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Advances in pathogen diagnostics
Dr Rick Mumford
Over the last 15 years, Fera has
established a world‐class reputation in
the development of plant diagnostics. Key achievements include
establishing real‐time PCR (polymerase chain reaction) as a routine tool
for plant diagnosticians and taking lateral flow devices (LFDs) through
from concept to a product; manufactured in the UK and used by
inspectors and growers around the world. More recently, research has
focused in two key areas: the development of sensitive DNA diagnostics
for field use and the use of cutting‐edge genomics to identify unknown
pathogens associated with new diseases.
While LFDs are the ultimate simple‐to‐use field diagnostic, they have limitations, for example, they
sometimes lack the required sensitivity to detect low concentration pathogens. To overcome these
disadvantages, Fera has developed DNA‐based field detection methods. This has included the field
deployment of portable real‐time PCR with the Plant Health & Seeds Inspectorate (PHSI), as part of
the Phytophthora ramorum eradication campaign. More recently, the focus has shifted towards
even simpler and faster technology, using a different DNA amplification technology: ‘Loop‐mediated
isothermal amplification’ (LAMP). While this newer technology is as specific and sensitive as existing
methods like PCR, it does not require multiple cycles of cooling and heating to amplify a specific
target gene sequence (ie it is ‘isothermal’). It is also less influenced by inhibitors found in plant
material that can stop PCR from working unless removed by DNA clean‐up methods. As a result,
LAMP assays can be performed on simpler equipment and often without the need for multi‐step
sample processing. After extensive trials, this device is now being deployed; both with PHSI at
Heathrow airport, to support import inspections, and with tree health inspectors, to aid field
monitoring for tree diseases, including ash dieback. It allows results to be obtained in minutes,
rather than waiting days for lab diagnosis.
The application of genomics for the rapid identification of new or unusual pathogens is another area
of significant progress. This approach uses next generation sequencing (NGS), an incredibly powerful
tool that combines advances in sequencing technology (hardware and chemistry) and DNA data
analysis (‘bioinformatics’), to allow the generation of massive amounts of sequence information in
days, where previously it took months. Now researchers can compare whole genomes of organisms,
rather than just a handful of genes or study whole populations of organisms such as soil microbes
(‘metagenomics’). Fera has applied NGS to diagnose new diseases. By taking samples from diseased
and healthy plants, sequencing and comparing the DNA in them, it is possible to identify viruses and
other pathogens that are the potential cause of the disease. As this approach is non‐targeted (ie not
looking for specific organisms), it is an excellent way of identifying ‘unknown’ pathogens, which are
difficult to identify using traditional diagnostics. This new approach has proved critical in identifying
the viruses causing a wide range of new crop diseases in the UK and abroad.
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Advances in Plant Diagnostics
Rick Mumford
Head of Plant Science
The Food & Environment Research Agency (Fera)
Three major challenges for diagnostics
Identifying new
pathogens
Supporting sustainable
intensification
Enhancing national
biosecurity
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Tree health: our greatest current biosecurity challenge
TRADE
NATURAL
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3
Detection in the field is not easy
Detecting pathogens in the lab
HTP real-time PCR Diagnostic arraysDNA bar-coding
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Rapid testing e.g. Lateral Flow Devices
• Antibody-based
• Single step
• Rapid (2-3 minutes)
• Genuine field testing
PCR methods in the field
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LAMP: Loop-mediated isothermal AMPlification
• 3 pairs of primers (internal, external, loop)
• Bst DNA polymerase with strand-displacing activity
• Internal primers generate product containing single-stranded loops
F3c
B2cF3
B2 3’
5’
5’
3’
F1cF2
B2B1c
F1c
F1
B1
B1c
FL
FLc
BLc
BL
Internal primer FIP
Internal primer BIP
B3
B3c
F2c
F2
Loop primer F-loopExternal primer F3
Loop primer B-loop External primer B3
Next generation portable DNA detectione.g. Guignardia citricarpa LAMP
Results in
10-15 mins
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Trial of methods with inspectors
“...not much more involved than using a LFD kit...”
