Detection Of Plant Pathogens Research Articles

DNA Extraction from Plant Leaves Using a Microneedle Patch.

Isolation of high-quality DNA from infected plant specimens is an essential step for the molecular detection of plant pathogens. However, DNA isolation from plant cells surrounded by rigid polysaccharide cell walls involves complicated steps and requires benchtop laboratory equipment. As a result, plant DNA extraction is currently confined to well-equipped laboratories and sample preparation has become one of the major hurdles for on-site molecular detection of plant pathogens. To overcome this hurdle, a simple DNA extraction method from plant leaf tissues has been developed. A microneedle (MN) patch made of polyvinyl alcohol (PVA) can isolate plant or pathogenic DNA from different plant species within a minute. During DNA extraction, the polymeric MN patch penetrates into plant leaf tissues and breaks rigid plant cell walls to isolate intracellular DNA. The extracted DNA is polymerase chain reaction (PCR) amplifiable without additional purification. This minimally invasive method has successfully extracted Phytophthora infestans DNA from infected tomato leaves. Moreover, the MN patch could be used to isolate DNA from other plant pathogens directly in the field. Thus, it has great potential to become a rapid, on-site sample preparation technique for plant pathogen detection. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Microneedle patch-based DNA extraction Support Protocol 1: Microneedle patch fabrication Support Protocol 2: Real-time PCR amplification of microneedle patch extracted DNA.

Technological Advances in Phytopathogen Detection and Metagenome Profiling Techniques.

The use of advanced molecular methods in plant pathology and applied microbiology has necessitated for more accurate, rapid detection and identification of plant pathogens. This is particularly significant given accelerated emergence of virulence that leads to increased prevalence of plant pathogens. Thus, the capacity to contain plant pathogens and ultimately disease progression is key to ensuring crop biosecurity and overall food security. Of recent, research on pathogens utilizes a holistic approach focusing on elucidating growth dynamics within the entire biome rather than studying individual or closely related isolates in unison. This has advanced knowledge and information of microbial ecosystem within natural environments in the twenty first century. Applied technological platforms used for rapid detection and profiling microbial biomes in this regard include digital PCR, pyrosequencing, Illumina, DNA microarray and barcoding, Ion torrent, and nanopore. These technologies have been applied in various fields including human health and medicine, marine and animal biology, crop production and water quality research, to mention but a few. Although much has been done and achieved through the development of several technologies, more accuracy is required to circumvent the shortfalls still experienced. This includes integrating existing methods with new applications such as viability PCRs and microbial viability testing. Hence, this review provides critical analysis of some widely used latest technologies in rapid detection and identification of plant pathogens, and profiling plant associated microbiomes that reveal growth dynamics and population diversity. The advantages and limitations of the technologies are also discussed.

Affimer-Enzyme-Inhibitor Switch Sensor for Rapid Wash-free Assays of Multimeric Proteins.

Robust technology is required to underpin rapid point-of-care and in-field diagnostics to improve timely decision making across broad sectors. An attractive strategy combines target recognition and signal generating elements into an "active" enzyme-switch that directly transduces target-binding into a signal. However, approaches that are broadly applicable to diverse targets remain elusive. Here, an enzyme-inhibitor switch sensor was developed by insertion of non-immunoglobulin Affimer binding proteins, between TEM1-β-lactamase and its inhibitor protein, such that target binding disrupts the enzyme-inhibitor complex. Design principles for a successful switch architecture are illustrated by the rapid (min), simple (wash-free), and sensitive (pM) quantification of multimeric target analytes in biological samples (serum, plasma, leaf extracts), across three application areas. A therapeutic antibody (Herceptin), protein biomarker (human C-reactive protein), and plant virus (cow pea mosaic virus) were targeted, demonstrating assays for therapeutic drug monitoring, health diagnostics, and plant pathogen detection, respectively. Batch-to-batch reproducibility, shelf-life stability, and consistency with validated enzyme-linked immunosorbent assay analysis confirm that the principle of an Affimer-enzyme-inhibitor switch provides a platform for point-of-care and in-field diagnostics.

