Development of a Real-Time Polymerase Chain Reaction Assay for Quantifying Verticillium albo-atrum DNA in Resistant and Susceptible Alfalfa

ABSTRACT A precise real-time polymerase chain reaction (PCR) assay was developed for quantifying Verticillium albo-atrum DNA. The assay was used in a repeated experiment to examine the relationship between the quantity of pathogen DNA detected in infected leaves and shoots and the severity of Verticillium wilt symptoms in several alfalfa cultivars expressing a range of disease symptoms. Plants were visually inspected for symptoms and rated using a disease severity index ranging from 1 to 5, and the quantity of pathogen DNA present in leaves and stems was determined with real-time PCR. No significant differences in pathogen DNA quantity or disease severity index were observed for experiments or for cultivar-experiment interactions. Significant differences were observed between cultivars for the quantity of pathogen DNA detected with real-time PCR and also for disease severity index ratings. In both experiments, the highly resistant check cultivar Oneida VR had significantly less pathogen DNA, and significantly lower disease severity index ratings than the resistant cultivar Samauri, the moderately resistant cultivar Vernema, and the susceptible check cultivar Saranac. In both experiments, the Spearman rank correlation between the amount of V. albo-atrum DNA detected in leaves and stems with real-time PCR and disease severity index ratings based on visual examination of symptoms was positive (>0.52) and significant (P < 0.0001). These results suggest that resistance to Verticillium wilt in alfalfa is characterized by a reduced colonization of resistant genotypes by the fungus.     

Histopathological Study of Barley Cultivars Resistant and Susceptible to Rhynchosporium secalis

ABSTRACT Differences in the penetration process by Rhynchosporium secalis were compared in resistant and susceptible barley cultivars at the seedling stage. Percent penetration and percent host cell wall alteration (HCWA) differed significantly among cultivars and isolates as revealed by light microscopy. Based on these two variables, the cultivars were statistically separated into two groups that corresponded to their disease reactions. The resistant cultivars, Johnston and CDC Guardian, showed 81.2 to 99.4% HCWA and 0.1 to 20.1% penetration at encounter sites, whereas the susceptible cultivars, Harrington, Argyle, and Manley, had 30.1 to 78.3% HCWA and 31.8 to 81.8% penetration. In the current study, cv. Leduc, which is susceptible at the seedling stage and resistant at the adult stage, showed the same percent HCWA and penetration as did susceptible cultivars. A significant negative correlation (P < 0.01) was found between percent penetration and percent HCWA for cultivars inoculated with two isolates of the pathogen. Isolate 1 was less virulent than isolate 2 with respect to percent penetration and induced significantly fewer HCWA. Scanning electron microscopy showed various shapes of fungal appressoria but no apparent difference in host reaction between resistant and susceptible cultivars. Transmission electron microscopy revealed interactions between the host and pathogen at various stages of penetration. The resistant cv. Johnston responded by producing appositions, as evidenced by a layer of compact osmiophilic material deposited on the inner side of the cell wall. Infection pegs produced by conidia were unable to penetrate the cuticle where an apposition had formed inside. When penetration occurred in the susceptible cv. Argyle, cytoplasmic aggregates and separation of the plasmalemma were visible from the host cell wall, but the layer of compact osmiophilic material was not always present. Data based on light microscopic observations suggested that HCWA may be one of the mechanisms responsible for resistance that is characterized as penetration prevention rather than as a slow rate of mycelial growth after successful penetration. HCWA occurred in response to attempted cuticle penetration, suggesting that HCWA may produce chemical barriers that help to prevent penetration.     

