Molecular Methods in Plant Disease Diagnostics

Principles and Protocols

Neil Boonham, Jenny Tomlinson, Rick Mumford, Neil Boonham, Jenny Tomlinson, Rick Mumford

• Publisher: CAB International
• Year: 2016
• Print ISBN: 9781780641478
• eBook ISBN: 9781780648262
• Topic: Biological Sciences
• Subtopic: Botany
• Index: Biological Sciences

Using molecular methods for plant disease diagnosis provides diagnosticians with a number of advantages over more traditional methods. They can allow the identification of morphologically similar species, for example, or the detection of infection prior to symptom formation. Not only can molecular tools help by increasing the efficacy, accuracy and speed of diagnosis; their common technological basis provides further benefits, especially where resources are limited and traditional skills are hard to sustain.This book provides protocols for nucleic acid-based methods currently applied to plant pathogen detection and identification. It takes the practitioner through the full range of molecular diagnostic and detection methods and, as these generic techniques are appropriate for use on any target with minimal modification, also provides a useful resource for students of plant pathology and plant pathologists. Beginning with the background and future directions of the science, it then addresses DNA barcoding, microarrays, polymerase chain reactions (PCR), quality assurance and more, forming a complete reference on the subject.

1 Introduction: advances in plant diagnostics – ­historical perspectives and future ­directions

Rick Mumford*

Fera, Sand Hutton, York, UK

*E-mail: [email protected]

1.1 Progress in recent years

In 2006, the editors of this book co-authored a review entitled ‘Advances in molecular phytodiagnostics – new solutions for old problems’ (Mumford et al., 2006). In this, they summarized the progress made during the first three decades of plant molecular diagnostics – since nucleic acid-based methods first started being used for the detection and identification of plant pathogens in the 1970s. In this introduction, we compare the state-of-the-art in the mid-2000s (as identified in the aforementioned review) with the current situation. This allows us to assess how this whole field of science has developed in the subsequent 9 years and where plant molecular diagnostics might be heading in the future.

1.2 The rise and rise of PCR

By 2006, the polymerase chain reaction (PCR) had become the dominant technology underpinning molecular plant diagnostics. Offering a whole raft of benefits including specificity, sensitivity, ease of assay design and applicability across a whole range of targets (pathogens and pests), PCR-based technology pushed other techniques such as dot-blot hybridization into the margins. By the mid-2000s, conventional PCR and, increasingly, real-time PCR were established within many diagnostic laboratories and were being used routinely. In subsequent years, this is a trend that has continued, especially the more wide-scale adoption of real-time PCR as a core diagnostic tool; despite having higher capital outlay and per-test costs, the advantages are sufficient to drive uptake, especially in larger, centralized laboratories. For example, if you look at national phytosanitary reference laboratories across Europe, all will now be equipped to perform methods based on this technology. This is also reflected in the number of standardized protocols that now feature real-time PCR methods, e.g. the EPPO Diagnostic Protocols (EPPO, 2010). Protocols for performing routine PCR-based tests, both conventional and real-time, are presented in this book (Chapters 2 and 3, respectively).

An essential part of the rise of PCR and real-time PCR and their adoption as core diagnostic technologies has been key developments that have occurred around the chemistry of nucleic acids (NAs), which had remained largely unchanged since it was first developed and introduced. Significant improvements have been made to the steps both pre- and post-PCR, with one such area being the development of better nucleic acid (NA) extraction methods. These have greatly enhanced both the quality and quantity of DNA and/or RNA obtained, with consequential improvements in test reliability. Advances have also improved the range of targets from which NAs can be effectively extracted, including difficult plant materials and other matrices, such as soil. This has greatly enhanced the range of ways in which PCR (and other molecular diagnostic methods) can be used, from the direct identification of isolated pests or pathogens through to the screening of bulk samples.

The other significant opportunity presented by PCR has been to provide the basis for DNA barcoding approaches. By combining broad-spectrum PCR primers (that amplify at taxonomic levels above species) with low-cost automated sequencing and better bioinformatics, routine sequencing-based identification of pests and pathogens is now both reliable and cost-effective. In 2015, this methodology has become established as a standard approach for both pest and pathogen identification, sitting alongside morphology-based methods and becoming increasingly essential where these traditional skills are either lacking or are unable to provide reliable taxonomic resolution; for example, in the identification of some juvenile life stages of pests. Methods for DNA barcoding both pathogens and pests are covered in this book in Chapters 5 and 6, respectively.

