New Insight Into DNA Isolation From Ceratocystis platani Conidia Using Automated Recovery

Single-cell technologies enable genome-level analysis of individual propagules, offering new insights into plant-pathogenic fungi. Such technology is a breakthrough approach to study blood cancers by characterising molecular information on a genome-wide scale at the single-cell level. These methods can be easily and successfully transferred to tracheomycotic plant pathogens, which cause host wilt. Ceratocystis platani is the causal agent of the canker stain disease of plane trees (Platanus spp.), a lethal wilt disease spreading within Europe. To displace and separate different types of C. platani conidia, a dielectrophoretic approach was tested. A total of 290 conidia were selected individually and grouped by DEPArray system according to different shapes: aleuroconidia, cylindrical endoconidia and clustered aleuroconidia. For each conidia population selected, 10 conidia were chosen randomly, and the DNA of each conidium was isolated and analysed. The target fungal DNA was identified by a specific C. platani quantitative PCR marker and by sequencing the amplicon. The fungal DNA was successfully extracted from single (aleuroconidia and cylindrical) or pooled conidia (clustered aleuroconidia) and identified by both methods after whole genome amplification. Our results showed that this technology is applicable to vascular plant pathogens. The use of single-cell technology will provide a new approach to the study of plant vascular diseases, allowing the analysis of single-spore molecular and physiological features not detectable in complex biological mixtures.