BSPP Report from undergraduate bursary:
Claviceps purpurea, which causes Ergot of wheat and Fusarium graminearum which causes Fusarium head blight (FHB) of wheat were studied. These two Ascomycete fungi can produce harmful mycotoxins in wheat grain and Fusarium can also reduce grain yield in infected crops.
Inoculated field experiments were carried out to assess the timing and quantity of spore release. Ergot fruiting bodies and inoculated black grass were planted in a wheat field as two sources of inoculum. Some of the fruiting bodies (stroma) were dissected and sectioned using a cryostat. The sections were stained and visualised under a light microscope to assess the sexual spore producing potential of a single stroma (Figure 1 and Figure 2).
Despite our inoculations, the incidence of ergot on the wheat in the inoculated field was surprisingly low, when compared to the high incidence observed on the grasses in the surrounding area. It is therefore hypothesised that insect vectoring of the asexual spores may be the more significant method of spore dispersal resulting in disease symptoms (rather than air borne sexual spores) and that either the timing of insect transmission did not coincide with wheat flowering (when the wheat is susceptible to infection) or the insects vectoring the pathogen did not visit the wheat.
Another wheat field was inoculated with Fusarium spp. by spreading leaf litter from infected wheat from the previous season. The field was misted to ensure the conditions were favourable for the production of the spores. A gradient of Fusarium incidence was observed over the Fusarium spp. inoculated wheat field which followed the direction of the prevailing winds, which were predominantly from one direction throughout the bulk of the flowering period. There was also a much higher incidence within the water misted part of the experiment than in the surrounding guard crop (Figure 3), but even here, the exceptionally wet June allowed a substantial amount of infection.
Burkhard spore traps were used to sample the air borne inoculum in both the Ergot and Fusarium fields. These sample air at 10 litres per minute with airborne particles impacted onto a wax-coated tape, which is moved by a clockwork mechanism past the air intake at 2 mm per hour. Microscopy and molecular techniques were employed to analyse the spore deposit on the tape. A considerable increase in detectable airborne F. graminearum spores was observed to coincide with the time of wheat flowering, when the wheat is most susceptible for infection.
Unfortunately the molecular analysis from the spore traps in the Ergot experimental field all came back negative suggesting a problem with the process of DNA extraction from the spore tape. DNA was extracted from spore tapes using a Microlysis buffer for the Fusarium experiment and using a Qiagen kit for the Ergot experiment. Although the Microlysis method yields a lower volume of DNA it seems to be a more reliable method for the purpose of spore tape extractions.
Spore tape samples analysed from both the Ergot and Fusarium fields for the presence of Fusarium pathogens showed a strong positive PCR band for the presence of Microdochium nivale cv. nivale. (another causative agent of FHB on wheat) on the same day in the two separate fields. This indicates that there was a considerable local presence of the pathogen in the air on this day.
Wheat grain harvested from the Fusarium experimental field was taken from each of the fungicide untreated plots and cultured on potato dextrose agar to assess the level of infection in the grain and to identify the pathogen species present (Figure 4). Over 96% of the grain tested was infected with Fusarium spp.
This project has provided me with some extremely valuable experiences in carrying out field experiments and I have learnt skills which I will take with me into my further studies as I embark on a PhD in plant pathology at Oregon State University.
Thanks are due to Jon West, Richard Gutteridge, Sarah Rogers, Simon Atkins, and Bruce Fitt for their support, advice and assistance in this project. Thanks also go to the British Society of Plant Pathology for funding this work and to Paul Nicholson at the John Innes Centre and David Kenyon at NIAB for assistance with molecular techniques.
Clare Elliott, Rothamsted Research.
Figure 1; 10µm section of C. purpurea stroma, stained using Toluidine blue and Ruthenium red
Figure 2; Thread like ascospores are visible in the ascocarp covering the surface of the C. purpurea stroma
Graph available on request
Figure 3; Gradient of FHB incidence assessment of fungicide untreated plots. Each line is a separate transect through the field with data collected at fungicide untreated plots only.
Figure 4; Fusarium infected wheat grain on PDA (5 days, 20˚C)
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