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. 

 

20/09/2013

1

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

20/09/2013

2

Tree health: our greatest current biosecurity challenge

TRADE

NATURAL

20/09/2013

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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4

Rapid testing e.g. Lateral Flow Devices

• Antibody-based

• Single step

• Rapid (2-3 minutes)

• Genuine field testing

PCR methods in the field

20/09/2013

5

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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6

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)

20/09/2013

7

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)

20/09/2013

8

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

20/09/2013

9

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

20/09/2013

10

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.

20/09/2013

11

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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12

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

20/09/2013

13

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”

20/09/2013

14

‘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

20/09/2013

15

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

20/09/2013

16

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?

20/09/2013

17

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

20/09/2013

18

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

20/09/2013

19

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