Metagenomic Sequencing for Identification of Xylella fastidiosa from Leaf Samples

Román-Reyna, Verónica; Dupas, Enora; Cesbron, Sophie; Marchi, Guido; Campigli, Sara; Hansen, Mary Ann; Bush, Elizabeth; Prarat, Melanie; Shiplett, Katherine; Ivey, Melanie L. Lewis; Pierzynski, Joy; Miller, Sally A.; Hand, Francesca Peduto; Jacques, Marie‐Agnès; Jacobs, Jonathan M.

doi:10.1128/msystems.00591-21

Cited by 15 publications

(14 citation statements)

References 40 publications

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“…Metagenomic sequencing of plant samples with the MinION sequencer or Illumina sequencing platforms had been used previously to detect viral, fungal and bacterial plant pathogens [16], including X. fastidiosa [17,45,46]. However, when the MinION was used previously to detect X. fastidiosa, the obtained sequencing reads were simply aligned to reference genomes to find the genome to which the highest number of reads mapped without further phylogenetic analysis [17]; identification was limited to subspecies, or sequencing was performed only after PCR of MLST loci making whole-genome-based phylogenetic analysis impossible [45].…”

Section: Discussionmentioning

confidence: 99%

“…More recently, the Illumina iSeq and MiSeq were used for detection and identification of X. fastidiosa as well [18]. Both Illumina platforms detected X.…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…Recently, culture-independent metagenomic sequencing (sequencing all the micro-organisms present in a sample simultaneously) has been explored for fast detection and identification of viral, fungal and bacterial plant pathogens [16], including X. fastidiosa [17, 18]. The Oxford Nanopore Technologies (ONT) MinION sequencer has attracted particular attention, because it requires minimal laboratory infrastructure for library preparation and sequencing, is of relatively low cost, and provides results almost in real-time [19].…”

Section: Introductionmentioning

confidence: 99%

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Investigating plant disease outbreaks with long-read metagenomics: sensitive detection and highly resolved phylogenetic reconstruction applied to Xylella fastidiosa

et al. 2022

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Early disease detection is a prerequisite for enacting effective interventions for disease control. Strains of the bacterial plant pathogen Xylella fastidiosa have recurrently spread to new crops in new countries causing devastating outbreaks. So far, investigation of outbreak strains and highly resolved phylogenetic reconstruction have required whole-genome sequencing of pure bacterial cultures, which are challenging to obtain due to the fastidious nature of X. fastidiosa . Here, we show that culture-independent metagenomic sequencing, using the Oxford Nanopore Technologies MinION long-read sequencer, can sensitively and specifically detect the causative agent of Pierce’s disease of grapevine, X. fastidiosa subspecies fastidiosa . Using a DNA sample from a grapevine in Virginia, USA, it was possible to obtain a metagenome-assembled genome (MAG) of sufficient quality for phylogenetic reconstruction with SNP resolution. The analysis placed the MAG in a clade with isolates from Georgia, USA, suggesting introduction of X. fastidiosa subspecies fastidiosa to Virginia from the south-eastern USA. This proof of concept study, thus, revealed that metagenomic sequencing can replace culture-dependent genome sequencing for reconstructing transmission routes of bacterial plant pathogens.

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Section: Discussionmentioning

confidence: 99%

“…More recently, the Illumina iSeq and MiSeq were used for detection and identification of X. fastidiosa as well [18]. Both Illumina platforms detected X.…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Investigating plant disease outbreaks with long-read metagenomics: sensitive detection and highly resolved phylogenetic reconstruction applied to Xylella fastidiosa

et al. 2022

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“…), bacteria ("Candidatus Liberibacter asiaticus"), and viruses (Carrot yellow leaf virus) [19]. Illumina sequencing was recently used to detect and identify particular subspecies of XF; although this method proved efficient, it involves the use of dedicated laboratory facilities and trained personnel [20]. The Oxford Nanopore Technologies (ONT) MinION device offers the same advantages as other NGS platforms but is also portable and inexpensive, allowing in-field deployment on a larger scale.…”

