Range of phytoplasma concentrations in various plant hosts as determined by competitive polymerase chain reaction

ABSTRACT For competitive polymerase chain reaction (PCR), an internal standard DNA template was developed that consisted of a highly conserved, internally deleted 16S rDNA fragment of an aster yellows phytoplasma. The internal standard was calibrated using a quantified culture of Acholeplasma laidlawii. Serial dilutions of the internal standard and fixed amounts of target templates from infected plants were coamplified with the same primers, and the products obtained were quantified using an enzyme-linked immunosorbent assay procedure. Analysis of the data revealed that the phytoplasma concentration in the plants examined differed by a factor of about 4 x 10(6). Phytoplasma concentrations of 2.2 x 10(8) to 1.5 x 10(9) cells per g of tissue were identified in periwinkles infected with various phytoplasmas. High to moderate concentrations were detected in Malus domestica (apple) genotypes infected with the apple proliferation phytoplasma, Alnus glutinosa (alder) genotypes infected with the alder yellows phytoplasma, and most aster yellows-infected Populus (poplar) genotypes examined. Very low phytoplasma concentrations, ranging from 370 to 34,000 cells per g of tissue, were identified in proliferation-diseased apple trees on resistant rootstocks 4551 and 4608, yellows-diseased Quercus robur (oak) trees, and Carpinus betulus (hornbeam) trees. Such low concentrations, which corresponded to about 4 to 340 cells in the reaction mixture, could only be detected and quantified by nested PCR.     

Detection and identification of Phytophthora fragariae Hickman by the polymerase chain reaction

Phytophthora fragariae Hickman, which causes strawberry red stele and raspberry root rot, is a quarantine organism for which specific and sensitive detection methods are required to test the health of planting material. Sequences of the internal transcribed spacer regions of the ribosomal gene repeat (rDNA) were used to develop primers for P. fragariae in a nested Polymerase Chain Reaction (PCR). The fungus was readily detected in infected but symptomless roots by nested, but not single-round, PCR. It was also detected in infested water samples obtained from the Dutch General Inspection Service by nested PCR. Detection of PCR products was at least 10-fold more sensitive by PCR-ELISA than by conventional visualisation on agarose gels.     

Detection of tobacco rattle virus in nematodes by reverse transcription and polymerase chain reaction

An assay, based on amplification of cDNA synthesized from genomic viral RNA, has been developed to detect tobacco rattle virus in infected plant material and viruliferous nematodes. A range of different TRV strains could be detected using the procedure developed. The presence of one to three viruliferous nematodes in a nematode suspension was sufficient for the detection of TRV. The minimum amount of purified virus detectable in the assay was 15 fg, indicating an increased sensitivity of the PCR-based assay as compared to serological detection methods, like ELISA. A dot-blot hybridization procedure was developed for the detection of the PCR products, making agarose gel electrophoresis dispensable.     

实时荧光RT-PCR方法检测香石竹环斑病毒

 本研究以香石竹环斑病毒(Carnation ringspot virus, CRSV)的5个分离物为研究对象,根据CRSV运动蛋白（MP）基因的保守序列设计一对特异性引物和TaqMan荧光探针,建立了检测CRSV 的实时荧光RT-PCR（real-time fluorescent RT-PCR）方法。该方法利用TaqMan探针水解产生的荧光信号实时监测目标基因的扩增,实现real-time fluorescent PCR扩增和检测同步进行。结果表明,本研究建立的实时荧光RT-PCR方法具有更快速、灵敏和特异的优点,与普通RT-PCR方法相比其灵敏度提高了100倍,适合于对进境种苗携带的CRSV的快速检测。     

香蕉黑腐病菌(Botryodiplodia theobromae)的PCR检测

 根据香蕉黑腐病菌可可球二孢菌(Botryodiplodia theobromae)与其它香蕉病原真菌核糖体基因转录间隔区(rDNA-ITS)ITS1和ITS2间序列差异,设计了特异引物Bth-S(5'-TCTCCCACCCTTTGTGAAC-3')和Bth-A(5'-AAAAGT-TCAGAAGGTTCGTC-3'),利用此引物对包括可可球二孢菌在内的21个菌株基因组DNA进行PCR扩增,结果只有4个可可球二孢菌菌株扩增到422bp特异带,其它17个菌株无扩增产物。灵敏度测试结果表明此特异引物能对1pg的可可球二孢菌基因组DNA进行扩增。对自然感染黑腐病的香蕉果实组织和接种可可球二孢菌或多种香蕉病原真菌混合接种的果实组织进行检测,Bth-S和Bth-A引物对不仅能够在自然感染黑腐病果实组织中特异检测到可可球二孢菌,而且能在未显症和发病的接菌香蕉果实组织中特异检测得到可可球二孢菌。这为香蕉可可球二孢菌潜伏侵染检测提供了技术支持。     

