瓜黑星病菌、枯萎病菌和蔓枯病菌的三重PCR检测

通过测定黄瓜黑星病菌(Cladosporium cucumerinum)rDNA的ITS序列,比对近缘种及瓜类几种重要病原菌的ITS序列,设计出特异性引物HX-1/HX-2,经过对引物HX-1/HX-2PCR条件的优化,可以扩增出1条190bp的黄瓜黑星病菌特异性DNA条带,灵敏度达到1pg/μL。进一步将引物HX-1/HX-2和瓜类枯萎病菌、瓜类蔓枯病菌特异检测引物Fn-1/Fn-2、Mn-1/Mn-2组合,建立三重PCR体系,可一次检测出瓜类黑星病菌、瓜类枯萎病菌、瓜类蔓枯病菌3种瓜类植物重要的病原菌。建立了可以应用于田间瓜类黑星病菌PCR检测技术和瓜类主要病害三重PCR检测技术,对瓜类病害的诊断和防治具有重要的指导作用。     

番石榴焦腐病菌的ITS分析及PCR检测

番石榴焦腐病是台湾入境大陆水果的重要植物病害,由葡萄座腔菌Botryosphaeria rhodina引起.为建立该病原菌快速、灵敏的检测技术,比较分析了葡萄座腔菌和葡萄座腔菌属其它种的ITS序列,在此基础上设计了1对检测番石榴焦腐病菌的特异性引物BF1/BR1,利用此引物从葡萄座腔菌中特异性扩增出287bp条带,而其余参试的菌株未能获得扩增条带.将真菌通用引物ITS1/ITS4和BF1/BR1进行巢式PCR扩增后,检测灵敏度提高1 000倍,可检测到葡萄座腔菌1pg的基因组DNA.结合快速碱裂解法提取发病组织的DNA,采用该PCR检测技术可从自然感染焦腐病果实中检测到葡萄座腔菌.     

利用双重PCR技术快速检测水稻细菌性谷枯病菌

根据水稻细菌性谷枯病ITS和gyrB基因,设计两对特异性PCR检测引物,建立了水稻细菌性谷枯病菌的双重PCR检测方法。用该方法对水稻细菌性谷枯病菌和其它植物源性细菌进行双重PCR扩增及灵敏度测试,并对采自不同地区的水稻样本进行水稻细菌性谷枯病菌的检测。结果显示,双重PCR方法能特异性地检测出8株水稻细菌性谷枯病菌,可从含水稻细菌性谷枯病菌浓度为102cfu/mL的菌液中检测出该病菌;采用该方法对我国不同地区的水稻材料进行检测,并未发现水稻细菌性谷枯病菌。     

基于TaqMan MGB探针的葡萄茎枯病菌实时荧光PCR检测方法

 葡萄茎枯病菌是我国进境植物检疫性有害生物。带菌植物材料是病害传播的重要载体,准确、灵敏、快速的检测方法是严格执行口岸检疫措施及研究病害防控措施的有力工具。根据葡萄茎枯病菌及其近似种的细胞骨架蛋白(Actin)基因序列差异,设计并合成1对引物和1条特异性TaqMan-MGB探针,建立了葡萄茎枯病菌的实时荧光PCR检测方法。通过对反应体系的优化,确定了葡萄茎枯病菌的实时荧光PCR最佳反应条件:引物终浓度为0.6 μmol·L^-1,探针终浓度为0.6 μmol·L^-1。灵敏度试验结果显示,最低检测限为总DNA含量20 pg(20 μL反应体系)。此方法快速灵敏,整个反应1 h即可完成,检测过程完全闭管,无需PCR产物后续处理,为快速检测葡萄茎枯病菌提供了重要参考。该方法用于口岸疑似菌株检测,可成功检测出葡萄茎枯病菌。本研究建立的基于TaqMan MGB探针的荧光定量PCR检测方法为葡萄茎枯病菌的早期快速检测监测提供了有力工具。     

芦笋茎枯病菌的鉴定及区域差异性分析

芦笋茎枯病是一种世界性分布的毁灭性病害,为准确鉴定其致病菌和探明不同区域菌株的分化程度,通过形态学观察和核糖体DNA内转录间区(rDNA ITS)序列比对进行形态学和分子生物学鉴定,比较五省份菌株的生物学特性和ITS序列上的差异,并进行系统发育分析。结果表明,芦笋茎枯病的致病菌为天门冬拟茎点霉Phomopsis asparagi。海南省菌株的菌丝生长较快,培养5天后的平均直径为8.5 cm;培养14天后,江西省菌株由白色变为淡绿色,其它各省菌株由白色变为灰白色,海南省菌株的菌落呈现同心轮纹状;福建省菌株产生的分生孢子器较多,平均60个/皿;五省菌株在1～238 bp和298～591 bp的ITS区段存在差异性碱基,其中河北省菌株的差异性碱基数最多;五省菌株大致聚为2个组群,河北省菌株单独聚为1个组群,其它省份菌株聚为1个组群,天门冬拟茎点霉P.asparagi与叶下珠生拟茎点霉P.phyllanthicola亲缘关系较近。     