“The instrument itself also seemed very simple and easy to use.”
“It was impressive how quickly you could get a result...”
2013 – complete deployment for identification of 5 priority quarantine targets
at Heathrow and Zurich airports
Ash Dieback (Chalara fraxinea)
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LAMP for detection of Chalara fraxinea
150 previously-tested samples tested by LAMP end
November: 34% infected, 66% C. fraxinea-negative
Primer design and initial test
end of October
Development of field
extraction method for ash
November
Improved assay design
mid-November
Finalised protocol for
validation
-10000
0
10000
20000
30000
40000
50000
60000
0 5 10 15 20 25 30
Flu
ore
sce
nce
Time (minutes)
NTC H. albidus
H. pseudoalbidus C.f-infected ash
TaqMan
+ -
LAM
P
+ 46 2
- 5 97
positive predictive
value = 96%
negative predictive
value = 95%
sensitivity
= 90%
specificity
= 98%
LAMP for detection of Chalara fraxinea
Take sample from leading
edge of lesion
Transfer approx. 1 µl per
LAMP reaction
Place in tube with PEG buffer
and shake for 1 minute
Transfer approx. 10 µl into
tube containing 90 µl water
2-5 minutes per sample
using innoculating
loop if in the field
1 minute up to 8 samples
2 minutes up to 14 samples
2 minutes up to 14 samples
Run LAMP on Genie II
instrument35 minutes up to 14 samples
Approx. 30 minutes hands-
on time to test 14 samples
(mostly sampling)
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LAMP for detection of Chalara fraxinea
http://www.bbc.co.uk/news/uk-20217453
LAMP is now being performed routinely by FC inspectors for
Ash die back pathogen
New virus first findings in the UK:1980-2013
Arboreal
Beet
Cereal
Field Veg
Grass
Ornamental
Potato
ProtectedEdible
Salad
Soft Fruit
Stone fruit Weed
Total = 55
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High-risk hosts e.g. petunia
More than 150 potential viral pathogens identified
New virus discovery: the impact of new technology
0
2
4
6
8
10
12
14
16
1980 1985 1990 1995 2000 2005 2010
Mean = 1.3 new viruses per year
Routine Molecular
diagnostics
Fera invests in
NGS
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Next-generation DNA sequencing technology at Fera
MiSeq (Illumina) GS-FLX-Titanium + (Roche)
250bp x 2 x 18 million 750bp x 1 million
Hands on time to run : 20 min Hands on time to run : 3.5 days
Cost per run: hundreds of pounds Cost per run: thousands of pounds
High error rate at end of reads Problems with homopolymers
Microbial genome sequencing, virus
sequencing
Amplicons.
The use of NGS technologies for diagnostics
Adams I. P., Miano D. W., Kinyua Z.M., A. Wangai A., Kimani E., Phiri N. ,Reeder R.,
Harju V., Glover R., Hany U., Souza-Richards R., Deb Nath P., Nixon T., Fox A., Barnes
A., Smith J., Skelton A., Thwaites R., Mumford R. And Boonham N. (2013). Use of next-
generation sequencing for the identification and characterization of Maize chlorotic mottle
virus and Sugarcane mosaic virus causing maize lethal necrosis in Kenya. Plant
Pathology
Harju V., Skelton A., Forde S., Bennett S., Glover R.H., Monger W.A., Adams I.P.,
Boonham N., Fox A. (2012) New virus detected on Nasturtium officinale, Watercress.
New Disease Reports
Monger W.A., Alicai T., Ndunguru J., Kinyua Z.M., Potts M., Reeder R.H., Miano D.W.,
Adams I.P., Boonham N., Glover R.H., Smith J.(2010). "The complete genome sequence
of the Tanzanian strain of Cassava brown streak virus and comparison with the Ugandan
strain sequence. Arch Virol 155(3): 429-433
Adam I.P, Glover R.H, Monger W., Mumford, R., Jackeviciene, E., Navalinskiene, M.,
Samuitiene, M., Boonham, N. (2009) Next generation sequencing and metagenomic
analysis: a universal diagnostic tool in plant virology. Molecular Plant Pathology.