Detection of Erysiphe necator in Air Samples Using the Polymerase Chain Reaction and Species-Specific Primers

ABSTRACT A polymerase chain reaction (PCR) assay employing species-specific primers was developed to differentiate Erysiphe necator from other powdery mildews common in the northwest United States. DNA was extracted from mycelia, conidia, and/or chasmothecia that were collected from grape leaves with a Burkard cyclonic surface sampler. To differentiate E. necator from other erysiphaeceous fungi, primer pairs Uncin144 and Uncin511 were developed to select unique sequences of the internal transcribed spacer regions of E. necator. Using these primers in PCR amplifications, a 367-bp amplicon specific to E. necator was generated, but no amplicons were generated from other erysiphaceous species collected from 48 disparate hosts representing 26 vascular plant families. The PCR limit of detection was one to five conidia of E. necator placed directly into reaction mixtures or 100 to 250 conidia placed on glass rods coated with silicon grease. During field studies, this PCR assay facilitated the detection of E. necator inoculum in air samples within hours of sample rod collection and prior to disease onset. Amplification of E. necator DNA did not occur when the PCR assay was conducted on vineyard air samples collected while grapes were dormant or during periods when vine growth occurred but E. necator remained dormant. The initial PCR detection of E. necator of the season occurred during seasonal ascospore releases caused by precipitation events between bud burst and the prebloom period during the 3 years of the study. Detection ceased for 7 to 11 days following ascospore release and then resumed several days prior to the observance of microscopic symptoms and signs of powdery mildew in the field. Results of this study represent the initial step toward the goal of incorporating an inoculum availability component into current and future grapevine powdery mildew risk assessment models.     

Detection of Eutypa lata and Eutypella vitis in Grapevine by Nested Multiplex Polymerase Chain Reaction

ABSTRACT Two fungi were isolated from grapevines in Michigan vineyards with Eutypa dieback symptoms: Eutypa lata and Eutypella vitis. These fungi are difficult to distinguish morphologically but are genetically distinct as determined by sequencing of the internal transcribed spacer (ITS) regions. The ITS regions of 25 Eutypa lata and 15 Eutypella vitis isolates were sequenced. Eutypa lata sequences were more variable than those of Eutypella vitis. Polymerase chain reaction (PCR) primers were designed for each species and evaluated against isolates of both fungi as well as 11 closely related Diatrypaceous fungi and 23 isolates of other fungi representing various pathogenic, saprophytic, and endophytic genera on grape and other small fruit crops. The primers were specific for their intended species. A nested multiplex PCR protocol was developed and used to successfully detect these fungi in wood samples from cankers with and without stromata from naturally infected vines as well as in artificially inoculated, potted canes. The primers developed in this study will assist in our abilities to diagnose and study the roles of Eutypa lata and Eutypella vitis in Eutypa dieback development.     

Specific Polymerase Chain Reaction Primers for the Detection of Plasmodiophora brassicae in Soil and Water

ABSTRACT The development of specific oligonucleotide primers for Plasmodiophora brassicae has led to a nested polymerase chain reaction (PCR) detection method for P. brassicae in soil and water. Initially, the PCR was used to amplify a section of the rDNA repeat. The PCR products were sequenced and the data used to design primers that were directed at the ribosomal RNA genes and internal transcribed spacer regions. Specificity was tested against more than 40 common soil organisms, host plants, and spore suspension contaminants, as well as P. brassicae isolates from around Australia and the world. Sensitivity was determined to be 0.1 fentograms (fg; 10(-15) g) for pure template and as low as 1,000 spores per g of potting mix. In soil, P. brassicae was detected in all soils where the inoculum was sufficient to result in clubroot symptoms. Also outlined is a simple method of DNA extraction from soil.     

A Species-Specific Polymerase Chain Reaction Assay for Rapid Detection of Phytophthora nicotianae in Irrigation Water

ABSTRACT Phytophthora nicotianae is a common and destructive pathogen of numerous ornamental, agronomic, and horticultural crops such as tobacco, tomato, and citrus. We have developed a species-specific polymerase chain reaction (PCR) assay for rapid and accurate detection of this pathogen in irrigation water, a primary source of inoculum and an efficient means of propagule dissemination. This PCR assay consists of a pair of species-specific primers (PN), customization of a commercial soil DNA extraction kit for purification of DNA from propagules in irrigation water, and efficient PCR protocols for primer tests and sample detection. The PN primers proved adequately specific for P. nicotianae in evaluations with 131 isolates of P. nicotianae, 102 isolates from 15 other species of Phytophthora, and 64 isolates from a variety of other oomycetes, true fungi, and bacteria. These isolates originated from a wide range of host plants, three substrates (plant tissue, soil, and irrigation water), and numerous geographic locations. The detection sensitivity is between 80 and 800 fg DNA/mul. The assay detected the pathogen in naturally infested water samples from Virginia and South Carolina nurseries more rapidly and accurately than standard isolation methods. Use of this PCR assay can assist growers in making timely disease management decisions with confidence.     

