Using molecular methods to study eyespot disease in wheat (PhD)

Eyespot is a damaging stem-base disease of cereals caused by the fungi Oculimacula acuformis and O. yallundae.  This project modelled the effects of agronomy (i.e. previous cropping, cultivation methods, soil texture, fungicide treatment, sowing date or place of winter wheat (WW)) in the rotation as 1st WW, 2nd WW or monoculture on DNA of Oculimacula spp. quantified in roots, 5 cm stem from the base and upper 10 cm stem sections. Fifty wheat fields were surveyed throughout England in 2012/13 season and in each field 36 randomly selected winter wheat plants were collected at growth stage (GS) 21-33, GS 39-44 and GS 66-77 over a one hectare area. DNA concentrations of O. yallundae and O. acuformis were quantified using Real-time PCR (qPCR) assays. PCR results indicated that O. acuformis was the predominant species causing the disease with the highest DNA concentrations of the pathogen measured at GS 65-77.  Previous crop had a significant effect on DNA of Oculimacula spp. and WW rotational sequence contributed significantly to it.  Seed treatment and fungicide application at GS 32 were found to significantly influence fungal biomass in plant tissues. In a separate study, a range of fungicide treatments were applied at GS 32 or GS 39 of the winter wheat crop and Oculimacula DNA was extracted from soil before fungicide application and at GS 51 and quantified using a qPCR assay.  In addition, the impact of the disease on plant physiology was investigated by probing photosystem II efficiency. Chlorophyll fluorescence induction kinetics were used to calculate the performance index of plants as an estimate of photosynthetic efficiency following treatment for disease control.  Boscalid application at GS 32 significantly increased performance index at GS 69, reduced disease severity, fungal biomass of O. acuformis in soil and DNA of O. yallundae in wheat stems. To quantify live, viable inoculum of individual Oculimacula spp in soil or in plants a two-step reverse transcriptase PCR assay was tested to synthesize and quantify cDNA from fungal mRNA obtained via RNA extraction. The assay was shown to efficiently quantify cDNA with traces of genomic DNA amplification beyond 35 cycles. Once validated in soil or in planta such an assay can potentially be utilized to increase our understanding of the epidemiology of eyespot disease and increase the accuracy of predicting disease risk associated with viable inoculum in field.