Specific detection of<Emphasis Type="Italic">Pythium aphanidermatum</Emphasis> from hydroponic nutrient solution by booster PCR with DNA primers developed from mitochondrial DNA

Pythium aphanidermatum causes damping-off and root rot of vegetable crops in hydroponic systems. A DNA probe was isolated and modified from a library ofHindIII-digested mitochondrial DNA ofP. aphanidermatum that strongly hybridized to DNA ofP. aphanidermatum and weakly hybridized to DNA ofPythium deliense. Cross-hybridizing sequences were absent from DNA of plants and other related fungi. The probe detected as little as 5 ng ofP. aphanidermatum DNA and 250 ng ofP. deliense DNA in slot-blot assays.P. aphanidermatum was detected by a hybridization assay of total DNA extracted directly from infected roots. A pair of oligonucleotide primers P1 and RP2, which allowed amplification of a specific 0.65 kb DNA fragment ofP. aphanidermatum using polymerase chain reaction (PCR), was designed from a specific DNA probe. Specific amplification of this fragment fromP. aphanidermatum was highly sensitive, detecting template DNA as low as 0.1 pg total DNA by booster PCR. Specific booster PCR amplification using P1 and RP2 was successful in detectingP. aphanidermatum in naturally infected nutrient solution and roots of vegetables in a field hydroponic system. http://www.phytoparasitica.org posting Sept. 22, 2002.     

Pcr-amplification of tomato yellow leaf curl virus (TYLCV) DNA from squashes of plants and whitefly vectors: Application to the study of TYLCV acquisition and transmission

DNA of tomato yellow leaf curl virus (TYLCV), a geminivirus transmitted by the whitefly Bemisia tabaci, was amplified from squashes of infected tomato plants and of viruliferous vectors using the polymerase chain reaction (PCR). Samples of infected tissues as small as 1 mm² were squashed onto a nylon membrane. A 1 × 2 mm strip containing the squash was introduced into a 25 µl PCR reaction mix. The reaction products were subjected to gel electrophoresis, blotted and hybridized with a radiolabeled virus-specific DNA probe. TYLCV DNA was amplified from squashes of leaves, roots, and stem of infected tomato and from individual viruliferous whiteflies. The same squash could be used several times to amplify different virus DNA fragments with various sets of primers. Thus plant and insect squashes can be used as templates for the amplification of geminiviral DNA with no need to prepare tissue extracts or purify nucleic acids. The squash-PCR procedure was applied to study whitefly transmission of TYLCV. Tomato plants were inoculated by placing a single viruliferous insect in the center of a young leaflet. In some plants TYLCV DNA was detected at the site of inoculation as early as 5 min after the beginning of the access feeding and in all plants after 30 min. The squash-PCR procedure also was applied to the study of TYLCV acquisition by the insect vector. TYLCV DNA was detected in the head of whiteflies as early as 5 min after the beginning of the access feeding on infected tomato plants. Viral DNA was detected in the thorax after 10 min and in the abdomen after 25 min.     

Bacterial DNA activates immunity in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

To initiate defense responses against invasion of pathogenic organisms, animals and plants must recognize microbe-associated molecular patterns (MAMPs). In this study, the elicitor activity of bacterial DNA on the model plant Arabidopsis thaliana was examined. EcoRI-digested plasmid DNA induced defense responses such as generation of reactive oxygen species and deposition of callose, whereas SmaI- and HapII-digested plasmid DNA and EcoRI-digested herring DNA did not remarkably induce these responses. Further, methylation of the CpG sequence of plasmid DNA and Escherichia coli DNA reduced the level of the defense responses. The endocytosis inhibitors wortmannin and amantadine significantly inhibited DNA-induced defense responses. These results suggest that non-methylated CpG DNA, as a MAMP, induced defense responses in Arabidopsis and that non-methylated DNA seems to be translocated into the cytoplasm by endocytosis.     