English

Detection and identification of plant pathogens is one of the most important strategies for sustainable plant diseases management. For this reason, the availability of fast, sensitive and accurate methods for detection and identification of plant pathogens is increasingly necessary to improve disease control decision making process. In other words, new technologies and improved methods with reduced/fair cost and improved speed, throughput, multiplexing, accuracy and sensitivity have emerged as an essential strategy for the control of both fungal and bacterial diseases. The development of recombinant DNA technology is also possible to isolate individual genes and incorporate resistance genes into otherwise agronomically acceptable cultivars to develop genetically resistance variety for a particular disease. These advances have been complemented by the development of new nucleic acids extraction methods, increased automation, reliable internal controls, multiplexing assays, online information and on site molecular diagnostics. The different types of polymerase chain reaction (PCR) are the most common DNA amplification technology used for detecting various plant pathogens. With the applications of bioinformatics as a modern technology in plant pathology, identification of specific motifs, DNA sequences has become possible, which ultimately increase the accuracy of modern techniques in plant disease diagnosis. The newly emerged proteomic technology is also a promising tool for providing information about pathogenicity and virulence factors that will open up new possibilities for plant disease diagnosis and appropriate protection measures. Key words: Biotechnology, molecular markers, marker assisted selection, quantitative trait loci (QTL), polymerase chain reaction (PCR), proteomics.

Development of a new methodology for the detection of Colletotrichum truncatum and Fusarium sp. in bell pepper seed

This study was carried out towards the development of rapid PCR-based assays for detection and identification of Colletotrichum truncatum, C. gloeosporioides sensu lato and Fusarium sp. infecting green and red bell pepper seeds in Trinidad using DNA templates produced by automated grinding and robotic paramagnetic particle capture. DNA was extracted from freshly collected symptomatic green and red bell pepper seeds using Promega’s Maxwell® 16 robotic system. The PCR assay of 220 seeds harvested from field-infected bell pepper fruit compared different optimization parameters including seeds that were symptomatic and asymptomatic of infection, the relative success of two different DNA polymerase enzymes, genus-specific and species-specific primers that were pre-existing in the literature in addition to primers that were newly designed for this study and duplex PCR for the simultaneous detection of DNA templates belonging to two different fungal species. The findings indicated that seeds carried dual infections of C. truncatum and Fusarium sp. Compared to other primers that were screened, our newly designed primers, ITS219-F/ITS419-R with GoTaq® Green Master Hot Start Taq DNA Polymerase, detected C. truncatum in the highest number of symptomatic seeds of infected green and red bell pepper fruit as well as in asymptomatic seeds of infected red bell pepper fruit. Fusarium sp. was detected only in red bell pepper seeds. Duplex PCR using ITS219-F/ITS419-R and Fa/Ra primers successfully simultaneously detected and identified C. truncatum and Fusarium sp. in symptomatic seeds of infected red bell pepper fruit. None of the seeds were infected with C. gloeosporioides sensu lato which is currently considered to be a minor pathogen in bell pepper in Trinidad. PCR-based detection methods are key tools in the detection and identification of fungal plant pathogens especially where seeds are an important inoculum source. The molecular approach as optimized in this study, is faster, more sensitive and the presence of single or dual fungal infections can be assessed in one assay.

Scanometric Detection of Tomato Leaf Curl New Delhi Viral DNA Using Mono- and Bifunctional AuNP-Conjugated Oligonucleotide Probes.

Scanometric detection of tomato leaf curl New Delhi viral DNA using AuNP-conjugated mono- and bifunctional oligo probes through direct DNA hybridization assay (DDH assay) and sandwich DNA hybridization assay (SDH assay) with silver enhancement was developed. Tomato leaf curl New Delhi virus (ToLCNDV) coat protein gene-specific thiol-modified ssoligo probes were used for the preparation of mono- and bifunctional AuNP-ssoligo probe conjugates (signal probes). ssDNA arrays were prepared using polymerase chain reaction (PCR), rolling circle amplification (RCA), genomic DNAs fragments, and phosphate-modified positive control/capture probes through 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/1-methylimidazole conjugation on the amine-modified glass slide (GS) surface. In the DDH assay, signal probes were directly hybridized with ssDNA array of positive control and ToLCNDV DNA samples and the detection signals were amplified by silver enhancement. Dark black/gray colors were developed on the GS by the result of Ag enhancement, which can be visualized and discriminated by the naked eye. The images were captured using a simple flatbed scanner, and the determined amounts of signal probes were hybridized with their target DNA. Similarly, the SDH assay also performed through two rounds of hybridization between capture probes and target DNA; target DNA and signal probes followed by silver enhancement. The detection signals were found higher in the PCR sample than the RCA and genomic DNA samples because of the presence of increased copy numbers of complementary DNAs in PCR samples. Further, bifunctional AuNP-ssoligo probe shows higher intensity of detection signal than monofunctional probes because it can be hybridized with both strands of dsDNA targets. Moreover, the DDH-based scanometric method showed higher detection sensitivity than the SDH assay-based scanometric method. Overall, bifunctional signal probes showed more detection sensitivity than monofunctional probes in scanometric methods based on both DDH and SDH assays. The limit of detection of this developed scanometric method was optimized (100 zM to 100 pM concentration). Further, DDH assay-based scanometric method shows significant advantages over the SDH assay method, such as cost-effectiveness, because it requires only single probes (signal probes), less time-consuming by the need of only single-step hybridization, and higher detection sensitivity (up to zM). To the best of our knowledge, this is the first attempt made to develop a scanometric-based nanoassay method for the detection of plant viral DNA. This approach will be a remarkable milestone for the application of nanotechnology in the development of nanobiosensor for plant pathogen detection.