1.3 Quality matters

One of the major differences between 2006 and the present day has been the increased focus on molecular diagnostics as an entire process, from sampling through to the provision of results. As new techniques were developed, efforts were understandably aimed at improvements in the component steps that constituted the test itself – namely extraction, assay and analysis – and significant improvements were made. Yet as these tests became more readily accepted and entered routine usage, it became clear that basic measures of test performance (for example, defining a limit of detection and a test’s cross-reactivity with related species) were not alone sufficient criteria for defining a robust test and delivering a consistent diagnostic performance. It is only in more recent years that terms such as repeatability and reproducibility have become standard words within the phytodiagnostician’s vocabulary. In order to overcome greater legal and regulatory scrutiny and challenge, laboratories have to provide greater quality assurance on the testing they carry out. This need for comparability is driving the development of international diagnostic protocols, both at a regional (e.g. EPPO, the European and Mediterranean Plant Protection Organization) and a global (e.g. IPPC, the International Plant Protection Convention) level. Internationally recognized quality standards such as ISO 17025 are also being adopted. As a requisite of this, laboratories are now having to invest in additional physical infrastructure and processes (for example, to prevent cross-contamination), alongside the need for better controls (such as certified reference materials) and additional validation studies. As a result, guidance on quality assurance and diagnostic processes is required to run a modern molecular diagnostics laboratory (see Chapter 10 of this book).

1.4 The move from laboratory to field

A decade ago, plant pathologists had begun to evaluate platforms that allowed real-time PCR to be performed in the field. Projects such as PortCheck took technologies such as SmartCycler (Cepheid, USA) and developed simplified protocols, adapted from those used in the laboratory, that ran reliably and permitted real-time PCR to be performed at sites away from centralized diagnostic laboratories for the first time. Indeed, by the late 2000s, the UK’s Plant Health and Seeds Inspectorate (PHSI) was using this technology at remote sites in the south-west of England as part of its Phytophthora ramorum eradication campaign.

However, despite this progress, it became apparent that PCR-based methods had significant limitations as a field-deployable technology, and the search for the next-­generation of on-site DNA testing began. A significant part of that investigation was to identify efficient isothermal chemistries that could act as effective alternatives to PCR. Of the many alternatives, loop-mediated isothermal amplification (LAMP) has emerged as the current preferred choice among plant diagnosticians and the chemistry that is being adopted for on-site testing. Together with the use of different chemistry, further advances have taken place based on simplifying extraction and developing better workflows and detection systems. In combination, these advances have made routine on-site molecular diagnostics a reality. A decade on from the first papers published that described the use of LAMP for plant pathogen detection, technology based on this chemistry is now being deployed with plant health inspectors and will start to become an integral part of quarantine monitoring and surveillance across Europe. Methods describing LAMP (Chapter 4) and on-site testing approaches (Chapter 9) are included in this book.

1.5 Identifying the unknown

While the methods described above offer solutions for specific target detection, in the laboratory or in the field, and approaches for more accurate identification, they all assume a level of understanding of what target is actually being tested. In many cases, the real challenge for diagnosticians is ‘unknowns’. These might be new diseases, for example, or known pathogens on a previously unrecorded host, complex disease syndromes where the causal agent is unclear or new pests and pathogens that have not been previously characterized. A classic example of this is blackcurrant reversion, where the virus causing the disease (Blackcurrant reversion virus, BRV) took decades to isolate and identify. In all of these scenarios, traditional approaches require multiple parallel tests to be performed, often based on a range of different techniques. This can be slow and frequently unsuccessful, e.g. where it proves impossible to isolate the causal agent. This challenge has led diagnosticians to investigate multiplex platforms that are cap­able of simultaneously detecting multiple targets in a single test. As described in a review by Boonham et al. (2007), microarray technology had become the main focus for this approach. Starting initially with viruses and glass slide arrays, over time this technology has developed to cover not only additional target taxa but also alternative formats such as tube arrays. An account of microarray detection procedures and protocols is provided in Chapter 7 of this book.

However, while arrays offer the potential to test for hundreds, if not thousands, of patho­gens in a single test, they use specific probes designed against known targets that have been previously identified and for which published genome data exist. Hence, while microarrays can help to identify known pathogens causing new diseases, they will still fail to identify new or highly distinct strains of pathogens. In 2005–2006, the solution to this problem remained elusive. Yet it was at that time that the first next-generation sequencing (NGS) platforms became available. With their huge de novo sequencing capacity, for the first time these new platforms offered a non-targeted means to generate sequences from the entire DNA complement present in a sample. Using advanced bioinformatics approaches, it would be possible to analyse the huge data sets generated and specifically identify those known or putative pathogen sequences (Studholme et al., 2011). By the late 2000s, this approach had been applied successfully for the detection of novel plant viruses. Further developments, new platforms, better bioinformatics and decreasing per run sequencing costs have seen this technology start to become adopted as a frontline diagnostic tool. As the technology progresses, this is a trend that will only continue to gain momentum. NGS diagnostic approaches and methods are covered in this book in Chapter 8.

1.6 Future challenges

In this short introduction, it has been possible to describe the huge progress made in plant molecular diagnostics since the 1970s and 1980s, when these methods first started to be used. The introduction has also highlighted the rapid progression made during the last decade or so in turning these methods from being specialist applications, carried out by experts in centralized laboratories, into routine methods that are everyday tools for diagnosticians and, increasingly, for non-specialists. The publication of this book provides clear evidence of that. None the less, for all of the progress made, challenges still remain and these will need to be addressed in future years if molecular diagnostics are to become even more widely adopted and the benefits offered by them fully realized (Boonham et al., 2014).