Studies on the interaction of aminocarb with calf thymus DNA by spectroscopic methods

The interaction between aminocarb and calf thymus DNA in physiological buffer (pH 7.4) was investigated by UV-vis spectrophotometry, fluorescence spectroscopy, viscosity measurements and DNA melting techniques. The absorption spectra of aminocarb with DNA showed a slight blue shift and hypochromic effect. Using ethidium bromide (EB) as a fluorescence probe, fluorescence quenching of the emission peak was seen in the DNA-EB system when aminocarb was added. The fluorescence polarization was gradually increased with increasing amounts of DNA. The value of melting temperature of DNA increased in the presence of aminocarb. Moreover, the relative viscosity of DNA increased with the addition of aminocarb. All the evidences indicated that the binding mode of aminocarb with DNA was an intercalative binding. The binding constants of aminocarb with DNA were determined. The calculated thermodynamic parameters suggested that the binding of aminocarb to DNA was driven mainly by hydrogen bond and van der Waals.     

样本保存方法及冷冻胁迫对蝗虫基因组DNA提取的影响

以亚洲小车蝗为研究材料,提出了使用液氮保存蝗虫样本的有效方法,并用基因组DNA提取试剂盒分别对液氮速冻后保存、直接冷冻、无水乙醇保存和干制蝗虫标本进行了基因组DNA的提取和电泳检测。结果表明,在保存3个月后,检测蝗虫样本直接冷冻法和干标本提取的总DNA浓度较低,因而琼脂糖电泳检测亮度低;液氮速冻后保存和无水乙醇保存的蝗虫样本提取的基因组DNA浓度大,琼脂糖电泳检测亮度高。表明液氮速冻后保存和无水乙醇保存的标本适合用于基因组学研究。通过对比,直接冷冻保存与液氮速冻保存结果得出,蝗虫在冷冻胁迫死亡的过程中有DNA降解发生。     

Inhibition of DNA polymerase β activity in isolated bovine liver nuclei by captan and related compounds

The effects on DNA synthesis of the fungicide captan and several structurally related compounds were investigated in isolated bovine liver nuclei. Captan, folpet, captafol, and trichloromethanesulfenyl chloride inhibited DNA synthesis to the same degree with ID₅₀ values of approximately 50 μM in a 40-min assay. The inhibition is concentration dependent and the degree of inhibition increases with time. Studies with structural analogs of captan indicated that inhibition of DNA synthesis by captan is mediated through the trichloromethylthio moiety of the captan molecule. In addition, the data indicate thiophosgene is probably not the toxic species involved in the inhibition of DNA synthesis. The isolated nuclei used in this study were shown to exhibit only a single DNA polymerase activity which was determined to be of the β or low-molecular-weight type. In addition to its inhibition in intact nuclei, captan inhibited the activity of the β polymerase in nuclear extracts as well as in partially purified enzyme preparations. These results indicate that captan inhibits DNA synthesis in our preparation of isolated nuclei by acting directly on the DNA polymerase-catalyzed reaction rather than by causing a nonspecific or indirect effect in the nuclear system such as alterations in the nuclear membrane or aggregation of the nuclei. The site of captan's inhibitory action is the DNA polymerase molecule. The interaction of captan with the polymerase results in irreversible inhibition of the enzyme. Interaction of captan with the template, if it occurs, does not appear to be involved in mediating the inhibition.     

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.     

3种根萤叶甲实时荧光PCR检测技术研究

根萤叶甲属(Diabrotica)被我国列为进境植物检疫性有害生物,该属中的玉米根萤叶甲(D.virgifera virgifera)、十一星根萤叶甲(D.undecimpunctata)、巴氏根萤叶甲(D.barberi)是北美的主要农业害虫。为了建立一种快速的分子鉴定方法以鉴定这3种根萤叶甲,本研究从田间采集的成虫样品中获得其部分mtDNA COI序列,与Gen Bank中的相关序列进行比对,设计筛选出3种根萤叶甲的特异性引物和TaqMan探针,并进行实时荧光PCR特异性和灵敏度检测。特异性检测结果表明,用目标种根萤叶甲DNA和其特异性探针和引物进行实时荧光PCR反应时,在30个循环反应内(Ct值30)有近S型扩增曲线出现,同时其他种均无荧光信号增长。此外,灵敏度结果显示,玉米根萤叶甲和巴氏根萤叶甲可检测的最小DNA模板浓度,即实时荧光PCR反应的灵敏度为0.1 ng/μL,十一星根萤叶甲为0.01 ng/μL。     