Real-time loop-mediated isothermal amplification: an early-warning tool for quarantine plant pathogen detection

An effective framework for early warning and rapid response is a crucial element to prevent or mitigate the impact of biological invasions of plant pathogens, especially at ports of entry. Molecular detection of pathogens by using PCR-based methods usually requires a well-equipped laboratory. Rapid detection tools that can be applied as point-of-care diagnostics are highly desirable, especially to intercept quarantine plant pathogens such as Xylella fastidiosa, Ceratocystis platani and Phytophthora ramorum, three of the most devastating pathogens of trees and ornamental plants in Europe and North America. To this aim, in this study we developed three different loop mediated isothermal amplification (LAMP) assays able to detect each target pathogen both in DNA extracted from axenic culture and in infected plant tissues. By using the portable instrument Genie® II, the LAMP assay was able to recognize X. fastidiosa, C. platani and P. ramorum DNA within 30 min of isothermal amplification reaction, with high levels of specificity and sensitivity (up to 0.02 pg µL−1 of DNA). These new LAMP-based tools, allowing an on-site rapid detection of pathogens, are especially suited for being used at ports of entry, but they can be also profitably used to monitor and prevent the possible spread of invasive pathogens in natural ecosystems.

Improved detection and identification of the sudden oak death pathogenPhytophthora ramorumand the Port Orford cedar root pathogenPhytophthora lateralis

Early detection provides the best way to prevent introduction and establishment of alien plant pathogens. Amplification ofDNAbyPCRhas revolutionized the detection and monitoring of plant pathogens. Most of those assays rely on the amplification of a fraction of the genome of the targeted species. With the availability of whole genomes for a growing number of fungi and oomycetes it is becoming possible to compare genomes and discover regions that are unique to a target organism. This study has applied this pipeline to develop a set of hierarchical TaqMan real‐timePCRdetection assays targetingDNAof all fourPhytophthora ramorumlineages, and a closely related species,P. lateralis. Nine assays were generated: three targetingDNAof allP. ramorumlineages, one for each lineage ofP. ramorum, one forP. lateralisand one targetingDNAofP. ramorumandP. lateralis. These assays were very accurate and sensitive, ranging from 98.7% to 100% detection accuracy of 2–10 gene copies of the targeted taxa from pure cultures or inoculated tissues. This level of sensitivity is within the lowest theoretical limit of detection ofDNA. It is expected that these assays will be useful because of their high level of specificity and the ease with which they can be multiplexed because of the inherent flexibility in primer and probe design afforded by their lack of conservation in non‐target species.

Loop-mediated Isothermal Amplification assay for Detection of Candidatus Liberibacter Asiaticus, a Causal Agent of Citrus Huanglongbing.

Huanglongbing (HLB, Citrus greening disease) is one of the most devastating diseases that threaten citrus production worldwide. Although HLB presents systemically, low titer and uneven distribution of these bacteria within infected plants can make reliable detection difficult. It was known loop-mediated isothermal amplification (LAMP) method has the advantages of being highly specific, rapid, efficient, and laborsaving for detection of plant pathogens. We developed a new LAMP method targeting gene contained tandem repeat for more rapid and sensitive detection of Candidatus Liberibacter asiaticus (CLas), putative causal agent of the citrus huanglongbing. This new LAMP method was 10 folds more sensitive than conventional PCR in detecting the HLB pathogen and similar to that of real-time PCR in visual detection assay by adding SYBR Green I to mixture and 1% agarose gel electrophoresis. Positive reactions were achieved in reaction temperature 57, 60 and 62°C but not 65°C. Although this LAMP method was not more sensitive than real-time PCR, it does not require a thermocycler for amplification or agarose gel electrophoresis for resolution. Thus, we expect that this LAMP method shows strong promise as a reliable, rapid, and cost-effective method of detecting the CLas in citrus and can be applied for rapid diagnosis is needed.

