Validation of a real-time PCR for the quantitative estimation of a G143A mutation in the cytochrome bc1 gene of Pyrenophora teres

A single nucleotide polymorphism (SNP) in the cytochrome b gene confers resistance to strobilurin fungicides for several fungal pathogens. Therefore, on the basis of a change at amino acid position 143 from glycine to alanine, a real-time PCR assay was established for the quantitative detection of the analogous SNP in the cytochrome b sequence of Pyrenophora teres Drechsler, which causes barley net blotch. Allelic discrimination was achieved by using allele specific primers with artificially mismatched nucleic acid bases and minor groove binding probes. Validation parameters for the lower limits of the working range, namely limits of detection (LOD) and limits of quantification (LOQ), were statistically determined by the variance of calibration data, as well as by the variance of the 100% non-strobilurin-resistant allele DNA sample (blank values). It was found that the detection was limited by the variance of blank values (five in 801 458 copies; 0.0006%), whereas the quantification was limited by the variance of calibration data (37 in 801 458 copies; 0.0046%). The real-time PCR assay was finally used to monitor strobilurin-resistant cytochrome b alleles in barley net blotch field samples, which were already classified in in vivo biotests to be fully sensitive to strobilurins. All signals for strobilurin-resistant cytochrome b alleles were below the LOD, and therefore the results are in total agreement with the phenotypes revealed by biotests.     

Population structure of South African and Australian Pyrenophora teres isolates

There are two recognized forms of the disease net blotch of barley: the net form caused by Pyrenophora teres f. teres (PTT) and the spot form caused by P. teres f. maculata (PTM). In this study, amplified fragment length polymorphism analysis was used to investigate the genetic diversity and population structure of 60 PTT and 64 PTM isolates collected across Australia (66 isolates) and in the south‐western Cape of South Africa (58 isolates). For comparison, P. tritici‐repentis, Exserohilum rostratum and Bipolaris sorokiniana samples were also included in the analyses. Both distance‐ and model‐based cluster analyses separated the PTT and PTM isolates into two strongly divergent genetic groups. Significant variation was observed both among the South African and Australian populations of PTT and PTM and among sampling locations for the PTT samples. Results suggest that sexual reproduction between the two forms is unlikely and that reproduction within the PTT and PTM groups occurs mainly asexually.     

Rapid-cycle PCR detection of Pyrenophora graminea from barley seed

Sixty RAPD primers were used to screen for a diagnostic marker that could be used to identify Pyrenophora graminea , a fungal seedborne pathogen that causes leaf stripe on barley. Primer pairs were designed to differentiate P. graminea from other Pyrenophora spp. using a sequence-characterized amplified region (SCAR) approach. A pair of P. graminea -specific primers (PG2 F/R) was obtained that amplified a single fragment from 37 isolates of P. graminea tested, but not from 29 isolates of other Pyrenophora spp. or 12 saprophytes isolated from barley seed. Rapid PCR detection was achieved using a LightCycler, in which the emission of fluorescence from the binding of SYBR Green I dye to the PCR products is measured. The P. graminea -specific product resulting from amplification with PG2 F/R can be distinguished from any nonspecific products by post-PCR melting point analysis. The PCR assay involves 40 amplification cycles of PCR, and the total PCR test including melting point analysis takes 25 min to complete. The rapidity of this test, combined with the closed 'in-tube' detection of PCR products, which reduces the potential for contamination, offers significant advantages compared with conventional laboratory and PCR analyses.     

Cytochrome b gene sequence and structure of Pyrenophora teres and P. tritici-repentis and implications for QoI resistance

Resistance to QoI fungicides in Pyrenophora teres (Dreschsler) and P. tritici-repentis (Died.) Dreschsler was detected in 2003 in France and in Sweden and Denmark respectively. Molecular analysis revealed the presence of the F129L mutation in resistant isolates of both pathogens. In 2004, the frequency of the F129L mutation in populations of both pathogens further increased. The G143A mutation was also detected in a few isolates of P. tritici-repentis from Denmark and Germany. In 2005, the F129L mutation in P. teres increased in frequency and geographical distribution in France and the UK but remained below 2% in Germany, Switzerland, Belgium and Ireland. In P. tritici-repentis, both mutations were found in a significant proportion of the isolates from Sweden, Denmark and Germany. The G143A mutation conferred a significantly higher level of resistance (higher EC50 values) to Qo inhibitors (QoIs) than did the F129L mutation. In greenhouse trials, resistant isolates with G143A were not well controlled on plants sprayed with recommended field rates, whereas satisfactory control of isolates with F129L was achieved. For the F129L mutation, three different single nucleotide polymorphisms (SNPs), TTA, TTG and CTC, can code for L (leucine) in P. teres, whereas only the CTC codon was detected in P. tritici-repentis isolates. In two out of 250 isolates of P. tritici-repentis from 2005, a mutation at position 137 (G137R) was detected at very low frequency. This mutation conferred similar resistance levels to F129L. The structure of the cytochrome b gene of P. tritici-repentis is significantly different from that of P. teres: an intron directly after amino acid position 143 was detected in P. teres which is not present in P. tritici-repentis. This gene structure suggests that resistance based on the G143A mutation may not occur in P. teres because it is lethal. No G143A isolates were found in any P. teres populations. Although different mutations may evolve in P. tritici-repentis, the G143A mutation will have the strongest impact on field performance of QoI fungicides.     

