Profiling grapevine trunk pathogens in planta: A case for community-targeted DNA metabarcoding

Authors: MORALES-CRUZ, ABRAHAM - University Of California; FIGUEROA-BALDERAS, ROSA - University Of California; GARCIA, JADRAN - University Of California; TRAN, ERIC - University Of California; ROLSHAUSEN, PHILIPPE - University Of California; Baumgartner, Kendra; CANTU, DARIO - University Of California, Davis

Submitted to: BMC Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/15/2018
Publication Date: 12/14/2018
Citation: Morales-Cruz, A., Figueroa-Balderas, R., Garcia, J.F., Tran, E., Rolshausen, P.E., Baumgartner, K., Cantu, D. 2018. Profiling grapevine trunk pathogens in planta: A case for community-targeted DNA metabarcoding. BMC Microbiology. 18:214. https://doi.org/10.1186/s12866-018-1343-0.
DOI: https://doi.org/10.1186/s12866-018-1343-0

Interpretive Summary: DNA metabarcoding, commonly used to identify all the species in a community of microbes, is a promising method for the simultaneous detecting within plant tissues the presence of more than one pathogen associated with disease complexes, such as the grapevine trunk diseases. Their detection is particularly challenging, due to the presence within an individual symptomatic vine of multiple pathogens, in addition to and other fungi that are not pathogens. As such, we designed a specific genetic marker, based on a region of the fungal genome called the internal transcribed spacer, for grapevine trunk-associated Ascomycete fungi (GTAA) and compared them to two ‘universal primers’, which are widely used in microbe studies. We first tested the primers by high-throughput amplicon sequencing of (i) multiple combinations of 'mock' communities, (ii) timecourse experiments with controlled inoculations, and (iii) diseased field samples from vineyards under natural levels of infection. All analyses showed that GTAA had greater affinity and sensitivity, compared to those of the universal primers. Importantly, with GTAA, profiling of mock communities and comparisons with shotgun-sequencing metagenomics of field samples gave an accurate representation of genera of important trunk pathogens, namely Phaeomoniella, Phaeoacremonium, and Eutypa, the abundances of which were greatly over- or under-estimated with universal primers. Overall, our findings not only demonstrate that DNA metabarcoding gives qualitatively and quantitatively accurate results when applied to grapevine trunk diseases, but also that primer customization and testing are crucial to ensure the validity of DNA metabarcoding results.

Technical Abstract: DNA metabarcoding, commonly used in exploratory microbial ecology studies, is a promising method for the simultaneous in planta-detection of multiple pathogens associated with disease complexes, such as the grapevine trunk diseases. Their detection is particularly challenging, due to the presence within an individual wood lesion of multiple co-infecting trunk pathogens and other wood-colonizing fungi, which span a broad range of taxa in the Fungal Kingdom. As such, we designed metabarcoding primers, using as template the ribosomal internal transcribed spacer of grapevine trunk-associated Ascomycete fungi (GTAA) and compared them to two universal primer widely used in microbial ecology. We first performed in silico simulations and then tested the primers by high-throughput amplicon sequencing of (i) multiple combinations of 'mock' communities, (ii) timecourse experiments with controlled inoculations, and (iii) diseased field samples from vineyards under natural levels of infection. All analyses showed that GTAA had greater affinity and sensitivity, compared to those of the universal primers. Importantly, with GTAA, profiling of mock communities and comparisons with shotgun-sequencing metagenomics of field samples gave an accurate representation of genera of important trunk pathogens, namely Phaeomoniella, Phaeoacremonium, and Eutypa, the abundances of which were greatly over- or under-estimated with universal primers. Overall, our findings not only demonstrate that DNA metabarcoding gives qualitatively and quantitatively accurate results when applied to grapevine trunk diseases, but also that primer customization and testing are crucial to ensure the validity of DNA metabarcoding results.