Diagnosis of plant diseases using the Nanopore sequencing platform

Reliable detection and identification of plant pathogens are essential for disease control strategies. Diagnostic methods commonly used to detect plant pathogens have limitations such as requirement of prior knowledge of the genome sequence, low sensitivity and a restricted ability to detect several pathogens simultaneously. The development of advanced DNA sequencing technologies has enabled determination of total nucleic acid content in biological samples. The possibility of using the single‐molecule sequencing platform of Oxford Nanopore as a general method for diagnosis of plant diseases was examined. It was tested by sequencing DNA or RNA isolated from tissues with symptoms from plants of several families inoculated with known pathogens (e.g. bacteria, viruses, fungi, phytoplasma). Additionally, samples of groups of 200 seeds containing one infected seed of each of two or three pathogens, as well as samples with symptoms but unidentified pathogens were tested. Sequencing results were analysed with Nanopore data analysis tools. In all the inoculated plants, pathogens were identified in real time within 1–2 h of running the Nanopore sequencer and were classified to the species or genus level. DNA sequencing or direct RNA sequencing of samples with unidentified disease agents were validated by conventional diagnostic procedures (e.g. PCR, ELISA, Koch test), which supported the results obtained by Nanopore sequencing. The advantages of this technology include: long read lengths, fast run times, portability, low cost and the possibility of use in every laboratory. This study indicates that adoption of the Nanopore platform will be greatly advantageous for routine laboratory diagnosis.

Label free detection of plant viruses with organic transistor biosensors

Plum Pox Virus (PPV) is the pathogen responsible for Sharka, a highly infectious disease affecting stone fruit trees and causing severe economic damages, which can be only contained through early-detection and frequent monitoring. We propose a bioelectronic PPV biosensor, based on a electrolyte-gated organic field-effect transistor (EGOFET), for the specific detection of PPV in plant extracts with a sub ng/ml detection limit. The sensing unit of the biosensor is based on anti-PPV antibodies, uniformly oriented on the gold gate electrode by using a sub-monolayer of Protein G. The sensitivity and dynamic range of the EGOFET-based biosensor are comparable to those of commercially available platforms for detection of plant pathogens. This novel electronic immunosensor is compatible with low-cost fabrication procedures and can be easily reconfigured into a fully portable device to be operated in greenhouse and in the field orchards.

Simple Detection of Cochliobolus Fungal Pathogens in Maize.

Northern corn leaf spot and southern corn leaf blight caused by Cochliobolus carbonum (anamorph, Bipolaris zeicola) and Cochliobolus heterostrophus (anamorph, Bipolaris maydis), respectively, are common maize diseases in Korea. Accurate detection of plant pathogens is necessary for effective disease management. Based on the polyketide synthase gene (PKS) of Cochliobolus carbonum and the nonribosomal peptide synthetase gene (NRPS) of Cochliobolus heterostrophus, primer pairs were designed for PCR to simultaneously detect the two fungal pathogens and were specific and sensitive enough to be used for duplex PCR analysis. This duplex PCR-based method was found to be effective for diagnosing simultaneous infections from the two Cochliobolus species that display similar morphological and mycological characteristics. With this method, it is possible to prevent infections in maize by detecting infected seeds or maize and discarding them. Besides saving time and effort, early diagnosis can help to prevent infections, establish comprehensive management systems, and secure healthy seeds.

Thiol-Capped Gold Nanoparticle Biosensors for Rapid and Sensitive Visual Colorimetric Detection of Klebsiella pneumoniae.

