Faster, simpler, more-specific methods for improved molecular detection of Phytophthora ramorum in the field

Phytophthora ramorum is the causal agent of sudden oak death. The pathogen also affects a wide range of tree, shrub, and herbaceous species in natural and landscaped environments as well as plants in the nursery industry. A TaqMan real-time PCR method for the detection of this pathogen in the field has been described previously; this paper describes the development of a number of assays based on this method which have various advantages for use in the field. A scorpion real-time PCR assay that is twice as fast as TaqMan was developed, allowing the detection of P. ramorum in less than 30 min. Also designed was a loop-mediated isothermal amplification (LAMP) assay, which allowed sensitive and specific detection of P. ramorum in 45 min using only a heated block. A positive reaction was identified by the detection of the LAMP product by color change visible to the naked eye.     

Composting is an effective treatment option for sanitization of Phytophthora ramorum-infected plant material

AIMS: To determine the effects of heat and composting treatments on the viability of the plant pathogen Phytophthora ramorum grown on both artificial and various natural substrates. METHODS AND RESULTS: Phytophthora ramorum was grown on V8 agar, inoculated on bay laurel leaves (Umbellularia californica) and on woody tissues of coast live oak (Quercus agrifolia). Effects on growth, viability and survival were measured as a result of treatment in ovens and compost piles. Direct plating onto PARP medium and pear-baiting techniques were used to determine post-treatment viability. No P. ramorum was recovered at the end of the composting process, regardless of the isolation technique used. By using a PCR assay designed to detect the DNA of P. ramorum, we were able to conclude the pathogen was absent from mature compost and not merely suppressed or dormant. CONCLUSIONS: Some heat and composting treatments eliminate P. ramorum to lower than detectable levels on all substrates tested. SIGNIFICANCE AND IMPACT OF THE STUDY: Composting is an effective treatment option for sanitization of P. ramorum-infected plant material. Assaying for pathogen viability in compost requires a direct test capable of differentiating between pathogen suppression and pathogen elimination.     

Molecular detection of Puccinia horiana Henn. the causal agent of Chrysanthemum white rust

Chrysanthemum white rust is one of the most important foliar diseases of pot chrysanthemum and is a quarantine pathogen in many countries. Under conducive environmental conditions, it has the potential to completely destroy susceptible cultivars. This is mainly avoided through frequent preventive fungicide applications. As part of a research program to develop a disease warning system, a molecular detection method was developed. To determine the nucleotide sequence of the nuclear rDNA-ITS (internal transcribed spacer) region of P. horiana, 56 isolates were collected between 2003 and 2006 from diseased commercial chrysanthemum plants from different national and international geographical areas. DNA was isolated from the basidiospores or teliospores from several isolates and the rDNA-ITS region was cloned and sequenced. Based on the limited variability in rDNA-ITS sequence between these isolates, several primer pairs were designed and tested for detection through conventional and real-time PCR. Specificity of detection was cross-checked against a variety of other fungi (saprophytes and other rusts) that may occur in the same environment, and against DNA of healthy chrysanthemum leaves. Using the best primers, the PCR-based methods successfully detected all the P. horiana isolates tested, while no signal was observed with other rust species up to 1 ng non target genomic DNA template. The limit of detection of P. horiana DNA in conventional, nested and real-time PCR was 10 pg, 10 fg and 10 fg, respectively. The DNA extraction method and PCR template concentration were optimized to maximize the recoverability of the pathogen from infected plant tissue. Using the optimized real-time PCR method, the pathogen could be detected in washed plant tissue, 9 hours after inoculation. Hence, this method allows detection of the P. horiana in any part of its latent stage and will also serve as a tool for studying the biology and epidemiology of the pathogen.     

Faster, Simpler, More-Specific Methods for Improved Molecular Detection of Phytophthora ramorum in the Field

Phytophthora ramorum is the causal agent of sudden oak death. The pathogen also affects a wide range of tree, shrub, and herbaceous species in natural and landscaped environments as well as plants in the nursery industry. A TaqMan real-time PCR method for the detection of this pathogen in the field has been described previously; this paper describes the development of a number of assays based on this method which have various advantages for use in the field. A scorpion real-time PCR assay that is twice as fast as TaqMan was developed, allowing the detection of P. ramorum in less than 30 min. Also designed was a loop-mediated isothermal amplification (LAMP) assay, which allowed sensitive and specific detection of P. ramorum in 45 min using only a heated block. A positive reaction was identified by the detection of the LAMP product by color change visible to the naked eye.     

