Iodixanol Discontinuous Density Gradients as an Effective Phytoplasma Enrichment Approach to Improve Whole Genome Sequencing

Phytoplasmas are unculturable bacteria, residing only as intracellular organisms. they are insect vectored phytopathogens of many plant species and, when infection is widespread, can significantly affect the yield and economic potential of crops. Their genomes are therefore invaluable to improve our understanding of their biology, evolution, and epidemiology. However, assembling phytoplasma genomes with a high contiguity and completeness is challenging due to high levels of contaminating host chromosomal and organellar genomes in total DNA extracts. Developing a repertoire of methods suitable for phytoplasma enrichment from a range of different hosts has been a focus in phytoplasma genomics since the early 2000s. We developed a phytoplasma enrichment method based on centrifugation through an iodixanol density gradient, and systematically quantified the level of DNA derived from phytoplasma or host throughout the processing steps. The method was effective at reducing host chromosomal DNA and enriching phytoplasma DNA for three hosts harbouring variable phytoplasma titres. This work highlights the value of microbial enrichment or host depletion methods for better outcomes in phytoplasma comparative genomics. The method is expected to be broadly applicable and will improve whole genome sequencing of phylogenetically diverse phytoplasmas in a range of naturally infected hosts. It should be effective for other obligate plant pathogenic bacteria.