Marcela Aguilera Flores from Virginia Tech presented at the Nanopore Community Meeting 2019 on “Culture-free detection of boxwood blight to improve disease diagnosis and prevention.” Aguilera Flores spoke about the use of the MinION for metagenomics to detect the fungi that cause blight in the boxwood. According to Aguilera Flores, boxwood is an important ornamental bush and the number one woody plant sold in the USA. Boxwood blight is caused by Calonectria pseudonaviculata (Cps) and Calonectria henricotiae in Europe. This is the most aggressive disease of this plant. The current method of diagnosis requires growth of spores on leaves in plant chambers under high humidity for several days and expertise by plant pathologists. The project aimed to evaluate the use of the MinION and Nanopore sequencing for the detection of fungal DNA. Several different DNA extraction methods were used. The team used a MinION Mk1B and MIN 106 R9.4 flow cells with the ligation sequencing kit (108 at the time) and native barcoding kit. EPI2ME WIMP did not classify this fungal pathogen. Therefore, the team used BLAST and MetaMaps to build a custom database with five fungal genomes. With this approach, they were able to detect Cps in all samples. The team is assembling the boxwood genome to determine if the unclassified reads are from the plant. This challenge is one we have encountered. The research team wants to improve fungal DNA extraction to improve detection even in asymptomatic plants.