Molecular detection of Puccinia horiana in Chrysanthemum x morifolium through conventional and real-time PCR

Puccinia horiana Henn. is a quarantine organism and one of the most important fungal pathogens of Chrysanthemum x morifolium cultivars grown for cut flower or potted plant production (florist's chrysanthemum) in several regions of the world. Highly specific primer pairs were identified for conventional, nested, and real-time PCR detection of P. horiana based on the specific and sensitive PCR amplification of selected regions in the internal transcribed spacers (ITS1 and ITS2) of the nuclear ribosomal DNA (rDNA). Using these different PCR versions, 10 pg, 10 fg, and 5 fg genomic DNA could be detected, respectively. When using cloned target DNA as template, the detection limits were 5000, 50, and 5 target copies, respectively. These detection limits were not affected by a background of chrysanthemum plant DNA. The DNA extraction method was optimized to maximize the recoverability of the pathogen from infected plant tissue. A CTAB extraction protocol or a selection of commercial DNA extraction methods allowed the use of 10 ng total (plant+pathogen) DNA without interference of PCR inhibitors. Due to the specificity of the primers, SYBR Green I technology enabled reliable real time PCR signal detection. However, an efficient TaqMan probe is available. The lowest proportion of infected plant material that could still be detected when mixed with healthy plant material was 0.001%. The real-time PCR assay could detect as few as eight pure P. horiana basidiospores, demonstrating the potential of the technique for aerial detection of the pathogen. The amount of P. horiana DNA in plant tissue was determined at various time points after basidiospore inoculation. Using the real-time PCR protocol, it was possible to detect the pathogen immediately after the inoculation period, even though the accumulation of pathogen DNA was most pronounced near the end of the latent period. The detection system proved to be accurate and sensitive and could help not only in pathogen diagnosis but also in pathogen monitoring and disease forecasting systems.     

Development and evaluation of a point-of-care test with a combination of EZ-Fast DNA extraction and real-time PCR and LAMP detection: evaluation using blood samples containing the bovine leukaemia DNA

Along with progress in globalization of society, the spread of infectious diseases has accelerated worldwide. The deployment of highly sensitive genetic tests is essential for early diagnosis and early containment of potential outbreaks and epidemics, as well as routine surveillance, although tedious and expensive nucleic acid extraction steps represent a major drawback. Here we developed a simple and rapid DNA extraction method, named as an EZ-Fast kit, applicable to the field setting. The kit does not require advanced laboratory equipment or expensive DNA extraction kits and achieves crude DNA extraction within 10 min at extremely low cost and can easily be performed in field settings. When combined with real-time PCR and LAMP analyses, the performance of the POCT, using 183 bovine blood samples, was similar to that of the existing DNA extraction method: 92·5% (135/146) (real-time PCR) and 93·7% (133/142) (LAMP) diagnostic sensitivities, and 100% diagnostic specificities. The developed POCT provides a powerful tool to facilitate on-site diagnosis in a field setting.     

Real-time PCR detection of Biscogniauxia mediterranea in symptomless oak tissue

AIMS: Real-time PCR, based on TaqMan chemistry, was used to detect Biscogniauxia mediterranea, a fungal pathogen that after a long endophytic phase may cause charcoal disease in oak trees. METHODS AND RESULTS: Specific primers and probe were designed and tested on axenic cultures of B. mediterranea and other fungi commonly colonizing oaks. Twig samples were collected in Tuscany from apparently healthy oaks (Quercus cerris, Quercus ilex and Quercus pubescens) growing near trees infected with the fungus. Twigs were divided into two groups: one for isolation in agar plates, and one for real-time PCR after DNA extraction. The detection limit of the assay was 0.01 pg/DNA, whereas the amounts of fungal DNA detected in asymptomatic tissue were >0.5 pg microg(-1) total DNA extracted. In the apparently healthy twigs the frequency of isolation found on agar was 25.0%, much lower than that with real-time PCR (96.4%). CONCLUSIONS: Real-time PCR is a sensitive and fast technique able to specifically detect and quantify the DNA of B. mediterranea in oak tissue. SIGNIFICANCE AND IMPACT OF THE STUDY: This diagnostic method is a precise tool to localize fungi in symptomless plant tissues and promises to advance our understanding of fungal infection during their latent phase.     