人工培养液在植原体昆虫介体筛选中的适用性

研究了一种人工培养液对各种常见的昆虫(主要是叶蝉)的亲和性和适用性.结果表明,该人工培养液适于本试验中大多数昆虫的人工饲养.用此方法,悬钩子广头叶蝉Macropsis.ftscula Zetterstedt和桤树广头叶蝉Oncopsis alniSchrank分别被再次确认为悬钩子矮化植原体和桤树黄化植原体的传播介体;田旋花麦蜡蝉Hyalesthes obsoletus Signoret再次被确认为葡萄黄化(stolbur)植原体的传播介体.此前,上述三种叶蝉已被传统的人工接种方法鉴定为相应植原体的传播介体.危害桤树的河谷树叶蝉Allygus modestus Scott尽管虫体DNA检测结果经常为阳性,但迄今其人工培养液的检测结果都是阴性,因此,我们认为河谷树叶蝉不是桤树黄化植原体的传播介体.Eppendorf管人工培养液饲养法不仅适用于潜在的植原体介体昆虫的筛选鉴定,而且可用于介体昆虫的生物防治研究.此外,本研究首次发现自然感染了葡萄上的一种被德国人称为"Vergi-lungskrankheit"植原体(AY组)的草地脊冠叶蝉Aprodes makarovi Zachvatkin.     

大豆携带菜豆晕疫病菌PCR检测技术研究

晕疫病是豆科作物上重要的检疫性细菌病害。本研究在传统 PCR 检测技术的基础上,分析了细菌洗涤液、洗涤时间以及洗涤液容量对一粒带菌大豆检出率的影响,以及有效检出不同带菌率大豆的最佳检测时间。结果表明,洗涤液种类、洗涤时间以及洗涤液容量对检测结果影响不大,但对不同带菌率大豆洗涤时间的长短直接影响病菌的检出率,实验表明振荡洗涤时间8 h,可检测到的大豆种子最低带菌率为0.0625%。本研究所建立的检测方法简便易行,可有效应用于口岸检测进口大豆中的菜豆晕疫病菌。     

Detection of Fusarium oxysporum f.sp. vasinfectum in cotton tissue by polymerase chain reaction

A polymerase chain reaction assay was developed for the detection of Fusarium oxysporum f.sp. vasinfectum (FOV), a serious wilt pathogen of cotton in many parts of the world. Based on small nucleotide differences in internal transcribed spacer sequences between 18S, 5.8S and 28S ribosomal DNAs, primers Fov1 (5'-CCCCTGTGAACATACCTTACT-3') and Fov 2 (5'-ACCAGTAACGAGGGTTTTACT-3') were selected. These primers unambiguously amplified a 400-bp DNA fragment of all the FOV isolates tested (from Angola, Brazil, China and the USA) but did not amplify any other isolates of mycoflora associated with cotton, such as F. moniliforme , Verticillium albo-atrum , V. dahliae , Aspergillus sp., F. oxysporum , F. sambucinum or F. solani . A control PCR assay was developed employing the universal primer pair ITS1 and ITS2 which amplified a fragment of approximately 220 bp from all isolates tested. This control assay demonstrated that all fungal DNAs were readily amplifiable, thus confirming that the lack of amplification with Fov1 and Fov2 primers was a result of primer specificity and not of other possible causes, such as DNA degradation or the presence of PCR inhibitors. The assay was effective on samples from the stems, leaves, roots and calli, and from plant tissues both with and without symptoms. This detection system proved to be accurate and sensitive and could aid not only diagnosis but also disease monitoring and forecasting.     