In the last few years, gold nanoparticle biosensors have been developed for rapid, precise, easy and inexpensive with high specificity and sensitivity detection of human, plant and animal pathogens. Klebsiella pneumoniae serotype K2 is one of the common gram-negative pathogens with high prevalence. Therefore, it is essential to provide the effective and exclusive method to detect the bacteria. Klebsiella pneumoniae serotype K2 strain ATCC9997 genomic DNA was applied to establish the detection protocol either with thiol-capped oligonucleotide probes and gold nanoparticles or polymerase chain reaction based on K2A gene sequence. In the presence of the genomic DNA and oligonucleotide probes, a change in the color of gold nanoparticles and maximum changes in wavelength at 550-650nm was achieved. In addition, the result showed specificity of 15 × 105CFU/mL and 9pg/μL by gold nanoparticles probes. The lower limit of detection obtained by PCR method was 1pg/μL. Moreover, results demonstrated a great specificity of the designed primers and probes for colorimetric detection assay and PCR. Colorimetric detection using gold nanoparticle probe with advantages such as the lower time required for detection and no need for expensive detection instrumentation compared to the biochemical and molecular methods could be introduced for rapid, accurate detection of the bacteria.

Nanopore Sequencing as a Surveillance Tool for Plant Pathogens in Plant and Insect Tissues

Plant pathogens are constantly emerging and spreading into new areas and there are often limited postdiagnosis treatment options for infection, making surveillance key to their control. Here we present results from a study testing the efficacy of a portable nanopore-based massively parallel sequencing (MPS) technology for use in the detection of diverse plant pathogens in selected samples. The Oxford MinION device was coupled with whole transcriptome amplification (WTA) to sequence the metatranscriptome of plant and insect tissues infected with either Candidatus Liberibacter asiaticus or plum pox virus. Results showed that this methodology is useful for detecting unsuspected viral and bacterial pathogens in plant and insect tissues. The percentage of generated reads assigned to plum pox virus was 95% from infected tissue and 3% from the viruliferous insect, Myzus persicae. Diaphorina citri sequencing led to 22% of the reads mapping as Ca. L. asiaticus. Plum pox virus and Ca. L. asiaticus were detected in both tissue and insect samples near the beginning of each sequencing run, demonstrating the capability of this methodology to obtain results rapidly. This approach also proved the capability of this system to determine the major components of the insect vector's microbiome and the specific strain of small-genome, high-titer pathogens.

Detection and Identification of Fungal Infections in Intact Wheat and Sorghum Grain Using a Hand-Held Raman Spectrometer.

Global population growth drives increasing food demand, which is anticipated to increase by at least 20% over the next 15 years. Rapid detection and identification of plant pathogens allows for up to a 50% increase in the total agricultural yield worldwide. Current molecular methods for pathogen diagnostics, such as polymerase chain reaction (PCR), are costly, time-consuming, and destructive. These limitations recently catalyzed a push toward developing minimally invasive and substrate general techniques that can be used in the field for confirmatory detection and identification of plant pathogens. Raman spectroscopy (RS) is a noninvasive, nondestructive, and label-free technique that can be used to determine chemical structure of analyzed specimens. In this study, we demonstrate that by using a hand-held Raman spectrometer, we can identify whether wheat or sorghum grains are healthy or not and identify present plant pathogens. We show that RS enables diagnosis of simple diseases, such as ergot, that are caused by one pathogen, as well as complex diseases, such as black tip or mold, which are induced by several different pathogens. The combination of chemometric analysis and RS allows for distinguishing between healthy and infected grains with high accuracy. We also show that RS can be used to determine states of disease development on grain. These results demonstrate that Raman-based approach for disease detection on plants is sample agnostic.

Development of a quantitative loop-mediated isothermal amplification assay for the field detection of Erysiphe necator.

Plant pathogen detection systems have been useful tools to monitor inoculum presence and initiate management schedules. More recently, a loop-mediated isothermal amplification (LAMP) assay was successfully designed for field use in the grape powdery mildew pathosystem; however, false negatives or false positives were prevalent in grower-conducted assays due to the difficulty in perceiving the magnesium pyrophosphate precipitate at low DNA concentrations. A quantitative LAMP (qLAMP) assay using a fluorescence resonance energy transfer-based probe was assessed by grape growers in the Willamette Valley of Oregon. Custom impaction spore samplers were placed at a research vineyard and six commercial vineyard locations, and were tested bi-weekly by the lab and by growers. Grower-conducted qLAMP assays used a beta-version of the Smart-DART handheld LAMP reaction devices (Diagenetix, Inc., Honolulu, HI, USA), connected to Android 4.4 enabled, Bluetooth-capable Nexus 7 tablets for output. Quantification by a quantitative PCR assay was assumed correct to compare the lab and grower qLAMP assay quantification. Growers were able to conduct and interpret qLAMP results; however, the Erysiphe necator inoculum quantification was unreliable using the beta-Smart-DART devices. The qLAMP assay developed was sensitive to one spore in early testing of the assay, but decreased to >20 spores by the end of the trial. The qLAMP assay is not likely a suitable management tool for grape powdery mildew due to losses in sensitivity and decreasing costs and portability for other, more reliable molecular tools.