Emergence of the sudden oak death pathogen Phytophthora ramorum

The recently emerged plant pathogen Phytophthora ramorum is responsible for causing the sudden oak death epidemic. This review documents the emergence of P. ramorum based on evolutionary and population genetic analyses. Currently infection by P. ramorum occurs only in Europe and North America and three clonal lineages are distinguished: EU1, NA1 and NA2. Ancient divergence of these lineages supports a scenario in which P. ramorum originated from reproductively isolated populations and underwent at least four global migration events. This recent work sheds new light on mechanisms of emergence of exotic pathogens and provides crucial insights into migration pathways.     

Susceptibility to Phytophthora ramorum in a key infectious host: landscape variation in host genotype, host phenotype, and environmental factors

Sudden oak death is an emerging forest disease caused by the invasive pathogen Phytophthora ramorum. Genetic and environmental factors affecting susceptibility to P. ramorum in the key inoculum-producing host tree Umbellularia californica (bay laurel) were examined across a heterogeneous landscape in California, USA. Laboratory susceptibility trials were conducted on detached leaves and assessed field disease levels for 97 host trees from 12 225-m(2) plots. Genotype and phenotype characteristics were assessed for each tree. Effects of plot-level environmental conditions (understory microclimate, amount of solar radiation and topographic moisture potential) on disease expression were also evaluated. Susceptibility varied significantly among U. californica trees, with a fivefold difference in leaf lesion size. Lesion size was positively related to leaf area, but not to other phenotypic traits or to field disease level. Genetic diversity was structured at three spatial scales, but primarily among individuals within plots. Lesion size was significantly related to amplified fragment length polymorphism (AFLP) markers, but local environment explained most variation in field disease level. Thus, substantial genetic variation in susceptibility to P. ramorum occurs in its principal foliar host U. californica, but local environment mediates expression of susceptibility in nature.     

Development of a Real-Time Microchip PCR System for Portable Plant Disease Diagnosis

Rapid and accurate detection of plant pathogens in the field is crucial to prevent the proliferation of infected crops. Polymerase chain reaction (PCR) process is the most reliable and accepted method for plant pathogen diagnosis, however current conventional PCR machines are not portable and require additional post-processing steps to detect the amplified DNA (amplicon) of pathogens. Real-time PCR can directly quantify the amplicon during the DNA amplification without the need for post processing, thus more suitable for field operations, however still takes time and require large instruments that are costly and not portable. Microchip PCR systems have emerged in the past decade to miniaturize conventional PCR systems and to reduce operation time and cost. Real-time microchip PCR systems have also emerged, but unfortunately all reported portable real-time microchip PCR systems require various auxiliary instruments. Here we present a stand-alone real-time microchip PCR system composed of a PCR reaction chamber microchip with integrated thin-film heater, a compact fluorescence detector to detect amplified DNA, a microcontroller to control the entire thermocycling operation with data acquisition capability, and a battery. The entire system is 25×16×8 cm³ in size and 843 g in weight. The disposable microchip requires only 8-µl sample volume and a single PCR run consumes 110 mAh of power. A DNA extraction protocol, notably without the use of liquid nitrogen, chemicals, and other large lab equipment, was developed for field operations. The developed real-time microchip PCR system and the DNA extraction protocol were used to successfully detect six different fungal and bacterial plant pathogens with 100% success rate to a detection limit of 5 ng/8 µl sample.     

A five-minute DNA extraction method for expedited detection of Phytophthora ramorum following prescreening using Phytophthora spp. lateral flow devices

In a direct comparison with established methods for Phytophthora ramorum detection (isolation followed by morphological identification, or conventional DNA extraction followed by TaqMan real-time PCR) a rapid, simplified detection method in which membranes of lateral flow devices (LFDs) are added directly to TaqMan real-time PCR reactions was used to test 202 plant samples collected by plant health inspectors in the field. P. ramorum prevalence within the 202 samples was approximately 40% according to routine testing by isolation or TaqMan real-time PCR. The diagnostic sensitivity and specificity of the rapid detection method were 96.3% and 91.2%, respectively. This method can be used in conjunction with Phytophthora spp. lateral flow devices to reduce the number of samples requiring testing using more laborious conventional methods. The effect of combining prescreening for Phytophthora spp. with P. ramorum-specific tests is discussed in terms of the positive and negative predictive values of species-specific detection when testing samples collected in different inspection scenarios.     