Detection of Xylophilus ampelinus in grapevine cuttings using a nested polymerase chain reaction

A sensitive and specific assay was developed to detect bacterial blight of grapevine caused by Xylophilus ampelinus (Panagopoulos, 1969) comb. nov. in grapevine cuttings. The 16S−23S rDNA intergenic spacer region of X. ampelinus was sequenced and pathogen-specific primers were designed from a region in the spacer between the tRNA (Ala) and the 23S genes. A nested PCR (n-PCR) reaction was applied with a first-stage PCR using universal primers within the ends of the 16S and 23S genes, followed by a second-stage PCR with nested primers specific to the X. ampelinus spacer region. A 277-bp fragment was amplified from 38 Xylophilus strains tested, but not from saprophytes associated with grapevine or phylogenetically related phytobacteria. The 277-bp product was shown to be derived from the X. ampelinus spacer region by restriction with Dra I, Sau 3AI, Taq I and Msp I, Southern hybridization and genomic DNA dot blots. When the (n-PCR) procedure was applied in the absence of nontarget DNA, the limit of detection was less than 10 colony-forming units (CFU) per µ L. The same number of  X. ampelinus CFU could be detected in the presence of 1·5 × 10⁵ CFU  µ L⁻¹ of Erwinia herbicola cells using the n-PCR procedure.     

Detection of the mycoplasma-like organism associated with lethal yellowing disease of palms in Florida by polymerase chain reaction

DNA amplification by polymerase chain reaction (PCR) was used specifically to detect the mycoplasma-like organism (MLO) associated with lethal yellowing disease of palms in Florida. For PCR, a pair of oligonucleotide primers was synthesized according to partial sequences of a cloned 1·3 kbp fragment of lethal yellowing MLO-specific genomic DNA isolated from a diseased windmill palm ( Trachycarpus fortunei ). A DNA product of about 1 kbp was specifically amplified by PCR in reaction mixtures containing template DNA derived from either heart, inflorescence or leaf tissues of lethal yellowing-affected palms. PCR performed for 35 cycles with as little as 5 pg of DNA template, in some instances, was sufficient consistently to amplify the same lethal yellowing MLO DNA product from hearts of 11 species comprising 30 symptomatic palms. Similar reliable and reproducible detection of the lethal yellowing MLO in palm inflorescence spikelets was also achieved after 35 cycles of PCR. When template DNA for PCR was derived from tissues of the the most immature emerging leaf, a 40-cycle reaction was sufficient for consistent foliar detection of the pathogen in all coconut palms including palms with earliest visible symptoms of disease.     

Detection and characterization of plum pox potyvirus isolates from southern Germany by polymerase chain reaction (PCR)1

Plum pox potyvirus (PPV) isolates show quantitative and qualitative differences in host plants, symptomatology, aphid transmissibility and molecular biology. In order to gain an overview of the different PPV strains that exist in Germany, several PPV isolates, mainly deriving from southern Germany, were analysed by polymerase chain reaction (PCR), the reaction yielding a 243 bp product. All PPV isolates tested were amplified and the amplified fragments were analysed by restriction endonuclease digestion. An Rsa I restriction site polymorphism in the amplified fragments allowed the discrimination of two groups of isolates.     

Detection of Erwinia carotovora subsp. atroseptica and Erwinia chrysanthemi in potato tubers using polymerase chain reaction

Soft rot erwiniae are a group of notorious plant pathogens for which currently available detection methods are inadequate. Based on the polymerase chain reaction, specific and sensitive detection of Erwinia carotovora subsp. atroseptica and E. chrysanthemi in potato tubers has been achieved. The composition of the PCR primers used in two specific detection systems is based on identification of the consensus of sequences of metalloprotease-coding genes present in soft rot erwiniae. Bacterial DNA was extracted from the potato tuber matrix by differential centrifugation in order to avoid interference of potato-derived compounds with the performance of the PCR assay. The PCR assay jjerformed with the E. carotovora subsp. atroseptica specific primer set was found to be capable of distinguishing E. carotovora subsp. atroseptica from all other Erwinia species and the closely related subspecies E. carotovora subsp. carotovora. With the E. chrysanthemi specific primer set, agarose gel electrophoresis is required for unequivocal differentiation between E. chrysanthemi and other erwiniae. Combined with the efficient extraction procedure, the assay allowed specific detection of less than 10³ culturable erwiniae per tuber. The specificity and sensitivity of the assay were not reduced in the presence of a 100-fold excess of DNA from both related and unrelated bacteria. This PCR-based method for detection of erwiniae in potato tubers provides a relatively fast and sensitive alternative to routinely applied serological methods.     

