Comparison of rooftop and field-based air samplers for early detection and population monitoring of plant pathogens

Pest and Pathogen Surveillance

Jon West, Gail Canning, Agata Kaczmarek & Kevin King

jon.west@rothamsted.ac.uk

Microscopy

- Lab-based and difficult to identify to species level

Post-capture detection and quantification

Immunological techniques – rapid, on-site test but often difficult to design specific antibodies

DNA-based methods

LAMP and RPA methods can now be automated for on-site use

West & Kimber (2015) Annals of Applied Biology 166: 4–17

Air Samplers

• Epidemiology & forecasting • Monitoring • Real-time Detection

3

• Sampler includes DNA release,

isothermal amplification &

quantification, wireless reporting.

• Result integrated with infection

models & risk prediction sent to

end-users.

Current work: sporadic arable

crop pathogens (Septoria,

Fusarium graminearum, Yellow

rust), potato pathogens, and

sugar beet pathogens

Real-time monitoring of airborne pathogen spores for disease risk alerts

Screen-shot of the Burkard web-portal

• User selects data from an individual site

• This example shows a positive fluorescence result

• Assays for up to 3 different pathogens can be made per sample

• Compatible with RPA or LAMP reagents

• Will email or textthe user when reagents run low

Auto-sampler being tested at Rothamsted, 2017

Change in spore concentration with height above a canola crop

0

1

2

3

4

5

1 10 100 1000Concentration (spores m-3)

He

igh

t (m

)

BotrytisPyrenopezizaDidymella

• Spore numbers decline to a regional background level within 200-2000m of the source

Spore thresholds depend on sampler location

West et al (2008)

6

Rotorod spore traps being used to measure Sclereotinia spore dispersal gradients at different heights

Results – more spores close to the ground near the source (A) fewer but evenly distributed once the spores had travelled a few tens of metres (B)

Spore numbers

he

igh

t @ A

@ B

Side View

Testing rotorodsampler on drone at Passo Fundo, Brazil, 2015

Viewing the spore trap sample back at the lab of Mauricio Fernandez – Pyrenophora and rust spores, grass and pine pollen

Sampling spores at 20m height. 7 minute flight time. 10m and ground level also sampled separately

8

› Fungal diversity in air during spring, early summer and autumn

› Relationship between spores, weather and local disease severity

› Compared rooftop sites: Wageningen (NL), Slagelse (DK) & Rothamsted (UK)and field location (Rothamsted)

Annemarie F. Justesen, Rumakanta Sapkota & Mogens Nicolaisen, Aarhus University, Dk;Cor Schoen, Wageningen NL; Gail Canning & Jon West, Rothamsted UK

Burkard 7-day spore trap

Relative abundance of individual species in air

1

2

3

4

5

6

7

8

9

10

11

DNA extraction & NGS

Nicolaisen M et al. (2017) Frontiers in Microbiology

Monitoring of plant pathogens in air samples using Next generation sequencing

PURE Task 11.2

Annemarie F. Justesen

AARHUSUNIVERSITET

Known Plant Pathogens Detected› Didymella exitialis › Mycosphaerella graminicola› Botryotinia fuckeliana› Microdochium nivale› Ramularia collo-cygni› Verticillium dahliae› Blumeria graminis› Fusarium oxysporum› Itersonillia perplexans› Lewia infectoria (Alternaria)› Epicoccum nigrum

• Rusts were not found by sequencing but were found by qPCR

Species composition – 30 species make up 70% of fungal air-spora

• Generally, samples taken above the field site had lower richness (44.2–67.3) than samples taken from roof tops (94.7–107.3).

Nicolaisen M et al. (2017) Frontiers in Microbiology

• Different genera containing important plant pathogens showed different patterns of relative read abundances during the season, but were strikingly similar across the three locations and in many cases peaking on approximately the same days.

• Some taxa were highly specific for some locations

Principal component analysis: distribution by rooftop location (8% variation explained, although highly different for some individual taxa) and by season (explains 25% of variation: p<0.001)

LocationRothamstedSlagelseWageningen

SeasonAutumn 2012Early Spring 2013Late Spring 2012late Spring 2013Early Spring 2012

Nicolaisen M et al. (2017) Frontiers in Microbiology

Fungal communities clustered according to both year and also season of the year

Seasonal and spatial differences in abundance of pathogens

Nicolaisen M et al. (2017) Frontiers in Microbiology

Rel

ativ

e A

bu

nd

ance

Blumeria

Rel

ativ

e A

bu

nd

ance

d)

Comparison of Sclerotinia DNA at Worcester (city roof) and ADAS Rosemaund (field) sites(about 50 km apart)

Worcester

Contour plots of spore concentrations at crop level (simulations)

What is the optimal sampling strategy considering different densities of sources and different source strengths for different pathogens in a landscape ?

Wind

• Sampling position is important for rare pathogens but for common pathogens (right-hand situation), one location can act as a ‘barometer’ for the entire region

• Spore thresholds depend on sampler location

• Spore numbers decline to a regional background level within 200 - 2000 m of the source

• Rooftop sampling gives a smoothed sample of ‘mixed air’ giving a better representation of the regional air spora

• High volume spore traps can boost sensitivity, allowing detection at rooftop sites

• Common, widespread pathogens can be monitored from relatively few sites

Summary

jon.west@rothamsted.ac.uk