基于TaqMan MGB探针的葡萄茎枯病菌实时荧光PCR检测方法

 葡萄茎枯病菌是我国进境植物检疫性有害生物。带菌植物材料是病害传播的重要载体,准确、灵敏、快速的检测方法是严格执行口岸检疫措施及研究病害防控措施的有力工具。根据葡萄茎枯病菌及其近似种的细胞骨架蛋白(Actin)基因序列差异,设计并合成1对引物和1条特异性TaqMan-MGB探针,建立了葡萄茎枯病菌的实时荧光PCR检测方法。通过对反应体系的优化,确定了葡萄茎枯病菌的实时荧光PCR最佳反应条件:引物终浓度为0.6 μmol·L^-1,探针终浓度为0.6 μmol·L^-1。灵敏度试验结果显示,最低检测限为总DNA含量20 pg(20 μL反应体系)。此方法快速灵敏,整个反应1 h即可完成,检测过程完全闭管,无需PCR产物后续处理,为快速检测葡萄茎枯病菌提供了重要参考。该方法用于口岸疑似菌株检测,可成功检测出葡萄茎枯病菌。本研究建立的基于TaqMan MGB探针的荧光定量PCR检测方法为葡萄茎枯病菌的早期快速检测监测提供了有力工具。     

基于小麦白粉病菌rDNA ITS序列的PCR分子检测

 Wheat powdery mildew(Blumeria graminis f.sp.tritici) is the one of main wheat diseases in China.Based on the internal transcribed spacer(ITS) sequences of ribosome of B.graminis f.sp.tritici,three molecular primer pairs(F1/R,F2/R and F3/R) were designed to detect the fungal pathogen of wheat powdery mildew.The species specificity of these primers was confirmed.F1/R was demonstrated a higher sensitivity than the other two primer pairs,and could detect as low as 1 pg DNA of B.graminis f.sp.tritici.Furthermore,F1/R primer pair was used to detect the pathogen DNA extracted from wheat leaves showing chlorosis and typical symptoms of powdery mildew caused by artificial inoculation with B.graminis f.sp.tritici.The preliminary results demonstrated the usefulness of this primer pair and its potential applications in efficient detection of wheat powdery mildew pathogen from leaves with latent infections at early growth stages of wheat.     

水稻细菌性谷枯病菌和水稻白叶枯病菌荧光RPA快速检测方法的建立

本研究分别针对我国进境植物检疫性有害生物水稻细菌性谷枯病菌及水稻白叶枯病菌建立了光RPA检测方法,并对其灵敏度、特异性以及对实际种子样本的检测能力进行了测试评价。结果表明,本研究建立的两种方法均能够在20 min内特异性检测到目标菌株,对水稻细菌性谷枯病菌(Burkholderia glumae)菌液的检测下限达到了11.4 CFU/反应,DNA检测下限达到了4.83×10^-5ng/反应;对水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae)菌液的检测下限达到了4.42 CFU/反应,DNA检测下限达到了3.83×10^-4ng/反应。两种方法均能够成功应用于带菌种子样品的快速检测。     

烟草根黑腐病菌的PCR分子检测

 根据烟草根黑腐病菌(Thielaviopsis basicola)与其它烟草病原真菌核糖体基因转录间隔区(internal transcribed spa-cer,ITS)序列间的差异,设计了一对特异性引物TB-5/TB-3,用于T. basicola的分子检测。利用该对引物对包括T. basicola在内的13个烟草病原菌菌株的基因组DNA进行PCR扩增,结果表明:只有T. basicola能扩增到一条400bp左右的特异性条带,其它菌株及阴性对照均无扩增产物。对烟草组织和土壤的检测结果也表明,该对引物能特异性的检测到T. basicola基因组DNA的存在。该引物对T. basicola基因组DNA检测的灵敏度为100fg/μL。     

鲜食马铃薯中青枯病菌的检测

在口岸送检的鲜食马铃薯上分离到一株细菌分离物0297,对该分离物在薯块上的为害症状和菌落形态特征进行观察,并经特异引物PCR检测、多位点序列分析、致病性测定.结果表明:患病薯块切面维管束变褐,按压分泌乳白色菌脓,分离物菌落在NA培养基上呈有光泽的乳白色;青枯病菌特异引物RS32/RS37扩增分离物0297得到预期583...     

