Deep Sequencing for Trunk Disease Diagnostics

The aim of this multi-year project was 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 sequencing based 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, meta-genome sequencing and metatranscriptome 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 second phase on 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 (Morales-cruz et al., 2018).

In summary, in these five 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).

We publish all protocols, which can be used freely by diagnostic and research labs. We are now seeking funds to survey propagation material and determine the association between pathogen contamination in propagation material and trunk disease incidence in young vineyards.