Identification of Molecular Genetic Markers in Pyrenophora teres f. teres Associated with Low Virulence on 'Harbin' Barley

ABSTRACT Two isolates of the barley net blotch pathogen (Pyrenophora teres f. teres), one possessing high virulence (0-1) and the other possessing low virulence (15A) on the barley cultivar Harbin, were crossed and the progeny of the mating were isolated. Conidia from cultures of the parent and progeny isolates were used as inoculum to determine the inheritance of virulence in the pathogen. Of the 82 progeny tested, 42 exhibited high virulence and 40 exhibited low virulence on 'Harbin' barley. The data support a model in which a single, major gene controls virulence in P. teres f. teres on this barley cultivar (1:1 ratio; chi(2) = 0.05, P = 0.83). Preparations of DNA were made from parental and progeny isolates, and the DNA was subjected to the random amplified polymorphic DNA (RAPD) technique in a search for molecular genetic markers associated with the virulence phenotype. Five RAPD markers were obtained that were associated in coupling with low virulence. The data indicate that the RAPD technique can be used to tag genetic determinants for virulence in P. teres f. teres.     

Identification of Molecular Markers Linked to a Pyrenophora teres Avirulence Gene

ABSTRACT Genetic control of avirulence in the net blotch pathogen, Pyrenophora teres, was investigated. To establish an appropriate study system, a collection of 10 net form (P. teres f. teres) and spot form (P. teres f. maculata) isolates were evaluated on a set of eight barley lines to identify two isolates with differential virulence on an individual host line. Two net form isolates, WRS 1906, exhibiting avirulence on the cv. Heartland, and WRS 1607, exhibiting high virulence, were mated and 67 progeny were isolated and phenotyped for reaction on Heartland. The population segregated in a 1:1 ratio, 34 avirulent to 33 virulent (chi(2) = 0.0, P = 1.0), indicating single gene control of WRS 1906 avirulence on Heartland. Bulked segregant analysis was used to identify six amplified fragment length polymorphism markers closely linked to the avirulence gene (Avr(Heartland)). This work provides evidence that the P. teres-barley pathosystem conforms to the gene-for-gene model and represents an initial step toward map-based cloning of this gene.     

Proteinaceous Metabolites from Pyrenophora teres Contribute to Symptom Development of Barley Net Blotch

ABSTRACT Pyrenophora teres, the causal agent of net blotch of barley (Hordeum vulgare L.), induces a combination of necrosis and extensive chlorosis in susceptible barley cultivars. Cell-free filtrates from both net and spot forms of P. teres; P. teres f. sp. teres, and P. teres f. sp. maculata were found to contain phytotoxic low molecular weight compounds (LMWCs) and proteinaceous metabolites which appear to be responsible for different components of the symptoms induced by the two forms of the pathogen in a susceptible cultivar of barley (cv. Sloop). Proteins induced only brown necrotic spots or lesions similar to those induced by the pathogens 72 h after inoculation. In contrast, LMWCs induced general chlorosis seen 240 h after inoculation but not the localized necrosis. Neither hydrolyzed or heat- or protease-treated proteinaceous metabolites induced the symptoms. This is the first report of the involvement of proteins produced by P. teres in symptom development during net blotch disease of barley.     

