Phytoplasma DNA Enrichment from Sugarcane White Leaves for Shotgun Sequencing Improvement.

Abstract

Sugarcane white leaf (SCWL) disease, caused by Candidatus Phytoplasma sacchari, poses a significant threat to sugarcane cultivation. An obligate parasite, phytoplasma is difficult to culture in laboratory conditions, making the isolation of its DNA from the massive amount of plant host DNA extremely challenging. Yet, the appropriate amount and quality of plant microbiome-derived DNA are key for high-quality DNA sequencing data. Here, a simple, cost-effective, alternative method for DNA isolation was applied using a guanidine-HCl-hydroxylated silica (GuHCl-Silica)-based method and microbiome DNA enrichment based on size-selective low-molecular-weight (LMW) DNA by PEG/NaCl precipitation. qPCR analysis revealed a significant enrichment of phytoplasma DNA in the LMW fraction. Additionally, the NEBNext Microbiome DNA enrichment kit was utilized to further enrich microbial DNA, demonstrating a remarkable increase in the relative abundance of phytoplasma DNA to host DNA. Shotgun sequencing of the isolated DNA gave high-quality data on the metagenome assembly genome (MAG) of Ca. Phytoplasma sacchari SCWL with completeness at 95.85 and 215× coverage. The results indicate that this combined approach of PEG/NaCl size selection and microbiome enrichment is effective for obtaining high-quality genomic data from phytoplasma, surpassing previous methods in efficiency and resource utilization. This low-cost method not only enhances the recovery of microbiome DNA from plant hosts but also provides a robust framework for studying plant pathogens in complex plant models.