Section: Introductionmentioning

confidence: 99%

Real-Time On-Site Diagnosis of Quarantine Pathogens in Plant Tissues by Nanopore-Based Sequencing

et al. 2022

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Rapid and sensitive assays for the identification of plant pathogens are necessary for the effective management of crop diseases. The main limitation of current diagnostic testing is the inability to combine broad and sensitive pathogen detection with the identification of key strains, pathovars, and subspecies. Such discrimination is necessary for quarantine pathogens, whose management is strictly dependent on genotype identification. To address these needs, we have established and evaluated a novel all-in-one diagnostic assay based on nanopore sequencing for the detection and simultaneous characterization of quarantine pathogens, using Xylella fastidiosa as a case study. The assay proved to be at least as sensitive as standard diagnostic tests and the quantitative results agreed closely with qPCR-based analysis. The same sequencing results also allowed discrimination between subspecies when present either individually or in combination. Pathogen detection and typing were achieved within 13 min of sequencing owing to the use of an internal control that allowed to stop sequencing when sufficient data had accumulated. These advantages, combined with the use of portable equipment, will facilitate the development of next-generation diagnostic assays for the efficient monitoring of other plant pathogens.

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Comparison of iSeq and MiSeq as the two platforms for 16S rRNA sequencing in the study of the gut of rat microbiome

Salamon

Zapała

Krawczyk

et al. 2022

Appl Microbiol Biotechnol

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Amplicon-based next-generation sequencing (NGS) of the 16S ribosomal RNA (16S) regions is a culture-free method used to identify and analyze Procaryota occurring within a given sample. The prokaryotic 16S rRNA gene contains conserved regions and nine variable regions (V1-V9) frequently used for phylogenetic classification of genus or species in diverse microbial populations. This work compares the accuracy and efficacy of two platforms, iSeq and MiSeq from Illumina, used in sequencing 16S rRNA. The most important similarities and differences of 16S microbiome sequencing in 20 fecal rat samples were described. Genetic libraries were prepared according to 16S Metagenomic Sequencing Library Preparation (Illumina) for the V3 and V4 regions of the 16S. The species richness obtained using iSeq technology was lower compared to MiSeq. At the second taxonomy level (L2), the abundance of taxa was comparable for both platforms. At the L7, the taxa abundance was significantly different, and the number of taxa was higher for the MiSeq. The alpha diversity was lower for iSeq than for MiSeq, starting from the order to the species level. The beta diversity estimation revealed statistically significant differences in microbiota diversity starting from the class level to the species level in samples sequenced on two investigated platforms. This work disclosed that the iSeq platform could be used to evaluate the bacterial profile of the samples to characterize the overall profile. The MiSeq System seems to be better for a detailed analysis of the differences in the microbiota composition. Key points • iSeq platform allows to shorten the sequencing time three times compared to the MiSeq. • iSeq can only be used for an initial and quick microbiome assessment. • MiSeq is better for a detailed analysis of the differences in the microbiota composition.

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Metagenomic Sequencing for Identification of Xylella fastidiosa from Leaf Samples

Abstract: Destructive Xylella fastidiosa ( Xf ) outbreaks in Europe highlight this pathogen’s capacity to expand its host range and geographical distribution. The current disease diagnostic approaches are limited by a multiple-step process, biases to known sequences, and detection limits.

Cited by 15 publications

References 40 publications

Investigating plant disease outbreaks with long-read metagenomics: sensitive detection and highly resolved phylogenetic reconstruction applied to Xylella fastidiosa

Investigating plant disease outbreaks with long-read metagenomics: sensitive detection and highly resolved phylogenetic reconstruction applied to Xylella fastidiosa

Real-Time On-Site Diagnosis of Quarantine Pathogens in Plant Tissues by Nanopore-Based Sequencing

Comparison of iSeq and MiSeq as the two platforms for 16S rRNA sequencing in the study of the gut of rat microbiome

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