Development and application of a PCR-based 'molecular tool box' for the identification of Phytophthora species damaging forests and natural ecosystems

A PCR-based 'molecular tool box', based on a region of the ras-related protein gene Ypt 1, was developed for the identification of 15 Phytophthora species that damage forests and trees: P. cactorum , P. cambivora , P. cinnamomi , P. citricola , P. europaea , P. inundata , P. lateralis , P. megasperma , P. nemorosa , P. kernoviae , P. pseudosyringae , P. psychrophila , P. quercina , P. ramorum and P. ilicis . Most primers proved highly specific in blast analyses and in tests with DNA from 72 isolates of 35 species of Phytophthora and nine species representative of Pythium . Exceptions were primers designed for P. cactorum and P. ilicis , which cross-reacted with P. idaei and P. nemorosa , respectively. Amplification with Phytophthora -genus-specific primers before amplification with the various species-specific primers (nested PCR) increased the sensitivity of detection over amplification with species-specific primers only: detection limits ranged between 100 and 10 pg target DNA µ L⁻¹ in the latter, compared with 100 fg µ L⁻¹ in nested PCR. Using existing methods for rapid extraction and purification of DNA, single-round amplification was appropriate for detection of target Phytophthora species in leaves, but nested PCR was required for soil and water samples. The quarantine pathogens P. ramorum and P. kernoviae were detected in a number of naturally infected leaves collected in England and Wales, whereas P. citricola was commonest in water and soil samples from natural Scottish ecosystems.     

Molecular detection of Fusarium solani f. sp. glycines in soybean roots and soil

A polymerase chain reaction (PCR)-based method was developed to detect DNA of Fusarium solani f. sp. glycines , the cause of soybean sudden death syndrome. Two pairs of primers, Fsg1/Fsg2 designed from the mitochondrial small subunit ribosomal RNA gene, and FsgEF1/FsgEF2 designed from the translation elongation factor 1-α gene, produced PCR products of 438 and 237 bp, respectively. Primer specificity was tested with DNA from 82 F. solani f. sp. glycines , 55 F. solani non-SDS isolates, 43 isolates of 17 soybean fungal pathogens and the oomycete Phytophthora sojae , and soybean. The sensitivity of primer Fsg1/Fsg2 was 10 pg while that of FsgEF1/FsgEF2 was 1 ng when using F. solani f. sp. glycines total genomic DNA or down to 10³ macroconidia g⁻¹ soil. Nested PCR increased the sensitivity of the PCR assay 1000-fold to 10 fg using primers Fsg1/Fsg2, and 1 pg using primers FsgEF1/FsgEF2. F. solani f. sp. glycines DNA was detected in field-grown soybean roots and soil by PCR using either single pairs of primers or the combination of two pairs of primers. The occurrence of F. solani f. sp. glycines was determined using nested PCR for 47 soil samples collected from soybean fields in 20 counties of Illinois in 1999. F. solani f. sp. glycines was detected in soil samples from all five Illinois Agricultural Statistic Districts including 100, 89, 50, 92 and 50% of the samples from East, Central, North-east and West Districts, respectively.     

香蕉枯萎病菌生理小种鉴定及其SCAR标记

 通过室内人工接种蕉类鉴别寄主,对采集于广东蕉区的18个蕉类枯萎病菌菌株进行鉴定,KP021、KP022、GZ981和JL021 4个菌株属Racel,其余14个菌株属Race4,说明广东蕉区同时存在尖孢镰刀菌古巴专化型Race1和Race4。用RAPD技术对上述18个菌株进行分析,从200条随机引物中筛选出8条引物可产生生理小种RAPD标记12个,其中标记Racel的8个,标记Race4的4个。对这些RAPD标记带分别进行回收、克隆、测序,根据这些特异片段序列分别设计相应的SCAR引物,通过对18个菌株的PCR扩增检验,有4个RAPD标记成功地转化为SCAR标记,其中Race1-SCAR标记1个、Race4-SCAR标记2个、同时能鉴定出2个小种的SCAR标记1个。应用这4个SCAR标记同时对采自田间的9个病菌分离物进行检测,能够准确地鉴定出广东蕉区的尖孢镰刀菌古巴专化型Racel和Race4,这为下一步开展香蕉枯萎病菌生理小种的分子鉴定及各生理小种田间流行动态监测奠定了基础。     

基于Ypt1基因为靶标的木香疫病病组织及病田土壤分子检测技术

A species-specific PCR assay was established for rapid and accurate detection of the oomycete pathogen Phytophthora tentaculata in diseased plant tissues and infected soil.A pair of species-specific primers Pt1/Pt2 were designed on the basis of Ras-related protein(Ypt1) gene sequences of the Phytophthora species.PCR amplification with the Pt primers resulted in a 386 bp product only from isolates of P.tentaculata.The detection threshold with Pt primers was 100 pg of genomic DNA.A nested PCR procedure was developed using Ypt1F/Ypt1R as the first-round amplification primers and Pt1/Pt2 as the second-round primers,which increased the detection sensitivity 100-fold to 1 pg.PCR using these Pt primers can also be used to detect P.tentaculata in naturally infected plant tissues and soil.The PCR-based method developed in this study provides a rapid and sensitive tool for detection of P.tentaculata.     