Harpin蛋白基因的克隆及序列分析

 以梨火疫病细菌基因组DNA为模板,通过合成5'-端及3'-端一对特异引物,利用多聚酶链式反应(PCR)扩增获得harpin蛋白基因。将其克隆到E.coli质粒上进行序列分析。结果表明:基因由1155个核苷酸组成,编码385个氨基酸残基组成的多肽。与发表序列相比较,核苷酸序列及推导出的氨基酸序列的同源性分别为99.31%和98.96%。     

几种植物类菌原体(MLOs)的分子检测及其遗传相关性比较

 以密西根翠菊黄化病MLO 16S rDNA序列为依据,参照有关引物序列,改进合成寡核苷酸R₁和L₁作为引物对,应用聚合酶链式反应(PCR)技术,以甘薯丛枝病(SPWB),泡桐丛枝病(PWB),枣疯病(JWB)和枣疯病长春花(JPWB)罹病植株中提取的DNA为模板,扩增出了1.2 Kb的MLO 16S rDNA片段。该方法所需罹病植株的总DNA量分别为:甘薯丛枝病2×10^-2 ng/μl,泡桐丛枝病0.4×10^-2 ng/μl,枣疯病0.8×10^-2 ng/μl,感染枣疯病的长春花0.3×10^-3 ng/μl。对1.2kb 16S rDNA进行限制性片段长度多态性(RFLP)分析,首次确证枣疯病的罹病植株中存在枣疯病MLO和泡桐丛枝病MLO混合侵染的现象。     

Plant MetGenMAP数据库分析水稻表达谱芯片数据的方法初探

基因芯片数据分析是利用基因芯片技术进行各项研究的关键。本文以水稻Affymetrix表达谱芯片的数据为例,利用Plant MetGenMAP数据库,对水稻受病原菌侵染后基因表达量及代谢途径的变化情况进行了初步分析,同时,提供了一种利用Plant MetGenMAP数据库分析水稻基因芯片数据的方法。     

Development of specific PCR primers for identification and detection of <Emphasis Type="Italic">Phytophthora capsici</Emphasis> Leon

A PCR-based method was developed for the identification and detection of Phytophthora capsici in pepper plants. Three PCR primers (CAPFW, CAPRV1 and CAPRV2) specific for P. capsiciwere designed based on the sequence of its internal transcribed spacer regions. CAPFW/CAPRV1 amplify a 452 bp product from P. capsici DNA whereas CAPFW/CAPRV2 a 595 bp fragment; neither set amplifies DNA from pepper or several fungi pathogenic to pepper. In conventional (single-round) PCR, the limit of detection was 5 pg DNA for both primer sets, whereas in nested PCR the detection limit for both was of 0.5 fg. However, when the dilution series of target DNA were spiked with plant DNA, amplification declined two-fold in both conventional and nested PCR. The CAPFW/CAPRV2 set in conventional PCR was used to detect P. capsici DNA in inoculated plants. Detection occurred as soon as 8h post-inoculation in stem samples from infected but still symptomless plants. The method was also tested to detect fungal DNA in infected soils.     

Characterization of Verticillium dahliae Isolates from Potato on Hokkaido by Random Amplified Polymorphic DNA (RAPD) and REP-PCR Analyses

Verticillium dahliae isolates from potato on the island of Hokkaido (potato isolates) and those belonging to pathotypes A (eggplant pathotype), B (tomato pathotype) and C (sweet pepper pathotype) were divided into three distinct groups by RAPD and REP-PCR. The three DNA groups I, II, III consisted of pathotypes A and C, pathotype B and potato isolates, respectively. The potato isolates were assigned to pathotype A on the basis of pathogenicity. Another set of potato isolates was further collected from eight potato cropping regions on Hokkaido to further examine the relationships among them in detail. Only one of these isolates was identified as DNA group II, but all the others were classified as DNA group III. Isolates from daikon, eggplant, and melon on Hokkaido also belonged to DNA group III. These results suggest that V. dahliae isolates from Hokkaido are unique at the DNA level and different from other pathotype A isolates in Japan. Received 28 February 2000/ Accepted in revised form 6 November 2000     

