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

ABSTRACT: BACKGROUND: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. Profiling of pathogen communities associated with grapevine trunk diseases 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. RESULTS:We first performed in silico simulations and then tested the primers by high-throughput amplicon sequencing of (i) multiple combinations of mock communities, (ii) time-course 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 over- or under-estimated with universal primers. CONCLUSIONS: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.

SUBMITTER: Morales-Cruz A

PROVIDER: S-EPMC6295080 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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

Morales-Cruz Abraham A Figueroa-Balderas Rosa R García Jadran F JF Tran Eric E Rolshausen Philippe E PE Baumgartner Kendra K Cantu Dario D

BMC microbiology 20181214 1

<h4>Background</h4>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. Profiling of pathogen communities associated with grapevine trunk diseases 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 broa ...[more]

PMID: 30547761

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Project description:BackgroundGrapevine trunk diseases (GTDs) is a disease complex caused by wood pathogenic fungi belonging to genera like Phaeomoniella, Phaeoacremonium, Fomitiporia, Eutypa and members of the family Botryosphaeriaceae. However, the co-occurrence of these fungi in symptomatic and asymptomatic vines at equivalent abundances has questioned their role in GTDs. Hence, we still lack a good understanding of the fungi involved in GTDs, their interactions and the factors controlling their assemblage in vines. We determined the fungal and bacterial microbiome in wood tissues of asymptomatic and symptomatic vines of three main Greek cultivars (Agiorgitiko, Xinomavro, Vidiano), each cultivated in geographically distinct viticultural zones, using amplicon sequencing.ResultsWe noted that cultivar/biogeography (lumped factor) was the strongest determinant of the wood fungal microbiome (p < 0.001, 22.7%), while GTD symptoms condition had a weaker but still significant effect (p < 0.001, 3.5%), being prominent only in the cultivar Xinomavro. Several fungal Amplicon Sequence Variants (ASVs), reported as GTD-associated pathogens like Kalmusia variispora, Fomitiporia spp., and Phaemoniella chlamydosporα (most dominant in our study), were positively correlated with symptomatic vines in a cultivar/viticultural zone dependent manner. Random Forest analysis pointed to P. chlamydosporα, K. variispora, A. alternata and Cladosporium sp., as highly accurate predictors of symptomatic vines (0% error rate). The wood bacterial microbiome showed similar patterns, with biogeography/cultivar being the main determinant (p < 0.001, 25.5%) of its composition, followed by the GTD status of vines (p < 0.001, 5.2%). Differential abundance analysis revealed a universal positive correlation (p < 0.001) of Bacillus and Streptomyces ASVs with asymptomatic vines. Network analysis identified a significant negative co-occurrence network between these bacterial genera and Phaemoniella, Phaeoacrominum and Seimatosporium. These results point to a plant beneficial interaction between Bacillus/Streptomyces and GTD pathogens.ConclusionsOur study (a) provides evidence that GTD symptomatic plants support a wood fungal microbiome, showing cultivar and biogeography-dependent patterns, that could be used as a proxy to distinguish between healthy and diseased vines, (b) points to strong interactions between the bacterial and fungal wood microbiome in asymptomatic vines that should be further pursued in the quest for discovery of novel biocontrol agents.

Project description:Grapevine trunk diseases (GTDs) are a serious problem of grapevines worldwide. The microbiota of the grapevine endosphere comprises prokaryotic and eukaryotic endophytes, which may form varied relationships with the host plant from symbiotic to pathogenic. To explore the interaction between grapevine endophytic bacteria and GTDs, the endomicrobiome associated with grapevine wood was characterized using next-generation Illumina sequencing. Wood samples were collected from grapevine trunks with and without external symptoms of GTD (cankers) from two vineyards in the Hunter Valley and Hilltops, NSW, Australia and metagenomic characterization of the endophytic community was conducted using the 16S rRNA gene (341F/806R) and ITS (1F/2R) sequences. Among the important GTD pathogens, Phaeomoniella, Phaeoacremonium, Diplodia and Cryptovalsa species were found to be abundant in both symptomatic and asymptomatic grapevines from both vineyards. Eutypa lata and Neofusicoccum parvum, two important GTD pathogens, were detected in low numbers in Hilltops and the Hunter Valley, respectively. Interestingly, Pseudomonas dominated the bacterial community in canker-free grapevine tissues in both locations, comprising 56-74% of the total bacterial population. In contrast, the Pseudomonas population in grapevines with cankers was significantly lower, representing 29 and 2% of the bacterial community in Hilltops and the Hunter Valley, respectively. The presence of Pseudomonas in healthy grapevine tissues indicates its ability to colonize and survive in the grapevine. The potential of Pseudomonas spp. as biocontrol agents against GTD pathogens was also explored. Dual culture tests with isolated fluorescent Pseudomonas against mycelial discs of nine Botryosphaeria dieback, three Eutypa dieback, and two Esca/Petri disease pathogens, revealed antagonistic activity for 10 Pseudomonas strains. These results suggest the potential of Pseudomonas species from grapevine wood to be used as biocontrol agents to manage certain GTD pathogens.

Project description:DNA metabarcoding is used to generate species composition data for entire communities. However, sequencing errors in high-throughput sequencing instruments are fairly common, usually requiring reads to be clustered into operational taxonomic units (OTUs), losing information on intraspecific diversity in the process. While Cytochrome c oxidase subunit I (COI) haplotype information is limited in resolving intraspecific diversity it is nevertheless often useful e.g. in a phylogeographic context, helping to formulate hypotheses on taxon distribution and dispersal.This study combines sequence denoising strategies, normally applied in microbial research, with additional abundance-based filtering to extract haplotype information from freshwater macroinvertebrate metabarcoding datasets. This novel approach was added to the R package "JAMP" and can be applied to COI amplicon datasets. We tested our haplotyping method by sequencing (i) a single-species mock community composed of 31 individuals with 15 different haplotypes spanning three orders of magnitude in biomass and (ii) 18 monitoring samples each amplified with four different primer sets and two PCR replicates.We detected all 15 haplotypes of the single specimens in the mock community with relaxed filtering and denoising settings. However, up to 480 additional unexpected haplotypes remained in both replicates. Rigorous filtering removes most unexpected haplotypes, but also can discard expected haplotypes mainly from the small specimens. In the monitoring samples, the different primer sets detected 177-200 OTUs, each containing an average of 2.40-3.30 haplotypes per OTU. The derived intraspecific diversity data showed population structures that were consistent between replicates and similar between primer pairs but resolution depended on the primer length. A closer look at abundant taxa in the dataset revealed various population genetic patterns, e.g. the stonefly Taeniopteryx nebulosa and the caddisfly Hydropsyche pellucidula showed a distinct north-south cline with respect to haplotype distribution, while the beetle Oulimnius tuberculatus and the isopod Asellus aquaticus displayed no clear population pattern but differed in genetic diversity.We developed a strategy to infer intraspecific genetic diversity from bulk invertebrate metabarcoding data. It needs to be stressed that at this point this metabarcoding-informed haplotyping is not capable of capturing the full diversity present in such samples, due to variation in specimen size, primer bias and loss of sequence variants with low abundance. Nevertheless, for a high number of species intraspecific diversity was recovered, identifying potentially isolated populations and taxa for further more detailed phylogeographic investigation. While we are currently lacking large-scale metabarcoding datasets to fully take advantage of our new approach, metabarcoding-informed haplotyping holds great promise for biomonitoring efforts that not only seek information about species diversity but also underlying genetic diversity.

Dataset Information

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

Publications

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

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