小麦白粉菌ISSR分子标记体系构建及其分离菌株的多样性分析

为了对小麦白粉菌群体的多样性进行研究,构建了其ISSR分子标记体系。以小麦白粉菌基因组DNA为模板,用正交设计和单因素水平优化的方法对ISSR反应程序中的一些重要参数进行摸索和优化,建立了小麦白粉菌ISSR-PCR反应的优化反应体系;对20个ISSR引物的退火温度进行了优化,并筛选出一些多态性较好的ISSR引物。对33个分离菌株的ISSR扩增表明,ISSR标记在我国小麦白粉菌中存在较高的多态性;对ISSR标记揭示的白粉菌的遗传多样性和毒性多样性进行了比较,结果表明两者之间存在一定程度的相关性。     

Specific Oligonucleotide Primers Based on Sequences of the 16S-23S rDNA Spacer Region for the Detection of <Emphasis Type="Italic">Burkholderia gladioli </Emphasis>by PCR

Specific primers were designed based on the sequences of the spacer region between the 16S and 23S ribosomal DNA (rDNA) for direct, rapid and specific detection of Burkholderia gladioli. These primers were named GLA-f and GLA-r. PCR performed on boiled bacterial suspensions yielded an amplification product of approximately 300 bp. No products from other bacterial species, including B. glumae were amplified, even after complete DNA extraction by the cetyltrimethyl-ammonium bromide (CTAB) method. Using the specific primers designed in this study, the PCR method can detect B. gladioli in plant samples within 6 hr. These data demonstrate the potential of specific PCR for the detection of B. gladioli. Received 10 December 2001/ Accepted in revised form 15 April 2002     

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。     

Quantification of <Emphasis Type="Italic">Pyrenophora graminea</Emphasis> in barley seed using real-time PCR

A real-time PCR assay was designed to quantify seed-borne infection of Pyrenophora graminea in barley (Hordeum vulgare). Conventional tests such as the freezing blotter method cannot distinguish P. graminea from the closely related P. teres. The seed infection threshold for P. graminea is lower than the one for P. teres and is therefore applied for both species although P. graminea may be absent. This results in unnecessary rejections of seed lots. PCR primers and a TaqMan probe were designed to target a P. graminea-specific DNA sequence. The potential of the real-time PCR assay for quantifying seed-borne infection of P. graminea was investigated by examining seed lots harvested from P. graminea-infected fields. The major part (84%) of the variation in the amount of P. graminea DNA measured by real-time PCR could be attributed to variation between seed lots while only about 8% was due to variation within seed lots. DNA quantities of P. graminea were positively correlated with seed infection incidence detected by the freezing blotter method as well as with the infection incidence of plants examined in the greenhouse. Both correlations were highly significant (P < 0.001) but the DNA quantities accounted only for 59% (R ² = 0.59) and 56% (R ² = 0.56), respectively, of the variation in the results obtained by the two conventional methods. Seed lots of varieties resistant to P. graminea contained considerable amounts of P. graminea DNA but showed no or only few leaf symptoms in the greenhouse test suggesting that the recommended seed infection thresholds could be raised for resistant varieties.     

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.     

双重real-time PCR定量测定小麦条锈菌潜伏侵染方法的建立与应用

 小麦条锈病是我国小麦生产上的重要病害之一。为高效准确地定量检测处于潜伏侵染阶段的小麦条锈菌,本研究根据已发表的小麦条锈菌和寄主小麦的引物,设计了各自的探针,建立了双重real-time PCR检测方法。为排除多个引物互作造成的干扰,对小麦条锈菌引物探针体系、小麦体系以及二者的双重real-time PCR体系进行了比较。CT值相关线性回归分析证明,引物之间互作很小,对定量检测无影响。已知浓度样品经梯度稀释,进行灵敏度检测,确定了双重real-time PCR对小麦条锈菌DNA和小麦DNA准确定量测定的最小检测限为0.4 pg和0.5 ng。同时建立了小麦条锈菌和小麦各自的标准曲线。用此方法检测来自两个不同地区的田间样本,得到的分子病情指数(MDI)与随后的发病趋势一致。本研究建立的双重real-time PCR分析方法可靠、高效、低误差,是对本实验室已有小麦条锈菌潜伏期分子检测方法的进一步优化。     