Specific detection of Phytophthora cactorum in diseased strawberry plants using nested polymerase chain reaction

Validated protocols for DNA purification and PCR amplification are reported for detection of Phytophthora cactorum in diseased strawberry plants. To remove PCR inhibitors, necrotic strawberry tissues were soaked in 5% alconox solution for >12 h before DNA extraction, and the extracted genomic DNA was embedded in an agarose gel chamber and subjected to electrophoresis. The purified DNA was amplified reliably by PCR. Nested PCR was used to detect a portion of the rRNA gene of P. cactorum in samples. In the first round of PCR, primers ITS1 and ITS4 amplified fragments of varying sizes from total genomic DNA from diseased strawberry plants. In the second round of PCR, a 1:25 dilution of the first-round PCR products was used as template with two P. cactorum- specific primer pairs (BPhycacL87FRG and BPhycacR87RRG, which amplified a 340-bp fragment and a 480-bp fragment from the rRNA gene; and BPhycacL89FRG and BPhycacR176RRG, which amplified a 431-bp fragment). Validation tests using culture-based isolations as a standard for comparison indicated that the DNA purification and PCR primers and amplification protocols were reliable and specifically amplified a portion of the rRNA gene of P. cactorum from necrotic root, crown and petiole tissues of strawberry naturally infected by the pathogen.     

牛筋草ISSR-PCR反应体系的建立与优化

以牛筋草地上组织为材料,采用正交优化和单因子试验两种方法对影响牛筋草ISSR-PCR体系的Mg2+浓度、dNTP浓度、Taq DNA聚合酶浓度、引物浓度和DNA模板浓度5个因素进行优化试验,建立适合牛筋草ISSR-PCR的反应体系.结果表明,牛筋草ISSR-PCR反应体系的最佳条件为:Mg2+1.75 mmol/L,dNTP 0.4 mmol/L,Taq DNA聚合酶1 U/25 μL,引物0.3μmol/L,模板DNA 80 ng/25μL,10×PCR Buffer 2.5 μL/25 μL.利用优化体系进行牛筋草的ISSR-PCR反应,可获得稳定性高、重复性好、背景清晰的电泳结果.     

Genome characterization of a new strain of peanut chlorotic streak virus causing chlorotic vein banding disease of groundnut (Arachis hypogaea L.) in India

The double-stranded DNA of the chlorotic vein banding isolate of peanut chlorotic streak virus (PCISV-CVB), isolated from purified virus, resolved into circular and linear molecules similar to those of other caulimoviruses. A physical map of viral DNA was constructed, which showed the PCISV-CVB DNA to be circular and composed of approximately 8·2 kbp. A number of restriction sites were found to be shared with a similar caulimovirus, PCISV, Nevertheless, several differences between physical maps of the two viruses suggest that PCISV-CVB should be considered as a distinct strain of PCISV. Bam HI-cleaved PCISV-CVB DNA was cloned into pUC 118 and was infectious when cleaved from the cloning vector and inoculated onto Vigna unguiculata.     

Detection and quantification of airborne inoculum of Sclerotinia sclerotiorum using quantitative PCR

This paper reports the development of a new specific diagnostic technique to accurately quantify airborne inoculum of Sclerotinia sclerotiorum and discusses its potential use in disease-forecasting schemes, using examples of three contrasting epidemic seasons: 2007, when there was a severe epidemic of sclerotinia stem rot (SSR) in England and high numbers of airborne ascospores were trapped at Rothamsted, and, in contrast, 2003 and 2004, when the incidence of SSR in England was low and low numbers of airborne ascospores were trapped at Rothamsted. DNA was extracted from wax-coated plastic tapes, such as those used in Burkard (Hirst-type) spore traps and rotating-arm traps. A SYBR-green quantitative PCR (qPCR) method produced a linear relationship between ascospore numbers and S. sclerotiorum DNA (mean 0·35 pg DNA per spore) and was able to detect DNA representing as few as two ascospores. The technique was insensitive to DNA of the host plant, Brassica napus , and other plant pathogens, including Sclerotinia minor , S. trifoliorum and Botrytis cinerea , and common airborne fungal genera such as Cladosporium and Penicillium . There was no relationship between rainfall and numbers of airborne ascospores of S. sclerotiorum present at Rothamsted during the period of infection in the severe SSR season (2007).     