Quantitative PCR detection of Batrachochytrium dendrobatidis DNA from sediments and water

The fungal pathogen Batrachochytrium dendrobatidis (Bd) causes chytridiomycosis, a disease implicated in amphibian declines on 5 continents. Polymerase chain reaction (PCR) primer sets exist with which amphibians can be tested for this disease, and advances in sampling techniques allow non-invasive testing of animals. We developed filtering and PCR based quantitative methods by modifying existing PCR assays to detect Bd DNA in water and sediments, without the need for testing amphibians; we tested the methods at 4 field sites. The SYBR based assay using Boyle primers (SYBR/Boyle assay) and the Taqman based assay using Wood primers performed similarly with samples generated in the laboratory (Bd spiked filters), but the SYBR/Boyle assay detected Bd DNA in more field samples. We detected Bd DNA in water from 3 of 4 sites tested, including one pond historically negative for chytridiomycosis. Zoospore equivalents in sampled water ranged from 19 to 454 1(-1) (nominal detection limit is 10 DNA copies, or about 0.06 zoospore). We did not detect DNA of Bd from sediments collected at any sites. Our filtering and amplification methods provide a new tool to investigate critical aspects of Bd in the environment.     

Quantification of conidial density of Aspergillus flavus and A. parasiticus in soil from almond orchards using real-time PCR

Aims:  To design the Aspergillus flavus and Aspergillus parasiticus -specific primers and a real-time PCR assay for quantification of the conidial density in soil.
Methods and Results:  Aspergillus flavus and A. parasiticus -specific DNA primers were designed based on internal transcribed spacer sequences to distinguish these two species and from other Aspergillus and other fungal species. A method of pathogen DNA extraction directly from soil samples was developed. Using the designed primers, a real-time PCR assay was developed to quantitatively determine the conidial density of each A. flavus and A. parasiticus in soil, after generating corresponding standard curves. Known conidial densities of each A. flavus or A. parasiticus in soil significantly correlated with those tested with the real-time PCR.
Conclusions:  This study demonstrated the applicability of the real-time PCR assay in studies of quantifying A. flavus and A. parasiticus in soil as inoculum sources.
Significance and Impact of the Study:  The A. flacus and A. parasitic -specific primers can be widely used in aflatoxin research. The real-time PCR assay developed in this study provides a potential approach to quantify the plant pathogen density from not only soil but also other sources in relation to aflatoxin contamination from environment, food and feed commodities.     

A rapid DNA extraction method for PCR identification of fungal indoor air contaminants

Following air sampling fungal DNA needs to be extracted and purified to a state suitable for laboratory use. Our laboratory has developed a simple method of extraction and purification of fungal DNA appropriate for enzymatic manipulation and Polymerase Chain Reaction (PCR) applications. The methodology described is both rapid and cost effective for use with multiple fungal organisms.     

Ancient isolation and independent evolution of the three clonal lineages of the exotic sudden oak death pathogen Phytophthora ramorum

The genus Phytophthora includes some of the most destructive plant pathogens affecting agricultural and native ecosystems and is responsible for a number of recent emerging and re-emerging infectious diseases of plants. Sudden oak death, caused by the exotic pathogen P. ramorum , has caused extensive mortality of oaks and tanoaks in Northern California, and has brought economic losses to US and European nurseries as well due to its infection of common ornamental plants. In its known range, P. ramorum occurs as three distinct clonal lineages. We inferred the evolutionary history of P. ramorum from nuclear sequence data using coalescent-based approaches. We found that the three lineages have been diverging for at least 11% of their history, an evolutionarily significant amount of time estimated to be on the order of 165 000 to 500 000 years. There was also strong evidence for historical recombination between the lineages, indicating that the ancestors of the P. ramorum lineages were members of a sexually reproducing population. Due to this recombination, the ages of the lineages varied within and between loci, but coalescent analyses suggested that the European lineage may be older than the North American lineages. The divergence of the three clonal lineages of P. ramorum supports a scenario in which the three lineages originated from different geographic locations that were sufficiently isolated from each other to allow independent evolution prior to introduction to North America and Europe. It is thus probable that the emergence of P. ramorum in North America and Europe was the result of three independent migration events.     

