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

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1 Pest and Pathogen Surveillance Comparison of rooftop and field-based air samplers for early detection and population monitoring of plant pathogens Jon West, Gail Canning, Agata Kaczmarek & Kevin King

2 Air Samplers Post-capture detection and quantification Microscopy - Lab-based and difficult to identify to species level Immunological techniques rapid, on-site test but often difficult to design specific antibodies Epidemiology & forecasting Monitoring Real-time Detection 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

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

4 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 or text the user when reagents run low Auto-sampler being tested at Rothamsted, 2017

5 Height (m) Spore thresholds depend on sampler location Change in spore concentration with height above a canola crop 5 4 West et al (2008) Botrytis Pyrenopeziza Didymella Concentration (spores m -3 ) Spore numbers decline to a regional background level within m of the source

6 Rotorod spore traps being used to measure Sclereotinia spore dispersal gradients at different heights height 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) Side A Spore numbers 6

7 Testing rotorod sampler on drone at Passo Fundo, Brazil, 2015 Sampling spores at 20m height. 7 minute flight time. 10m and ground level also sampled separately Viewing the spore trap sample back at the lab of Mauricio Fernandez Pyrenophora and rust spores, grass and pine pollen

8 Monitoring of plant pathogens in air samples using Next generation sequencing Annemarie F. Justesen, Rumakanta Sapkota & Mogens Nicolaisen, Aarhus University, Dk; Cor Schoen, Wageningen NL; Gail Canning & Jon West, Rothamsted UK 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) DNA extraction & NGS Burkard 7-day spore trap Relative abundance of individual species in air Nicolaisen M et al. (2017) Frontiers in Microbiology 8

9 AARHUS Species UNIVERSITET composition 30 species make up 70% of fungal air-spora PURE Task 11.2 Annemarie F. Justesen Generally, samples taken above the field site had lower richness ( ) than samples taken from roof tops ( ). Rusts were not found by sequencing but were found by qpcr 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 Nicolaisen M et al. (2017) Frontiers in Microbiology 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

10 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) Location Rothamsted Slagelse Wageningen Season Autumn 2012 Early Spring 2013 Late Spring 2012 late Spring 2013 Early Spring 2012 Fungal communities clustered according to both year and also season of the year Nicolaisen M et al. (2017) Frontiers in Microbiology

11 Relative Abundance Relative Abundance Seasonal and spatial differences in abundance of pathogens Blumeria d) Nicolaisen M et al. (2017) Frontiers in Microbiology

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

13 What is the optimal sampling strategy considering different densities of sources and different source strengths for different pathogens in a landscape? Contour plots of spore concentrations at crop level (simulations) 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

14 Summary Spore thresholds depend on sampler location Spore numbers decline to a regional background level within 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 jon.west@rothamsted.ac.uk