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

Abstract

21DNA metabarcoding, commonly used in exploratory microbial ecology studies, is a promising 22 method for the simultaneous in planta-detection of multiple pathogens associated with disease 23 complexes, such as the grapevine trunk diseases. Their detection is particularly challenging, due 24 to the presence within an individual wood lesion of multiple co-infecting trunk pathogens and 25 other wood-colonizing fungi, which span a broad range of taxa in the Fungal Kingdom. As such, 26 we designed metabarcoding primers, using as template the ribosomal internal transcribed spacer 27 of grapevine trunk-associated Ascomycete fungi (GTAA) and compared them to two universal 28 primer widely used in microbial ecology. We first performed in silico simulations and then tested 29 the primers by high-throughput amplicon sequencing of (i) multiple combinations of mock 30 communities, (ii) time-course experiments with controlled inoculations, and (iii) diseased field 31 samples from vineyards under natural levels of infection. All analyses showed that GTAA had 32 greater affinity and sensitivity, compared to those of the universal primers. Importantly, with 33 GTAA, profiling of mock communities and comparisons with shotgun-sequencing metagenomics 34 of field samples gave an accurate representation of genera of important trunk pathogens, namely 35Phaeomoniella, Phaeoacremonium, and Eutypa, the abundances of which were greatly over-or 36 under-estimated with universal primers. Overall, our findings not only demonstrate that DNA 37 metabarcoding gives qualitatively and quantitatively accurate results when applied to grapevine 38 trunk diseases, but also that primer customization and testing are crucial to ensure the validity of 39 DNA metabarcoding results. 40 41 42 identification and, thus, require skilled expertise in mycology [20]. However, these approaches 66 still require an initial culturing step, which may limit the detection of slow-growing fungi. 67 Alternatively, with species or genus-specific markers, PCR could be used to determine in planta 68 the presence of certain species, thereby skipping the culturing step [21, 22]. One limitation of this 69 approach, however, is that it may not detect all trunk pathogens in a given sample [23, 24]. Indeed, 70 certain combinations of fungi may be important in the severity of symptom expression [25]. 71Because trunk pathogens cause mixed infections, attempts have been made to characterize the 72 composition of the trunk-pathogen community. For example, finger-printing techniques like 73 Automated Ribosomal Intergenic Spacer Analysis (ARISA) [26] and Single-Strand Conformation 74Polymorphism (SSCP) [27, 28] have been used to compare fungal communities among different 75 samples of grapevine wood, although these do not identify trunk pathogens to the species level. A 76 DNA macroarray system, based on reverse dot-blot hybridization containing oligonucleotides 77 complementary to portions of the -tubulin locus, was developed for species-level identification, 78 specifically for dete...