Culture-free detection of boxwood blight to improve disease diagnosis and prevention

The ornamental bush boxwood is the most sold woody plant in the USA, with a value of $126 million a year. Boxwood blight, caused by the fungal species Calonectria pseudovaniculata (Cps) and Calonectria henricotiae (only seen in Europe), is the most aggressive disease of boxwood, causing defoliation. It is easily spread by contact. It can be asymptomatic and is very hard to get rid of when the plant contracts the disease.

Marcela discussed how current methods for blight identification involve long incubation periods in high humidity environments, which mean that it could be too late to prevent the spread of disease. Fast and early diagnosis is needed to prevent, or at least reduce, its spread. The goal of Marcela’s work was to use the MinION for faster detection of Cps in metagenomic samples from Boxwood, with different levels of infection.

Five DNA extraction methods were tested initially, to determine the optimal method. Once DNA had been extracted and prepared for sequencing, samples were sequenced on the MinION. In terms of the bioinformatics analysis, using the EPI2ME workflow “What’s in my pot (WIMP)?" Marcela identified fungi in the samples, for analysis to be "as fast as possible", but greater resolution was achieved using MetaMaps, BLAST, and a custom database.This correctly identified the exact species C. pseudovaniculata in the infected samples.

In conclusion, Marcela stated how she was able to detect Cps in small quantities, in all of the metagenomic samples, and the MinION enabled rapid pathogen detection. Her future work will involve genome assembly to help determine if unclassified reads were from the plant, and improved fungal DNA extraction so that the workflow can be used even for asymptomatic plants.

Authors: Marcela Aguilera Flores