Chlorotoluron Metabolism in Leaves of Resistant and Susceptible Biotypes of the Grass Weed Alopecurus myosuroides

The metabolism of the herbicide chlorotoluron by susceptible and resistant biotypes of the grass weed, Alopecurus myosuroides, was examined. After administration of radiolabelled herbicide to leaves, metabolites were extracted and analysed. The metabolites identified consisted of mono-demethylated-, di-demethylated- and ring methyl-hydroxylated chlorotoluron. Metabolism was more extensive in the resistant biotype, yielding principally the non-phytotoxic ring methyl-hydroxylated metabolite. The metabolites observed are characteristic of the activity of cytochrome P450 mixed-function oxygenase action. The specific cytochrome P450 inhibitor, 1-aminobenzotriazole, reduced accumulation of the ring methyl-hydroxylated metabolite in the resistant biotype.     

Real-Time Fluorescent Polymerase Chain Reaction Detection of Phytophthora ramorum and Phytophthora pseudosyringae Using Mitochondrial Gene Regions

ABSTRACT A real-time fluorescent polymerase chain reaction (PCR) detection method for the sudden oak death pathogen Phytophthora ramorum was developed based on mitochondrial DNA sequence with an ABI Prism 7700 (TaqMan) Sequence Detection System. Primers and probes were also developed for detecting P. pseudosyringae, a newly described species that causes symptoms similar to P. ramorum on certain hosts. The species-specific primer-probe systems were combined in a multiplex assay with a plant primer-probe system to allow plant DNA present in extracted samples to serve as a positive control in each reaction. The lower limit of detection of P. ramorum DNA was 1 fg of genomic DNA, lower than for many other described PCR procedures for detecting Phytophthora species. The assay was also used in a three-way multiplex format to simultaneously detect P. ramorum, P. pseudosyringae, and plant DNA in a single tube. P. ramorum was detected down to a 10(-5) dilution of extracted tissue of artificially infected rhododendron 'Cunningham's White', and the amount of pathogen DNA present in the infected tissue was estimated using a standard curve. The multiplex assay was also used to detect P. ramorum in infected California field samples from several hosts determined to contain the pathogen by other methods. The real-time PCR assay we describe is highly sensitive and specific, and has several advantages over conventional PCR assays used for P. ramorum detection to confirm positive P. ramorum finds in nurseries and elsewhere.     

Detection and Quantification of Phytophthora ramorum from California Forests Using a Real-Time Polymerase Chain Reaction Assay

ABSTRACT The timely and accurate detection of pathogens is a critical aid in the study of the epidemiology and biology of plant diseases. In the case of regulated organisms, the availability of a sensitive and reliable assay is essential when trying to achieve early detection of the pathogen. We developed and tested a real-time, nested polymerase chain reaction (PCR) assay for the detection of Phytophthora ramorum, causal agent of sudden oak death. This technique then was implemented as part of a widespread environmental screen throughout California. The method here described is sensitive, detecting less than 12 fg of pathogen DNA, and is specific for P. ramorum when tested across 21 Phytophthora spp. Hundreds of symptomatic samples from 33 sites in 14 California counties were assayed, resulting in the discovery of 10 new host species and 23 infested areas, including 4 new counties. With the exception of a single host, PCR-based discovery of new hosts and infested areas always was confirmed by traditional pathogen isolations and inoculation studies. Nonetheless, molecular diagnostics were key in early pathogen detection, and steered the direction of further research on this newly discovered and generalist Phytophthora species.     

实时荧光RT-PCR方法检测黄瓜绿斑驳花叶病毒

根据黄瓜绿斑驳花叶病毒不同分离物外壳蛋白基因(CP)的保守序列,设计合成引物和TaqMan荧光探针,建立了黄瓜绿斑驳花叶病毒的实时荧光RT-PCR(Real time RT-PCR)检测方法.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PCR扩增与检测同步进行.结果表明,实时荧光RT-PCR方法检测灵敏度比普通RT-PCR高约10倍,并且具有快速、简便、准确的优点,适合于CGMMV的快速、高效检测.     