Detection and identification of four common rust pathogens of cereals and grasses using real-time polymerase chain reaction

ABSTRACT Puccinia spp. are widespread pathogens of cereals and grasses that annually cause significant yield losses worldwide, especially in barley, oat, and wheat. Urediniospore morphology and early symptom development have limited usefulness for distinguishing Puccinia spp. Therefore, we developed real-time polymerase chain reaction assays for rapid detection of the four rust pathogen species, Puccinia graminis (Pers.:Pers.), P. striiformis (Westend.), P. triticina (Eriks.), and P. recondita (Roberge ex Desmaz.). Duplex assays were constructed for the nuclear rDNA gene, using the variable internal transcribed spacer 1 (ITS1) region to distinguish between species, and the conserved 28S region as an internal control. Species-specific ITS1 primer/probe sets were highly specific and could detect <1 pg of DNA. The species-specific primer/probe sets showed positive results over a linear range of DNA five orders of magnitude or greater. Specificity of the assays was tested using multiple collections representing a range of races and formae speciales within a species. Additionally, assay specificity was evaluated by testing a range of other grass rust pathogens, as well as other fungi. The 28S primer/probe combination was successful in detecting all Puccinia spp. tested within the duplex assays, validating the integrity of each assay. Finally, the assays were used to identify unknown rust fungi infecting pasture grasses.     

Cloning of a specific DNA region of white top pathogen of pea and its detection by polymerase chain reaction

White top strain (WT strain) of Pseudomonas syringae pv. pisi (Ppi) is a variant strain causing white top disease of peas. The WT strain is distinguishable from common Ppi strains only by symptom expression chlorosis and whitening of apical shoots. To develop a specific detection method for the WT strain, we cloned a specific DNA region of the WT strain using transposon tagging. Five mutants defective in white top symptom expression were obtained. A part of the Tn5-flanking region was cloned and labeled as a hybridization probe. One clone, pAY3, gave two signal bands, one of which was detected from the genomic DNA of all the WT and the common Ppi strains; another was specific to WT strains. A restriction map of pAY3 showed that it contains two BamHI fragments; one is 5.0kb in length involving a part of Tn5, and the other is 1.5kb, did not carry Tn5, and may have been accidentally ligated into pAY3. The 1.5-kb band was subcloned as pAY13 and was used as a probe. It hybridized specifically to WT strains. These results suggested that the WT strains have a specific DNA region and that part of the region was successfully cloned. Sequence analysis of pAY13 showed that it is similar to part of nonribosomal peptide synthetases (NRPSs) genes. The deduced amino acid sequence of pAY13 suggested the existence of eight conserved motifs of NRPSs. WT strain-specific PCR primers, PS1 and PS2, were designed from the DNA sequence. These primers gave a specific amplification product of 981bp from both the genomic DNA and a direct cell preparation of WT strains. No specific amplicon was produced from Ppi strains that caused only water-soaked lesions or from strains of other P. syringae pathovars. A specific amplicon was not produced from four strains of the pea pathogen: P. marginalis pv. marginalis, P. viridiflava, Erwinia carotovora ssp. carotovora, Xanthomonas campestris pv. pisi. Using the primers, WT strain was detected from water-soaked lesions and green and white tissues without water soaking.The sequence reported in this paper has been deposited in the DDBJ database under accession no. AB117755     

Detection of airborne inoculum of Leptosphaeria maculans and Pyrenopeziza brassicae in oilseed rape crops by polymerase chain reaction (PCR) assays

The potential use of DNA-based methods for detecting airborne inoculum of Leptosphaeria maculans and Pyrenopeziza brassicae , both damaging pathogens of oilseed rape, was investigated. A method for purifying DNA from spores collected using Hirst-type spore samplers and detecting it using polymerase chain reaction (PCR) assays is described. For both pathogens, the sensitivities of the DNA assays were similar for spore-trap samples and pure spore suspensions. As few as 10 spores of L. maculans or P. brassicae could be detected by PCR and spores of both species could be detected against a background of spores of six other species. The method successfully detected spores of P. brassicae collected using spore traps in oilseed rape crops that were infected with P. brassicae. Leptosphaeria maculans spores were detected using spore traps on open ground close to L. maculans -infected oilseed rape stems. The potential use of PCR detection of airborne inoculum in forecasting the diseases caused by these pathogens is discussed.     

