Sequencing Herbarium Specimens of a Common Detrimental Plant Disease (Powdery Mildew)

Powdery mildew (Erysiphaceae) is a detrimental plant disease that occurs on a variety of economically important crops. Powdery mildew consists of over 873 species of fungal pathogens that affect over 10,000 plant species. Genetic identification of powdery mildew is accomplished using the internal transcribed spacer (ITS) and large subunit (LSU) regions of the nuclear ribosomal RNA gene cluster. The ITS and LSU regions of powdery mildews can be useful in ecological, epidemiological, phylogenetic, and taxonomic investigations. However, sequencing these regions is not without its challenges. For example, powdery mildew sequences are often contaminated with plant and/or fungal DNA. Also, there tends to be a limited amount and older specimens' DNA can fragment over time. The success of sequencing powdery mildew often depends on the primers used for running polymerase chain reaction (PCR). The primers need to be broad enough that they match the majority of powdery mildew DNA yet specific enough that they do not align with other organisms. A review of the taxonomy and phylogeny of the powdery mildews is presented with an emphasis on sequencing the ITS + LSU genomic regions. Additionally, we introduce a new nested primer protocol for sequencing powdery mildew herbarium samples that includes six new powdery mildew-specific primers. The new sequencing protocol presented allows specimens up to 130 years old to be sequenced consistently. Sequencing herbarium specimens can be extremely useful for addressing many ecological, epidemiological, phylogenetic, and taxonomic problems in multiple plant pathogenic systems including the powdery mildews.