PCR detection of Fusarium oxysporum f.sp. gladioli race 1, causal agent of Gladiolus yellows disease, from infected corms

Fusarium oxysporum f.sp. gladioli (FOG) race 1 infects both large- and small-flowered Gladiolus cultivars. Race 2 isolates infect only small-flowered cultivars but can be present as epiphytes on large-flowered plants. When 160 arbitrary 10-mer oligonucleotide primers were tested on FOG by PCR to find RAPD markers specific for race 1, the RAPD primer G12 amplified two discriminating DNA fragments, AB (609 bp) and EF (1196 bp), in race 1 isolates only. Both fragments were cloned and sequenced. Two pairs of race 1-specific primers for multiplex PCR were designed. Tests of 112 F. oxysporum isolates by PCR showed that, in almost all cases, race 1 isolates of vegetative compatibility group 0340 could be distinguished with these primers. Seven putative race 1 isolates did not react in multiplex PCR; hybridization studies with labelled AB and EF DNA fragments showed that these isolates belong to separate groups. A bioassay was developed to detect corms that were latently infected with FOG race 1. Gladiolus corms were homogenized and incubated for 5 days at 28°C in a semiselective medium to induce growth of Fusarium . Cultivated mycelium was isolated and subjected to the developed multiplex PCR after standard DNA isolation or disruption by microwave treatment.     

Rapid and Sensitive Detection of Phytophthora sojae in Soil and Infected Soybeans by Species-Specific Polymerase Chain Reaction Assays

ABSTRACT Root and stem rot caused by Phytophthora sojae is one of the most destructive diseases of soybean (Glycine max) worldwide. P. sojae can survive as oospores in soil for many years. In order to develop a rapid and accurate method for the specific detection of P. sojae in soil, the internal transcribed spacer (ITS) regions of eight P. sojae isolates were amplified using polymerase chain reaction (PCR) with the universal primers DC6 and ITS4. The sequences of PCR products were aligned with published sequences of 50 other Phytophthora species, and a region specific to P. sojae was used to design the specific PCR primers, PS1 and PS2. More than 245 isolates representing 25 species of Phytophthora and at least 35 other species of pathogens were used to test the specificity of the primers. PCR amplification with PS primers resulted in the amplification of a product of approximately 330 bp, exclusively from isolates of P. sojae. Tests with P. sojae genomic DNA determined that the sensitivity of the PS primer set is approximately 1 fg. This PCR assay, combined with a simple soil screening method developed in this work, allowed the detection of P. sojae from soil within 6 h, with a detection sensitivity of two oospores in 20 g of soil. PCR with the PS primers could also be used to detect P. sojae from diseased soybean tissue and residues. Real-time fluorescent quantitative PCR assays were also developed to detect the pathogen directly in soil samples. The PS primer-based PCR assay provides a rapid and sensitive tool for the detection of P. sojae in soil and infected soybean tissue.     

Development of an international standard set of barley differential genotypes for Pyrenophora teres f. teres

International comparison of virulence profiles of Pyrenophora teres f . teres (Ptt), the cause of barley net blotch, is seriously restricted by inconsistencies in differential testers used among researchers. This paper reports an attempt to develop an appropriate set of differentials to standardize characterization of Ptt populations globally. Fourteen barley genotypes (Canadian Lake Shore (CLS), Harbin, c-8755, c-20019, Manchurian, Tifang, CI 9825, CI 5791, CI 9819, Beecher, CI 9214, Skiff, Prior and Corvette) were selected from among genotypes previously used as Ptt differentials. Three cultivars (Pirkka, Haruna Nijo and Harrington) were included to identify a universally susceptible control. Genotypes were inoculated with approximately 1000 Ptt isolates from Russia, Europe, Australia and Canada. The mean reaction frequency of genotypes ranged from highly resistant (CI 9819, CI 5791, c-8755 and CI 9825) to highly susceptible (Harrington, Haruna Nijo and Pirkka). The best differential abilities were demonstrated by Harbin, CLS, c-20019, Manchurian and Prior. Application of cluster analyses identified genotypes with similar reaction patterns, which supported a reduction of genotypes in the set. When combined with an algorithm comparing the ability of individual genotypes to discriminate among Ptt isolates, a further reduction of genotypes was justified. A new, concise set of barley genotypes for differentiating virulences in Ptt was formulated. It is proposed that these genotypes be adopted as the standard, international differential set to characterize and identify the virulence properties of Ptt populations across environments. The new Ptt differential set consists of the genotypes c-8755, c-20019, CI 5791, CI 9825, CLS, Harbin, Prior, Skiff and Harrington.