应用TaqMan探针实时荧光定量PCR技术早期检测稻瘟病

 稻瘟病是一种严重危害水稻生产的真菌病害,早期监测和防治是关键。温室接种大田主栽品种合系39和感病水稻蒙古稻,定时观察发病症状。提取水稻病样总DNA,根据稻瘟菌28S rDNA基因序列,设计特异引物和TaqMan探针,进行荧光定量PCR检测。结果表明稻瘟菌在蒙古稻和大田主栽品种合系39上症状最初出现时间为接种后72 h,蒙古稻典型症状出现时间为接种后168 h,在合系39上出现典型症状的时间为接种后190 h以后。利用TaqMan探针荧光定量PCR技术,在接种12 h的蒙古稻和合系39上均能检测到稻瘟菌DNA,接种48 h,菌量拷贝数达到最高峰,接种72 h,菌量拷贝数开始下降。不同品种中病原菌拷贝数存在差别,在接种12 h的蒙古稻中稻瘟菌含量为7.2×103个拷贝数,在合系39中为4.9×103个拷贝数。本研究结果表明,应用实时荧光定量PCR技术可以在接种后12 h症状未显现之前检测到稻瘟菌,为稻瘟病流行监测和早期防治提供了科学依据。     

The use of polymerase chain reaction to detect latent infection of Clavibacter michiganensis subsp. michiganensis in tomato seedlings

A method was developed for routine analysis to detect latent infection of Clavibacter michiganensis subsp. michiganensis in samples from tomato seedling lots, before transplant, using polymerase chain reaction. In samples of 300 stem segments 1 cm long, the sensitivity threshold of the method was estimated at around 1.1 × 10³ corynebacteria or 0.33% of latently infected stems. This method was shown to have a good specificity.     

Detection of begomoviruses in clarified plant extracts: a comparison of standard, direct-binding, and immunocapture polymerase chain reaction techniques

ABSTRACT Three polymerase chain reaction (PCR) techniques-standard PCR (Std-PCR), direct-binding PCR (DB-PCR), and immunocapture PCR (IC-PCR)-using degenerate primers were optimized and evaluated for the detection of begomoviruses. Tomato leaf samples were ground in three different extraction buffers and subjected to Std-PCR. The effect of the buffers on the detection limits of amplification of the virus (detection of the initial and end points of dilution) was determined. With the optimal extraction buffer determined by the first experiment, the antibody concentration and incubation conditions for IC-PCR were evaluated to determine the requirements for maximum capture of antigens during the capture phase of the technique. The incubation conditions of DB-PCR were also investigated to determine the most favorable conditions for adsorption of the viral template. The reproducibility of all assays was evaluated. With the results of the optimization experiments, the applicability of the three techniques to different plant species was assessed. Extracts of plant species belonging to three families were subjected to the optimized Std-, DB-, and IC-PCR protocols. Std- and IC-PCR both achieved reproducible detection of begomoviruses, but the detection limits and amplified band intensity for all plant species tested were superior for the latter. DB-PCR was an unreliable method of detection, because of poor reproducibility and low intensity of amplified bands. These results indicate that the optimized IC-PCR detection system using degenerate primers is the most effective for the detection of begomoviruses in clarified plant extracts.     

Distribution ofLeptosphaeria maculans in two fields in southern Ontario as determined by the polymerase chain reaction

The distribution of the highly virulent and weakly virulent types ofLeptosphaeria maculans, causal agent of blackleg of oilseed rape, was studied in two fields in southern Ontario. Using a polymerase chain reaction-based assay with primers specific for these virulence types, plant tissues were directly examined for the occurrence of the pathogen. The highly virulent type was detected in leaf, stem and crown tissue at most of the sampling sites. The weakly virulent type was detected only in leaf lesions at 50% of the sites in field 1 and 30% of the sites in field 2. Of 96 leaf lesions examined, 48 contained the highly virulent type, 12 contained the weakly virulent type and 16 contained both the highly virulent and weakly virulent types.Sclerotinia sclerotiorum was isolated from all leaf lesion that did not react withL. maculans virulence type-specific primers. Coinfection of single blackleg leaf lesions by bothL. maculans virulence types thus occurred in oilseed rape plants in the field. Only the highly virulent type was detected in pseudothecia on stubble. Approximately 1% of the seed collected from these two fields containedL. maculans, and both the highly virulent and weakly virulent types were detected. The highly virulent type was more prevalent and non-specific in the kind of plant tissue it infected, whereas the weakly virulent type appeared to be limited to infecting leaves and seed. This study illustrates an application of polymerase chain reaction with virulence type-specific oligonucleotide primers to study the epidemiology of blackleg of oilseed rape.