Deep sequencing for trunk disease diagnostics

The aim of this multi-year project is to develop rapid and cost-effective diagnostic methods
for detection, identification, and quantification of trunk pathogens in asymptomatic and
symptomatic grape wood. Healthy vines are essential for the successful establishment and
sustainability of all grape production systems. Since wood pathogens may remain
asymptomatic in young, non-stressed vines, propagation material may contain latent fungal
infections and may become symptomatic after planting and serve as a source of inoculum
for further infections of potentially clean plants. Methods of virus detection and eradication
have been crucial in ensuring that the material in germplasm repositories and clean plant
programs is free of known viruses. There remains much to be developed in terms of fungal
pathogen detection. Our laboratories have developed comprehensive genomic information
on several ascomycetes associated with the most common and aggressive trunk diseases,
which provides the unprecedented opportunity for the implementation of new sequencingbased
diagnostic tools that take advantage of Next Generation Sequencing (NGS)
technologies. By allowing the testing of mother plants in foundation blocks and propagation
material in nurseries, we expect that the applications of deep sequencing diagnostics will
help establish a certification program for trunk pathogen-free germplasm and reduce the
amount of trunk pathogens introduced into vineyards at planting as well as the incidence of
young vine decline. Deep-sequencing diagnostics will also help identify disease-causing
organisms associated with diseased vines in older vineyards.
In the 1st year of the project (2015 – 2016) we collected diseased wood material from
commercial vineyards and characterized the associated fungal pathogen species using
traditional methods, such as morphological and sequence-based identification of purified
fungal colonies. We used these samples to determine how effective ITS-sequencing, metagenome
sequencing and meta-transcriptome sequencing approaches are in identifying and
quantifying pathogenic species directly in planta. Data simulations allowed us to determine
what mapping algorithm was the most specific and sensitive in detecting trunk pathogens
both qualitatively and quantitatively. All NGS methods we tested were in agreement with
traditional diagnostic methods, but also allowed us to detect simultaneously multiple
pathogen species with no need of hands-on sample culturing and colony purification.
Additionally, unlike traditional diagnostics, which are strictly qualitative, NGS approaches
allowed us to determine the relative abundances of the different infecting species. This
work was published in Molecular Plant Pathology (Morales-cruz et al., 2017). Among all
methods tested, ITS-seq is still the most cost-effective until library preparation costs for
RNA and DNA-seq do not decline significantly. For this reason, ITS-seq was chosen for
further protocol optimization to improve sensitivity and specificity for diagnostics purposes.
In the second year of the project (2016-2017), we (a) confirmed that NGS allows the
detection with high specificity of actively infecting pathogens when vines are experimentally
infected with individual pathogen strains; (b) established that NGS detection is quantitative
and allows to differentiate between diseased and healthy vines; (c) developed a protocol for
testing dormant cuttings and started testing cuttings provided by a commercial nursery. In
the 2016-2017 funding cycle, we also developed a new DNA extraction protocol that
reduced the time required for processing and the amounts of sample, reagents and waste.
In the 3rd and 4th year of the project, our effort focused on the development and
optimization of a new set of optimized primers for ITS-seq designed specifically to target
the ITS of grapevine trunk pathogens. The primers as well as the method are publicly
available and described in a peer-reviewed article published in December 2018 (Moralescruz
et al., 2018).
In summary, in these four years we have:
1. Applied NGS to trunk pathogen diagnostics and demonstrated that NGS provides
qualitatively and quantitatively accurate simultaneous identification of multiple trunk
pathogens directly from grapevine wood samples (Morales-Cruz et al., 2017 Mol
Plant Pathol).
2. Developed a new protocol with optimized diagnostic markers for NGS ITS-seq diagnostics of trunk diseases, which is publicly available and described in detail in
Morales-Cruz et al. (2018; BMC Microbiology).