Development of a sensitive assay for the detection of Pseudoloma neurophilia in laboratory populations of the zebrafish Danio rerio

The zebrafish Danio rerio is an increasingly important biological model in many areas of research. Due to the potential for non-protocol-induced variation, diseases of zebrafish, especially those resulting in chronic, sub-lethal infections, are of great concern. The microsporidium Pseudoloma neurophilia is a common parasite of laboratory zebrafish. Current methods for detection of this parasite require lethal sampling of fish, which is often undesirable with poorly spawning mutant lines and small populations. We present here an improved molecular-based diagnostic assay using real-time polymerase chain reaction (PCR), and including sonication treatment prior to DNA extraction. Comparisons of several DNA extraction methods were performed to determine the method providing the maximum sensitivity. Sonication was found to be the most effective method for disrupting spores. Compared to previously published data on PCR-based assay using a dilution experiment, sensitivity is increased. This shows that our assay, which includes sonication, is capable of detecting parasite DNA at 1 log higher dilution than the conventional PCR-based assay, which does not include sonication. Furthermore, we demonstrate the application of this method to testing of water, eggs, and sperm, providing a potential non-lethal method for detection of this parasite in zebrafish colonies with a sensitivity of 10 spores 1(-1) of water, 2 spores per spiked egg sample, and 10 spores microl(-1) of spiked sperm sample.     

Evaluating the effects of laboratory protocols on eDNA detection probability for an endangered freshwater fish

The effectiveness and accuracy of detection using environmental DNA (eDNA) is dependent on understanding the influence laboratory methods such as DNA extraction and PCR strategies have on detection probability. Ideally choice of sampling and extraction method will maximize eDNA yield and detection probability. Determining the survey effort required to reach a satisfactory detection probability (via increased PCR replicates or more sampling) could compensate for a lower eDNA yield if the sampling and extraction method has other advantages for a study, species or system. I analysed the effect of three different sampling and extraction methods on eDNA yield, detection probability and PCR replication for detecting the endangered freshwater fish Macquaria australasica from water samples. The impact of eDNA concentration, PCR strategy, target amplicon size and two marker regions: 12S (a mitochondrial gene) and 18S (a nuclear gene) was also assessed. The choice of sampling and extraction method and PCR strategy, rather than amplicon size and marker region, had the biggest effect on detection probability and PCR replication. The PCR replication effort required to achieve a detection probability of 0.95, ranged from 2 to 6 PCR replicates depending on the laboratory method used. As all methods yielded eDNA from which M. australasica was detected using the three target amplicons, differences in eDNA yield and detection probability between the three methods could be mitigated by determining the appropriate PCR replication effort. Evaluating the effect sampling and extraction methods will have on the detection probability and determining the laboratory protocols and PCR replication required to maximize detection and minimize false positives and negatives is a useful first step for eDNA occupancy studies.     

Rapid identification of Phytophthora ramorum using PCR-SSCP analysis of ribosomal DNA ITS-1

AIMS: The primary objectives of this study were to determine if a single-strand conformation polymorphism (SSCP) analysis can be used for rapid identification of Phytophthora ramorum, an important quarantine plant pathogen worldwide, and to further assess the potential of the SSCP technique as a taxonomic tool for the genus Phytophthora. METHODS AND RESULTS: SSCP of ribosomal DNA internal transcribed spacer 1 was characterized for 12 isolates of P. ramorum, using a recently reported protocol. The SSCP patterns of this species then were compared with those of 18 closely related Phytophthora species. Phytophthora ramorum had a unique pattern and was easily distinguished from genetically, morphologically and ecologically close relatives. CONCLUSION: An immediate benefit of this study is provision of a highly effective and efficient identification tool for P. ramorum in the quarantine process. SIGNIFICANCE AND IMPACT OF THE STUDY: This study also provides additional evidence demonstrating that the SSCP is an ideal DNA marker for species differentiation within the genus Phytophthora.     

Quantitative analysis of phytopathogenic ascomycota on leaves of pedunculate oaks (Quercus robur L.) by real-time PCR

Leaves of oak trees are often infected by various pathogenic fungi. As it is difficult to remove such organisms quantitatively from the leaf surface and as it is often impossible to grow these organisms independently from their host, there are almost no molecular data available from these oak leaf specific pathogens. For the quantitative removal of the microorganisms a procedure was developed combining a wax and microorganism freezing method with a DNA extraction technique. For the development of a species specific detection, DNA of pathogenic filamentous fungi was isolated from hyphae of the upper leaf surface of Quercus robur. Three different species could be identified as (i) Cladosporium sp., (ii) Ramularia sp. and (iii) Microsphaera alphitoides by amplifying and sequencing an 18S-28S segment of their rDNA. For the final quantification a real-time PCR protocol was established allowing the species specific quantification of the three pathogenic filamentous fungi. The whole procedure was successfully applied to quantify the amount of the three species on oak leaves collected in autumn.