10(4):537-45.
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Case Study: Internal browning of carrot (HDC-FV382a)
• Long standing industry issue (10 years +) of unidentified cause
• Carrots showing internal necrosis symptoms linked to viral infection
• Whole crop rejection on processing line due to >5% affected carrots
Conventional approaches
• Assessed 3300 carrots for browning symptoms
• 3% showed symptoms
• 100 affected and 100 unaffected screened for virus
• Conventional diagnostics by RT-PCR:
– Parsnip yellow fleck virus
– Carrot Motley Dwarf (Carrot red leaf virus, Carrot mottle
virus, Carrot red leaf associated viral RNA)
• No association between infection and necrosis was found
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MiSeq Sequencing
• Deep sequencing included in experimental plan
• 12 necrotic carrots & 12 healthy
• 2 MiSeq runs: 16 million reads
• Detailed bioinformatics analysis
Viruses found by NGS
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Summary of NGS
Complete genomes of:
• Carrot yellow leaf virus (most prevalent in affected)
• Carrot mottle virus
• Carrot red leaf virus
• Carrot red leaf associated virus
• Beet western yellows associated (unexpected)
• a novel virus in the same genus as Carrot yellow leaf
virus
• 2 novel Betaflexiviruses
• a novel carrot Torradovirus
Summary of Real-time PCR results
“Carrot yellow leaf virus
(CYLV) is strongly associated
with necrosis”
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‘Diagnosis is not the end, but the beginning of practice’ Martin H. Fischer
Crop losses from pests, diseases and weeds
• Global pre-harvest losses are estimated to be between
25-40% (depending on crop)
2001-03Figures
Weeds Pests Diseases Total(Global Mean)
Total (NW Europe)
Total (Central Africa)
Wheat 7.7 7.9 12.6 28.2 14 35
Potato 8.3 10.9 21.1 40.3 24 50
After Oerke, 2006
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Sustainable intensification
Fungicidetreatments on cereals
1990 2008
Area treated
(Ha)
16.6M 27.6M
No. of
applications:
1-2 65% 36%
3 or more 25% 51%
Wheat yields Fungicide usage
‘producing more output from the same area of land while reducing the negative
environmental impacts…………’
Common scab of potato (R448)
CUF
weeks post initiation
1 3 5 7 9 11
ratio txt/16S
-0.01
0.00
0.01
0.02
0.03
0.04
0.05
Unirrigated
Irrigated
Using molecular diagnostics to improve crop management
• Potato scab disease is controlled by
(often excessive) irrigation
• Molecular diagnostics used in field
experiments to inform
recommendations to farmers for
better targeted irrigation
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NGS analysis of microbial communities
Glasshouse experiments show
that, in absence of soil microbial
communities, irrigation has no
effect on scab level
NGS analysis of bacterial and
fungal communities in soil to
establish links between water-soil
microbes-disease suppression
Researchers Diagnosticians InspectorsIndustry &
general publicRemote & real-time monitoring
The future of diagnostics?
Future?
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Generic surveillance networks
e.g. Integrated
biosensor disease
risk management
system
To spray or
not to spray?
Earlier detection
Remote sensing
‘Citizen science’
Use existing
monitoring &
surveillance
systems
Smart traps
Acoustics
Better
detection
tools
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The further use of Omic technologies
GenomicsGenomics
ProteomicsProteomics
MetabolomicsMetabolomics
DNA/RNA
Proteins/peptides
Metabolites /
small molecules
Identification of non-DNA
markers of disease or
resistance
???
Innovation in platforms will drive future applications
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Acknowledgements
• Fera colleagues:
• Field diagnostics: Neil Boonham, Jenny Tomlinson, Sioban Ostoja-Starzewska, Paul Beales• Common scab work: Richard Thwaites, Melanie Sapp, John Elphinstone• Carrot virus work: Adrian Fox, Ian Adams, Toby Hodges, Anna Skelton, Roy Macarthur
Cambridge
University Farms
Thank you