The first major challenge is around sampling and nucleic acid extraction. As already noted, significant progress has been made in terms of moving to quicker and more reliable methods that avoid the use of noxious solvents such as phenol. Yet in the 21st century, we still rely predominantly on one single extraction chemistry, namely binding to silica in the presence of a chaotropic agent (the ‘Boom’ method). While different formats now exist, including columns and magnetic particles, and provide greater flexibility for automation, this extraction chemistry also presents limitations is terms of capacity and the ability to deal with inhibitors. New chemistries and innovative platforms could certainly create new opportunities for innovation in diagnostics. Current methods for the preparation of plant samples through grinding, milling or pulverization present a major bottleneck for high-throughput testing. The development of better sample preparation equipment, which combines a low per sample cost with reduced labour input and minimal cross-­contamination risk, would be a panacea for many diagnosticians. Going a step further back, the process of sampling prior to extraction continues to be a stage that is exclusively manual and very labour intensive. The development of better dedicated sampling devices would help to accelerate the testing process, aid consistency and benefit the staff carrying out this work. In the longer term, the integration of sampling, extraction and testing within a single platform or device is the ultimate goal for many working in this field. However, this will only be achieved through the implementation of a truly interdisciplin­ary science approach, combining the expertise of biologists, chemists and engineers alike.

Another key area where future efforts need to be focused is the development of integrated, quality-assured diagnostic workflows that cover the entire diagnostic process. Alongside the obvious advances in the testing methodology, there will need to be further developments in the diagnostic support services that work with these new technologies. Key examples would be the development of better proficiency testing schemes for plant pests and pathogens – ones that are not only applicable to laboratory testing but also to field testing. With new technologies such as NGS, new approaches will be required to verify not only the generation of sequence data sets but also their analysis – the design will be needed of new ways to verify and approve bioinformatics pipelines. As sequence-based identification becomes increasingly important, more effort will be required to establish verified databases of voucher sequences that have been confirmed as belonging to the species in question. This must build on approaches already being developed under initiatives such as the European initiative Q-Bank (a collection of comprehensive databases on quarantine plant pests and diseases available at www.
q-bank.eu). Finally, as we see the expanding devolution of diagnostics from the laboratory to the field, this further strengthens the need for expert reference laboratories with the ability not only to verify and confirm field diagnosis, but also to support the performance of field testing with advice, training and other diagnostic services, e.g. proficiency testing. These reference laboratories will in addition provide an essential role in the identification of new and emerging pests, a role which is unlikely to be easily achieved in the field using rapid, targeted diagnostics.

The final big challenge is data and data sharing. With the rise of new technologies such as NGS, diagnosticians will face new challenges in how to handle, analyse and store the huge data sets generated, which dwarf those generated by previous techniques. There will also be challenges linked to sharing data between field and laboratory as diagnostics become more portable. There is a drive to encourage better data sharing between organizations, both nationally and internationally, and this will require the adoption of standard data formats. All of these drivers will mean a much greater role for bioinformaticians, software designers and other knowledge management professionals working along with diagnosticians. So, as described earlier, this is another area of plant diagnostics where interdisciplinarity will need to become a major theme in the future.

References

Boonham, N., Tomlinson, J. and Mumford, R.A. (2007) Microarrays for rapid identification of plant viruses. Annual Review of Phytopathology 45, 307–328.

Boonham, N., Kreuze, J., Winter, S., van der Vlugt, R., Bergervoet, J., Tomlinson, J. and Mumford R. (2014) Methods in virus diagnostics: from ELISA to next generation sequencing. Virus Research 186, 20–31.

EPPO (2010) Standard PM 7/76: use of EPPO diagnostic protocols. EPPO Bulletin 40, 350–352.

Mumford, R.A., Tomlinson, J., Barker, I. and Boonham, N. (2006) Advances in molecular ­phytodiagnostics – new solutions for old problems. European Journal of Plant Pathology 116, 1–19.

Studholme, D.J., Glover, R.H. and Boonham, N. (2011) Application of high-throughput DNA sequencing in phytopathology. Annual Review of Phytopathology 49, 87–105.

2 Conventional PCR

Lisa I. Ward,* Tracey M. Immanuel, ­Subuhi Khan, Lia W. Liefting and Catia Delmiglio

Plant Health and Environment Laboratory, Ministry for Primary ­Industries, Auckland, New Zealand

*Corresponding author: [email protected]

2.1 Introduction

The discovery of DNA by James Watson and Francis Crick in 1953 founded the field of molecular biology (Watson and Crick, 1953); it took another 30 years of technologi...

Table of contents

• Cover
• Half Title
• Title
• Copyright
• Contents
• Editors and Contributors
• Foreword
• Chapter 1 Introduction: advances in plant diagnostics – historical perspectives and future directions
• Chapter 2 Conventional PCR
• Chapter 3 Real-time PCR
• Chapter 4 Loop-mediated isothermal amplification (LAMP)
• Chapter 5 DNA barcoding for identification of plant pathogens
• Chapter 6 DNA barcoding of invertebrate plant pests
• Chapter 7 Microarrays125
• Chapter 8 Next-generation sequencing
• Chapter 9 On-site testing for plant pathogens
• Chapter 10 Quality assurance for molecular testing in plant health
• Index
• Back