Quantifying trade-offs in the choice of ribosomal barcoding markers for fungal amplicon sequencing: a case study on the grapevine trunk mycobiome

Abstract

The evolution of sequencing technology and multiplexing has rapidly expanded our ability to characterize fungal diversity in the environment. However, obtaining an unbiased assessment of the fungal community using ribosomal markers remains challenging. Longer amplicons were shown to improve taxonomic resolution and resolve ambiguities by reducing the risk of spurious operational taxonomic units. We examined the implications of barcoding strategies by amplifying and sequencing two ribosomal DNA fragments. We analyzed the performance of the full internal transcribed spacer (ITS) and a longer fragment including also a part of the 28S replicated on 60 grapevine trunk core samples. Grapevine trunks harbor highly diverse fungal communities with implications for disease development. Using identical handling, amplification and sequencing procedures, we obtained higher sequencing depths for the shorter ITS amplicon. Despite the more limited access to polymorphism, the overall diversity in amplified sequence variants was higher for the shorter ITS amplicon. We detected no meaningful bias in the phylogenetic composition due to the amplicon choice across analyzed samples. Despite the increased resolution of the longer ITS-28S amplicon, the higher and more consistent yields of the shorter amplicons produced a clearer resolution of the fungal community of grapevine stem samples. Our study highlights that the choice of ribosomal amplicons should be carefully evaluated and adjusted according to specific goals.

Landscape-scale endophytic community analyses in replicated grapevine stands reveal that dieback disease is not caused by specific fungal communities

“…Upon examining the distribution of the Neosetophoma genus across various geographical regions, a notably higher occurrence was observed in the Valais region (Figure 5D). Valais is the region that exhibits the lowest incidence of esca impact (Monod et al, 2023). When we examined the presence of the Neosetophoma genus alongside the recorded mortality rates in each vineyard, we did not detect any correlation though (R=0.05 p=0.518) (Figure 5E).…”

“…Our study builds upon previous research by substantially increasing the number of samples, emphasizing that even sampling strategies with hundreds of data points may still be underpowered. Previous research conducted on the same vineyards has linked the incidence of esca symptoms to pedo-climatic factors (Monod et al, 2023), suggesting that soil water holding capacity is a key factor for disease development.…”

“…All plants originate from a single nursery to ensure standardization of both age and genetic makeup. The set of replicated Gamaret plots was tracked based on physiological indicators such as yield, must and leaf chemical composition, plant water status, along with meteorological and climatic recordings, soil analyses, and the incidence of esca (Monod et al, 2023).…”

“…Valais is the region that exhibits the lowest incidence of esca impact (Sup. Fig., (Monod et al, 2023).…”

“…A systematic investigation into esca symptom development using standardized grapevine genotypes planted in diverse environments revealed that soil water holding capacity is likely a factor favoring symptom development (Monod et al, 2023). Further studies have shown that water availability can influence symptom expression (Marchi et al, 2006;Sosnowski et al, 2007) and symptom development was likely favored by stronger amplitudes in wet/dry climate transitions (Monod et al, 2023). Highthroughput amplicon sequencing techniques can generate high-resolution assessments of fungal diversity within grapevine trunks (Dissanayake et al, 2018;Pacifico et al, 2019;Monod et al, 2022).…”