田间水稻单个病斑中稻瘟病菌的遗传多样性

根据93个单病斑中分离的227个单孢菌株样品的分析，研究了来自同一定殖系统(同一病斑)中稻瘟病菌不同单孢菌株(同源菌株)的遗传变异。在致病性分析的样品中，仅有5个病斑分离的10个单孢菌株在所有鉴别品种上表现同源菌株有完全相同的致病反应，并且有完全相同的DNA指纹图谱带型；另有13个病斑上分离的32个单孢菌株的同源菌株不仅属于不同的生理小种，而且在1～3个单基因品种上也有不同的致病反应。用rep—PCR指纹图谱和致病型分析67个病斑的157个单孢样品中的同源菌株具有完全相同的DNA指纹图谱；13个病斑中分离的27个单孢样品中同源菌株具有显著差异的指纹图谱；8个病斑分离的27个单孢菌株样品中同一来源样品中部分样品有相同的指纹图谱带型，部分样品却有不同的指纹图谱。同源菌株之间有完全相同致病性其指纹图谱亦相同或有很小的差异；通常，致病性差异很大的同源菌株其指纹图谱差异也很大。作者认为来自同一定殖系统中的不同单孢菌株存在的遗传变异与病菌的异核现象或自然状态下不同遗传背景菌株同时侵染造成同一病斑有关。     

Development of a PCR-based diagnostic test for Spongospora subterranea f.sp. nasturtii, the causal agent of crook root of watercress (Rorippa nasturtium-aquaticum)

The polymerase chain reaction (PCR) technique was utilized to obtain internal transcribed spacer ribosomal DNA (ITS rDNA) and small-subunit (18S) rDNA sequences from UK isolates of Spongospora subterranea f.sp. nasturtii , a plasmodiophorid pathogen of watercress ( Rorippa nasturtium-aquaticum ). ITS sequence data obtained from S. subterranea isolated from a range of UK sites were found to be identical. PCR primers were designed using these sequences and were shown to be capable of specific amplification of S. subterranea f.sp. nasturtii DNA from plant tissue and from water samples containing zoospores of the pathogen. As little as 5 ng total genomic DNA from infected plant material, or 1000 zoospores, was required for consistently successful amplification of DNA. A filtration-based method for obtaining pathogen DNA for PCR from watercress-bed water was developed.     

离体砂糖橘黄龙病病果中病菌浓度变化规律的探究

Early studies have revealed the uneven distribution of Candidatus Liberibacter asiaticus (CLas) in Huanglongbing (HLB)-affected citrus trees. Bacteria of CLas were found to be enriched in the fruit, especially in the pith. In this study, we further investigated the dynamics of CLas population in excised citrus fruits. The CLas-infected Shatangju fruits were collected and kept for 4 weeks under constant temperature and moisture. The concentrations of CLas in the pith of fruits were detected weekly, and the fruits on trees were detected at the same time as the control. Our results showed that the total DNA, citrus DNA and CLas DNA from the excised fruits all decreased during the storage, but the CLas concentration in the pith of excised fruit increased firstly, and then decreased with time. The CLas concentrations in the pith of diseased fruit at 14 d and 28 d after picking were higher than that of the first day and reached the highest at 14 d. Comparing with the fruits on the tree, CLas concentrations in the pith of excised fruits were also significantly higher at the 14 d and 28 d. With the degrading of total DNA in fruit pith, the proportion of CLas DNA was increased, which could be served as a suitable material for the DNA extraction and CLas genome sequencing.