双重PCR检测马铃薯晚疫病和环腐病方法的建立

通过克隆马铃薯环腐病菌和晚疫病菌转录间隔区(ITS)序列,并对测序结果进行同源性比较,选取差异位点分别设计了两对引物P.IN1/P.IN2和C.IN1/C.IN2,并检测了引物的特异性及方法的灵敏度。引物P.IN1/P.IN2可扩增出1条363bp马铃薯晚疫病菌的特异性条带,在DNA水平上其灵敏度达18fg/μL;引物C.IN1/C.IN2可扩增出1条218bp马铃薯环腐病菌的特异性条带,在细菌数上检测灵敏度为104 cfu/mL。混合这两对引物构建双重PCR反应体系,能从马铃薯环腐病菌和晚疫病菌的混合DNA及感染这两种菌的马铃薯植株中同时扩增到363bp和218bp的特异片段。实现了同时对马铃薯晚疫病菌和环腐病菌的快速可靠检测。     

密执安棒形杆菌的DNA条形码基因筛选与评价

密执安棒形杆菌是一种重要的植物病原细菌,其包含了能引起不同植物病害的5个亚种。近年来迅速发展起的DNA条形码技术给植物病原细菌快速准确的检测鉴定提供了新思路,本文对棒形杆菌属的4条候选DNA条形码基因进行PCR扩增测序,并从扩增及测序成功率、种内及种间遗传距离、barcoding gap及NJ树等几个方面进行比较,结果表明gyr B基因最适合进行该属内种及亚种的区分,cpn60基因可作为该属分类鉴定的有效补充。     

辽宁西瓜衰萎病的病原菌鉴定

近年来,辽宁省新民市西瓜上发生一种新病害,病株根系衰弱、根毛数量减少,根部表皮粗糙,韧皮部木栓化,成软木塞状.随病情发展,植株叶 片自茎基部向顶部逐渐褪绿、变黄、枯萎,重病植株逐渐衰败、凋萎、死亡,故称之西瓜衰萎病.国际上,许多国家有关于西瓜、甜瓜的衰萎病发生的报道[1-5],而我国未见相关研究报道.在辽宁省西瓜产区田间调查发现,该病发生普遍,造成不同程度产量损失,重病田发病率高达30％,且有逐年加重趋势.据此,作者采集病害植株根部组织,分离获得病原菌,通过柯赫氏法则证病试验、病菌培养性状观察以及病菌rDNA ITS序列分析等试验,旨在明确该病害的症状、病原菌种类,为病害的防治提供理论依据.     

Real-time PCR for detection and quantification of Erwinia amylovora, the causal agent of fireblight

Real-time PCR was used for quantitative detection of Erwinia amylovora , the causative agent of fireblight. Specific primers were created from a DNA fragment of the common plasmid pEA29, successfully used for standard PCR identification of the pathogen. The primers amplified DNA from various E. amylovora strains, but not from other plant-associated bacteria. DNA of E. amylovora was also amplified from field samples and from inoculated apple leaves or flowers. Neither the presence of other bacteria nor low amounts of tissue extracts from bark or leaves changed the signal threshold. Assays with SYBR Green I instead of the Taqman probe showed a similar sensitivity, detecting 50 cells per assay. Real-time PCR could be especially useful for mass screening of commercial products and for resistance studies of transgenic host plants, in breeding experiments and after treatments to control fireblight.     

Comparison of PCR, BIO-PCR, DIA, ELISA and isolation on semiselective medium for detection of Xanthomonas albilineans, the causal agent of leaf scald of sugarcane

Polymerase chain reaction (PCR) and newly designed primers, XAF1/XAR1, were tested for selective detection of the causal agent of leaf scald of sugarcane, Xanthomonas albilineans . The efficiency and reliability of PCR were compared with dot immunobinding assay (DIA), ELISA and classical isolation techniques for detecting X. albilineans in suspensions of pure cells and extracts of field-collected stalk and leaf samples of sugarcane. In addition, classical PCR and BIO-PCR (biological amplification followed by PCR) were compared with isolation on a semiselective agar medium. Classical PCR and BIO-PCR techniques had the advantage of not requiring pathogenicity tests to confirm the identity of colonies tentatively identified as X. albilineans on modified semiselective XAM agar medium. The m-XAM medium and BIO-PCR techniques were the most sensitive; however, the former required seven days whereas the latter required only four days. The BIO-PCR technique was as sensitive as the semiselective medium technique and eliminated the need to conduct any additional tests to confirm the identification.     

