PVY^O (EF026074). It was suggested that PVY-CP-4 derived from tobacco in Hefei might belong to PVY^O. PVY-CP-7 clustered together with PVYN and PVYNTN and formed another branch. Furthermore, PVY-CP-7 shared the highest sequence similarity (98.3%) with PVY^NTN(AJ890347), PVY^NTN (EF026075) and PVY^NTN(AJ585342). It was supposed that PVY-CP-7 derived from potato in Wuhe probably belonged to PVY^NTN.
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  小西葫芦黄花叶病毒山东南瓜分离物的分子特性

  刘金亮, 邵云华, 张广民, 竺晓平, 杨广玲, 时呈奎, 李向东

  植物病理学报. 2009, 39(5): 544-548.

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  Zucchini yellow mosaic virus (ZYMV) was detected by RT-PCR from pumpkin (Cucurbita moschata) plant showing yellowing and mosaic symptom from Liaocheng, Shandong Province. The 3'-termial 1 684 bp genomic sequence covered 633 bp of NIb encoding sequence, 840 bp of cp gene and 211 bp of 3'-untranslated region of the isolate ZYMV-Liaocheng was determined. The cp gene of ZYMV-Liaocheng shared identities of 81.4%-98.8% and 89.4%-99.5% at nucleotide and amino acid levels, respectively, with other ZYMV sequences available in the GenBank. Phylogenetic analysis indicated that ZYMV could be clustered to 6 genotypes. ZYMV-Liaocheng belonged to genotypeⅠ, which contained isolates from Asia, Europe and America. Genotypes Ⅲ and Ⅴ were unique and contained only isolates from East Asia. The isolates from East Asia had the highest variability.
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  防治苹果树腐烂病杀菌剂的室内筛选

  王磊, 郜佐鹏, 黄丽丽, 韦洁玲, 臧睿, 康振生

  植物病理学报. 2009, 39(5): 549-554.

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  The inhibitory effects of 10 fungicides on apple tree valsa canker were compared on dishes and twigs. The results showed that conidia germination and mycelial growth were inhibited by all fungicedes tested. The EC₅₀ value of Difenoconazole was 6. 1×10^-3μg·mL^-1, the lowest among tested fungicide in inhibiting conidia germination. Tebuconazole and Imazalil also showed obvious inhibition effect. Thiophanatemethyl was the least efficient with the EC₅₀ value 2.6×10¹ μg·mL^-1. Difenoconazole also showed the highest activity for inhibiting mycelial growth with ECho value of 2.3×10^-2 μg·mL^-1. Tebuconazole was better than other fungicide. Thiophanate-methyl and Propineb were the least efficient with quite high EC₅₀ value. Furthermore, the size of the lesion after inoculation on excised twigs also revealed that Difenoconazole was most efficient because it showed the smallest lesion of 394 mm² compared with other tested fungicides. So Difenoconazole and Tebuconazole could be used to control apple tree valsa canker in field to subsititute forbided fungicides such as asomate.
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  土壤处理对西洋参根际线虫数量动态的影响及对防治茎线虫根腐病的效果

  张雪松, 张国珍, 张海旺, 彭东文, 杜治俭, 唐春艳

  植物病理学报. 2009, 39(5): 555-560.

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  Root rot of American ginseng (Panax quinquefolium) caused by Ditylenchus destructor is a novel disease found recently in Beijing area. The effects of three soil treatments (fumigating with chloropicrin, chloropicrin + lvfeng organic manure and nematicide fosthiazate) on the number of rhizospheric nematodes, survived plants, root yield and root rot of 3-year ginseng plant were compared. The effects were also investigated at the second year after treatment. The results indicated that the number of parasitic nematodes in rhizosphere of treated soil reached the peak value in late June to early July as the soil temperature raised in the growing season. Compared with the regular treatment, the number of plant parasitic nematodes was reduced while non-plant parasitic nematodes increased. The number of non-plant parasitic nematodes in the soil treated with chloropicrin + lvfeng organic manure was 2 times than that treated with chloropicrin only. The ratio of non-parasitic to parasitic nematodes of three treatments was higher than the control. Percent of survived plants was 94.8%-96.4% and diseased root was decreased obviously. The control efficacy was more than 89% at the first year after treatment. The survived plants and plot yield of ginseng increased significantly and the control efficacy was around 40% at the second year. The best of the three treatments was by chloropicrin + organic manure.