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.     

A Polymerase Chain Reaction (PCR) Assay for the Detection of Inoculum of Sclerotinia sclerotiorum

The development of a polymerase chain reaction (PCR) assay for the detection of inoculum of the plant pathogenic fungus Sclerotinia sclerotiorum is described. The PCR primers were designed using nuclear ribosomal DNA internal transcribed spacer sequences. Specific detection of DNA from S. sclerotiorum was possible even in the presence of a 40-fold excess of DNA from the closely related fungus Botrytis cinerea. PCR products were obtained from suspensions of untreated S. sclerotiorum ascospores alone, but DNA purification was required for detection in the presence of large numbers of B. cinerea conidiospores. Specific detection of inoculum of S. sclerotiorum was possible in field-based air-samples, using a Burkard spore trap, and from inoculated oilseed rape petals. The assay has potential for incorporation into a risk management system for S. sclerotiorum in oilseed rape crops.     

Detection of the Pinewood Nematode, Bursaphelenchus xylophilus, Using a Real-Time Polymerase Chain Reaction Assay

ABSTRACT The pinewood nematode, Bursaphelenchus xylophilus, has caused significant damage to pine plantations both in East Asia and North America and is an important quarantine organism. A real-time polymerase chain reaction (PCR) assay was developed to detect B. xylophilus. A set of primers and probe specific for B. xylophilus was designed to target the ribosomal DNA internal transcribed spacer region. Optimal primer concentration, Mg(2+) concentration, and extension temperature were 400 nM, 3.0 mM, and 60 degrees C, respectively. The assay was highly specific and sensitive, detecting as little as 0.01 ng of B. xylophilus DNA. The real-time PCR assay also successfully detected B. xylophilus in field samples, and it should be very useful for quarantine purposes.     

Pheromone-Related Inhibitors of Ustilago hordei Mating and Tilletia tritici Teliospore Germination

ABSTRACT Ustilago hordei, the causal agent of barley covered smut, produces mating pheromones that break down to smaller peptide compounds that act as potent inhibitors of mating and germination in several fungi. The pheromones are members of the farnesylated family of proteins. Synthetic peptide analogs of the pheromone derivatives, ranging in size from 4 mers to full length pheromones, were farnesylated, methyl esterified, or both and tested for mating or teliospore germination inhibition with U. hordei or Tilletia tritici, respectively. N-Acetyl-S-farnesylcysteine, which inhibits processing of Ras, and other sulfur-containing compounds such as homocysteine or methionine, were likewise modified and tested. The most potent inhibitors were methionine methyl ester and modified 4-mer peptides from both pheromones. Alanine scanning of the inhibitory 4 mers determined that the native amino acid sequence was specific for a high level of activity. The sulfur amino acids appear to be required for inhibition. Glasshouse studies using selected antagonists of mating and teliospore germination as seed treatments inhibited covered smut of barley and common bunt of wheat, although the level of control was inconsistent. The use of pheromone-related antagonists to mating or teliospore germination is a promising, novel strategy for control of smut and bunt diseases.     

Real-Time Polymerase Chain Reaction Quantification of Phytophthora capsici in Different Pepper Genotypes

ABSTRACT Reliable and sensitive quantification of Phytophthora capsici in pepper plants is of crucial importance in managing the multiple syndromes caused by this pathogen. A real-time polymerase chain reaction (PCR) assay was developed for the determination of P. capsici in pepper tissues. DNA levels of a highly virulent and a less virulent isolate were measured in different pepper genotypes with varying degrees of resistance. Using SYBR Green and specific primers for P. capsici, the minimal amount of pathogen DNA quantified was 10 pg. Pathogen DNA was recorded as early as 8 h postinoculation. Thereafter, the increase was rapid in susceptible cultivars and slower in resistant ones. The amount of pathogen DNA quantified in each pepper genotype correlated with susceptibility to Phytophthora root rot. Likewise, there was a relationship between the virulence of the pathogen and the degree of colonization. Differences also were found in oomycete amount among pepper tissues, with maximal pathogen biomass occurring in stems. The real-time PCR technique developed in this study was sensitive and robust enough to assess both pathogen development and resistance to Phytophthora root rot in different pepper genotypes.     