Antagonistic effect of soil rhizosphere microorganisms on Bipolaris sorokiniana,the causal agent of wheat seedling blight

The biocontrol efficiency of Epicoccum purpurascens, Gliocladium roseum, three strains of Bacillus subtilis, and Pseudomonas fluorescens, isolated from the rhizosphere of wheat plants, was assessed in relation to seedling blight caused by Bipolaris sorokiniana. An in vitro study of the potential antagonist was performed using the dual culture technique and by ‘sowing’ wheat seeds pelleted with the saprophytes in plates with water agar?+?the pathogen. In vivo assays were carried out in the greenhouse and in the field with pelleted seeds sown in artificially infested soil. Both the number of living plants and the number of plants with necrosis on the leaves and the base of the stems and roots were assessed 15 days after sowing. Under greenhouse conditions, B. subtilis 3 and G. roseum reduced the level of infection of Buck Pucará and Trigomax 100 cultivars, respectively. In the field, biocontrol of the disease was not achieved.     

国外引进甘蔗材料白叶病植原体巢式PCR检测及其序列分析

为有效防止引起重要危险性检疫病害甘蔗白叶病(SCWL)的植原体病原随引进甘蔗材料侵入我国,确保我国甘蔗生产安全,本研究对从缅甸、菲律宾、法国和泰国引进的22个甘蔗材料进行了SCWL植原体巢式PCR检测,并对阳性样品的巢式PCR产物进行了测序及序列分析。结果表明,18个甘蔗材料呈SCWL植原体阳性,阳性检出率为81.8%。所有SCWL阳性样品的16S-23S基因间隔区片段长210bp,与GenBank中已有的其他SCWL植原体分离物(登录号HQ917068、AB646271)的同源性为99.8%~100%,并在系统发育树中聚为一个类群。根据SCWL的巢氏PCR检测及其序列分析结果,对呈阳性的材料及时进行了集中销毁处理。     

Sensitive and specific detection of Clavibacter xyli subsp. xyli, causal agent of ratoon stunting disease of sugarcane, with a polymerase chain reaction-based assay

A sensitive, specific polymerase chain reaction-based assay was developed for the detection of the causal agent of ratoon stunting disease of sugarcane, Clavibacter xyli subsp. xyli . This assay uses oligonucleotide primers derived from the internal transcribed spacer region between the 16S and 23S rRNA genes of the bacterial rRNA operon. The assay is specific for C. xyli subsp. xyli and does not produce an amplification product from the template of the closely related bacterium C. xyli subsp. cynodontis , nor from other bacterial species. The assay was successfully applied to the detection of C. xyli subsp. xyli in fibrovascular fluid extracted from sugarcane and was sensitive to approximately 22 cells per PCR assay. A multiplex PCR test was also developed which identified and differentiated C. xyli subsp. xyli and C. xyli subsp. cynodontis in a single PCR assay.     

茎基腐亚洲镰孢菌侵染抗感小麦品种的组织病理学观察

小麦茎基腐是由多种镰孢菌侵染的世界性土传病害,亚洲镰孢菌(Fusarium asiaticum)是我国冬小麦主产区茎基腐镰孢菌的优势种群,对小麦生产造成巨大损失。本研究利用绿色荧光蛋白报告基因标记亚洲镰孢菌,研究其侵染抗感小麦的病理组织学过程,建立了茎基腐病菌与寄主互作的直观性的研究体系,对病害防治及抗病育种具有重要意义。基于PEG-CaCl_2介导原生质体转化法将gfp导入亚洲镰孢菌株CF0915,对转化子进行荧光表达、PCR验证、遗传稳定性、生长特性及致病力分析,选取与野生型表现相近的转化子进行侵染分析。结果表明,绿色荧光蛋白基因(gfp)与潮霉素基因(hyg)PCR扩增表明gfp已整合入真菌基因组中,转化子菌丝与分生孢子表现强烈绿色荧光信号,gfp能够在转化子中稳定遗传,菌落形态、生长速度及致病力与野生型菌株无显著差异;将gfp标记病菌分生孢子接种感病品种1 d后,大量孢子附着于根毛及根表皮细胞开始萌发,接种2 d后观察到抗性品种分生孢子萌发;感病品种接种3 d后,菌丝直接侵入表皮细胞或沿表皮细胞间层定殖生长,扩展至皮层组织,8 d后菌丝从根部迅速扩展至茎基部,至第10 d大量菌丝充塞根皮层细胞,叶鞘维管束也被菌丝侵染,并产生大量大型分生孢子,植株表现褐色病斑,14 d后根部及茎维管束被大量菌丝体填充,而后产生大量厚垣孢子,至25 d大部分感病品种幼苗萎蔫死亡;与感病品种相比,抗性品种在整个侵染过程中表现时间滞后。本研究对引起茎基腐病的亚洲镰孢菌侵染小麦的组织学过程观察,为病菌致病机理的阐释及抗病资源的利用提供了重要理论依据。