In‐field molecular diagnosis of plant pathogens: recent trends and future perspectives

Accurate management practices in crop health and food safety are critical, especially regarding the detection of plant pathogens in the early stages of a disease. To date, specific, fast and sensitive technologies for point‐of‐care diagnosis and simple or grower‐friendly devices are very valuable, as no specialized staff are required for diagnosing a disease in the field. This is especially the case today, when factors such as climate change may cause the appearance of pathogens in areas where years ago they were unexpected. The aim of this research is to review some of the promising techniques that can be applied to in‐field molecular detection of plant pathogens and how these techniques can change the way farmers and pathologists are diagnosing plant diseases. Some of them, like loop‐mediated isothermal amplification and recombinase polymerase amplification, are already being successfully used for routine diagnosis. However, most technologies still need validation in the plant pathology field, where they have a promising future for in‐field diagnosis when combined with simple DNA extraction methods, reagent stabilization techniques and their integration into portable devices.

Specific and Sensitive Primers Developed by Comparative Genomics to Detect Bacterial Pathogens in Grains.

Accurate and rapid detection of bacterial plant pathogen is the first step toward disease management and prevention of pathogen spread. Bacterial plant pathogens Clavibacter michiganensis subsp. nebraskensis (Cmn), Pantoea stewartii subsp. stewartii (Pss), and Rathayibacter tritici (Rt) cause Goss’s bacterial wilt and blight of maize, Stewart’s wilt of maize and spike blight of wheat and barley, respectively. The bacterial diseases are not globally distributed and not present in Korea. This study adopted comparative genomics approach and aimed to develop specific primer pairs to detect these three bacterial pathogens. Genome comparison among target pathogens and their closely related bacterial species generated 15–20 candidate primer pairs per bacterial pathogen. The primer pairs were assessed by a conventional PCR for specificity against 33 species of Clavibacter, Pantoea, Rathayibacter, Pectobacterium, Curtobacterium. The investigation for specificity and sensitivity of the primer pairs allowed final selection of one or two primer pairs per bacterial pathogens. In our assay condition, a detection limit of Pss and Cmn was 2 pg/μl of genomic DNA per PCR reaction, while the detection limit for Rt primers was higher. The selected primers could also detect bacterial cells up to 8.8 × 103 cfu to 7.84 × 104 cfu per gram of grain seeds artificially infected with corresponding bacterial pathogens. The primer pairs and PCR assay developed in this study provide an accurate and rapid detection method for three bacterial pathogens of grains, which can be used to investigate bacteria contamination in grain seeds and to ultimately prevent pathogen dissemination over countries.

On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management strategies that reduce the impact of the disease. Presently in many diagnostic laboratories, the polymerase chain reaction (PCR), particularly real-time PCR, is considered the most sensitive and accurate method for plant pathogen detection. However, laboratory-based PCRs typically require expensive laboratory equipment and skilled personnel. In this study, soil-borne pathogens of potato are used to demonstrate the potential for on-site molecular detection. This was achieved using a rapid and simple protocol comprising of magnetic bead-based nucleic acid extraction, portable real-time PCR (fluorogenic probe-based assay). The portable real-time PCR approach compared favorably with a laboratory-based system, detecting as few as 100 copies of DNA from Spongospora subterranea. The portable real-time PCR method developed here can serve as an alternative to laboratory-based approaches and a useful on-site tool for pathogen diagnosis.

On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System.

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management strategies that reduce the impact of the disease. Presently in many diagnostic laboratories, the polymerase chain reaction (PCR), particularly real-time PCR, is considered the most sensitive and accurate method for plant pathogen detection. However, laboratory-based PCRs typically require expensive laboratory equipment and skilled personnel. In this study, soil-borne pathogens of potato are used to demonstrate the potential for on-site molecular detection. This was achieved using a rapid and simple protocol comprising of magnetic bead-based nucleic acid extraction, portable real-time PCR (fluorogenic probe-based assay). The portable real-time PCR approach compared favorably with a laboratory-based system, detecting as few as 100 copies of DNA from Spongospora subterranea. The portable real-time PCR method developed here can serve as an alternative to laboratory-based approaches and a useful on-site tool for pathogen diagnosis.