Molecular Detection of Phytophthora ramorum by Real-Time Polymerase Chain Reaction Using TaqMan, SYBR Green, and Molecular Beacons

ABSTRACT Sudden oak death, caused by Phytophthora ramorum, is a severe disease that affects many species of trees and shrubs. This pathogen is spreading rapidly and quarantine measures are currently in place to prevent dissemination to areas that were previously free of the pathogen. Molecular assays that rapidly detect and identify P. ramorum frequently fail to reliably distinguish between P. ramorum and closely related species. To overcome this problem and to provide additional assays to increase confidence, internal transcribed spacer (ITS), beta-tubulin, and elicitin gene regions were sequenced and searched for polymorphisms in a collection of Phytophthora spp. Three different reporter technologies were compared: molecular beacons, TaqMan, and SYBR Green. The assays differentiated P. ramorum from the 65 species of Phytophthora tested. The assays developed were also used with DNA extracts from 48 infected and uninfected plant samples. All environmental samples from which P. ramorum was isolated by PARP-V8 were detected using all three real-time PCR assays. However, 24% of the samples yielded positive real-time PCR assays but no P. ramorum cultures, but sequence analysis of the coxI and II spacer region confirmed the presence of the pathogen in most samples. The assays based on detection of the ITS and elicitin regions using TaqMan tended to have lower cycle threshold values than those using beta-tubulin and seemed to be more sensitive.     

Development of a High-Throughput Method for Quantification of Plasmopara viticola DNA in Grapevine Leaves by Means of Quantitative Real-Time Polymerase Chain Reaction

ABSTRACT Plasmopara viticola is a strictly biotrophic oomycete that causes downy mildew, which is one of the most important grapevine diseases. Control of the disease is most often achieved by fungicide applications, which may have severe environmental consequences. Therefore, alternative control strategies based on biocontrol agents (BCAs) are currently in development. Thousands of potential BCAs have to be screened for their antagonist efficacy against Plasmopara viticola. Evaluation of their effect on the pathogen can be achieved by detecting the amount of P. viticola DNA in leaves treated with potential antagonists and infected with the pathogen. In this study, a rapid high-throughput method was developed for relative quantification of P. viticola DNA directly from Vitis vinifera leaves by means of multiplex real-time quantitative polymerase chain reaction (PCR) with TaqMan chemistry. This method allows simultaneous amplification, but independent detection, of pathogen and host DNA by using species-specific primers and TaqMan probes that are labeled with different fluorescent dyes. Including detection of V. vinifera DNA in the tests is fundamental because it provides an endogenous reference and allows normalization for variations caused by sample-to-sample differences in DNA extraction, PCR efficiencies, and pipetting volumes. The developed method allows highly sensitive and specific detection of P. viticola DNA (minimal detectable quantity of 0.1 pg). Moreover, high precision and reproducibility of TaqMan assays were observed over a linear range of four orders of magnitude, confirming the reliability of the developed PCR assay. Potential applications range from screening for BCA efficiency to evaluation of fungicide efficacy, or assessment of host resistance.     

Enhanced Polymerase Chain Reaction Methods for Detecting and Quantifying Phytophthora infestans in Plants

ABSTRACT Sensitive and specific primer sets for polymerase chain reaction (PCR) for Phytophthora infestans, the oomycete that causes late blight of potato and tomato, were developed based on families of highly repeated DNA. The performance of these primers was compared to those developed previously for the internal transcribed spacer (ITS) of ribosomal DNA. The detection limit using the new primers is 10 fg of P. infestans DNA, or 0.02 nuclei. This is about 100 times more sensitive than ITS-directed primers. Nested polymerase chain reaction (PCR) allows the measurement of down to 0.1 fg of DNA using the new primers. To enhance the reliability of diagnostic assays, an internal positive control was developed using an amplification mimic. The mimic also served as a competitor for quantitative PCR, which was used to assess the growth of P. infestans in resistant and susceptible tomato. A key dimension of this study was that two laboratories independently checked the specificity and sensitivity of each primer set; differences were noted that should be considered when PCR is adopted for diagnostic applications in any system.