Diversity of Phytophthora Species Detected in Disturbed and Undisturbed British Soils Using High-Throughput Sequencing Targeting ITS rRNA and COI mtDNA Regions

Disease outbreaks caused by introduced Phytophthora species have been increasing in British forests and woodlands in recent years. A better knowledge of the Phytophthora communities already present in the UK is of great importance when developing management and mitigation strategies for these diseases. To do this, soils were sampled in “disturbed” sites, meaning sites frequently visited by the public, with recent and new plantings or soil disturbances versus more “natural” forest and woodland sites with little disturbance or management. Phytophthora diversity was assessed using high-throughput Illumina sequencing targeting the widely accepted barcoding Internal Transcribed Spacer 1 (ITS1) region of rRNA and comparing it with the mitochondrial cytochrome c oxidase I (COI) gene. Isolation of Phytophthora was run in parallel. Nothophytophthora spp. and Phytophthora spp. were detected in 79 and 41 of the 132 locations of the 14 studied sites when using ITS or COI, respectively. A total of 20 Phytophthora amplicon sequence variants (ASVs) were assigned to known Phytophthora species from eight clades (1a, 2, 2b, 3a, 5, 6b, 7a, 8b, 8c, 8d, 10a, and 10b) and 12 ASVs from six clades (1a, 2c, 3a, 3b, 6b, 7a, 8b, 8c, and 8d) when using ITS or COI, respectively. Only at two locations were the results in agreement for ITS, COI, and isolation. Additionally, 21 and 17 unknown Phytophthora phylotypes were detected using the ITS and COI, respectively. Several Phytophthora spp. within clades 7 and 8, including very important forest pathogens such as P. austrocedri and P. ramorum, were identified and found more frequently at “disturbed” sites. Additionally, eight ASVs identified as Nothophytophthora spp. were detected representing the first report of species within this new genus in Britain. Only three species not known to be present in Britain (P. castaneae, P. capsici, and P. fallax) were detected with the ITS primers and not with COI. To confirm the presence of these or any potential new Phytophthora species, sites should be re-sampled for confirmation. Additionally, there is a need to confirm if these species are a threat to British trees and try to establish any eradication measures required to mitigate Phytophthora spread in Britain.

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Microbial Communities Associated with Farmed Genypterus chilensis: Detection in Water Prior to Bacterial Outbreaks Using Culturing and High-Throughput Sequencing

Animals ◽

10.3390/ani10061055 ◽

2020 ◽

Vol 10 (6) ◽

pp. 1055 ◽

Cited By ~ 2

Author(s):

Arturo Levican ◽

Jenny C. Fisher ◽

Sandra L. McLellan ◽

Ruben Avendaño-Herrera

Keyword(s):

Bacterial Community ◽

High Throughput ◽

Native Species ◽

High Throughput Sequencing ◽

Disease Outbreaks ◽

Emerging Pathogens ◽

Prophylactic Measures ◽

Almost All ◽

Conger Eel ◽

Genypterus Chilensis

The red conger eel (Genypterus chilensis, Guichenot) is a native species included in the Chilean Aquaculture Diversification Program due to high commercial demand. In the context of intensified farming, prior reports link two disease outbreaks with emerging pathogens in the Vibrio and Tenacibaculum genera. However, the roles remain unclear for the bacterial community and each specific bacterium is associated with the rearing environment for healthy specimens. The success of red conger eel farming therefore warrants research into the bacterial composition of aquaculture conditions and the antimicrobial susceptibilities thereof. This study used culturing methods and high-throughput sequencing to describe the bacterial community associated with water in which G. chilensis was farmed. With culturing methods, the predominant genera were Vibrio (21.6%), Pseudolteromonas (15.7%), Aliivibrio (13.7%), and Shewanella (7.8%). Only a few bacterial isolates showed amylase, gelatinase, or lipase activity, and almost all showed inhibition zones to commonly-used antibiotics in aquaculture. By contrast, high-throughput sequencing established Paraperlucidibaca, Colwellia, Polaribacter, Saprospiraceae, and Tenacibaculum as the predominant genera, with Vibrio ranking twenty-seventh in abundance. High-throughput sequencing also established a link between previous outbreaks with increased relative abundances of Vibrio and Tenacibaculum. Therefore, monitoring the presence and abundance of these potential pathogens could be useful in providing prophylactic measures to prevent future outbreaks.

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Sorting things out - assessing effects of unequal specimen biomass on DNA metabarcoding

10.7287/peerj.preprints.2561v1 ◽

2016 ◽

Cited By ~ 1

Author(s):

Vasco Elbrecht ◽

Bianca Peinert ◽

Florian Leese

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Sequencing Depth ◽

West Germany ◽

Coi Gene ◽

Mountain Stream ◽

Detection Rates ◽

Size Classes ◽

Dna Metabarcoding ◽

Size Sorting

1) Environmental bulk samples often contain many taxa with biomass differences of several orders of magnitude. This can be problematic in DNA metabarcoding and metagenomic high throughput sequencing approaches, as large specimens contribute over proportionally much DNA template. Thus a few specimens of high biomass will dominate the dataset, potentially leading to smaller specimens remaining undetected. Sorting of samples and balancing the amounts of tissue used per size fraction should improve detection rates, but has not been systematically tested. 2) Here we tested the effects of size sorting on taxa detection using freshwater macroinvertebrates. Kick sampling was performed at two locations of a low-mountain stream in West Germany, specimens were morphologically identified and sorted into small, medium and large size classes (< 2.5x5, 5x10 and up to 10x20 mm). Tissue from the 3 size categories was extracted individually, and pooled to simulate bulk samples that were not sorted and samples which were sorted and then pooled proportionately by specimen size. DNA from all 5 extractions of both samples was amplified using 4 different freshwater primer sets for the COI gene and sequenced on a HiSeq Illumina sequencer. 3) Sorting taxa by size and pooling them proportionately according to their abundance lead to a more equal amplification compared to the processing of complete samples without sorting. The sorted samples recovered 30% more taxa than the unsorted samples, at the same sequencing depth. Our results imply that sequencing depth can be decreased ~ 5 fold when sorting the samples into three size classes. 4) Our results demonstrate that even a coarse size sorting can substantially improve detection rates. While high throughput sequencing will become more accessible and cheaper within the next years, sorting bulk samples by specimen biomass is a simple yet efficient method to reduce current sequencing costs.

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Sorting things out - assessing effects of unequal specimen biomass on DNA metabarcoding

10.7287/peerj.preprints.2561 ◽

2017 ◽

Cited By ~ 1

Author(s):

Vasco Elbrecht ◽

Bianca Peinert ◽

Florian Leese

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Size Fraction ◽

Sequencing Depth ◽

Coi Gene ◽

Mountain Stream ◽

Detection Rates ◽

Size Classes ◽

Dna Metabarcoding ◽

Size Sorting

Environmental bulk samples often contain many taxa that vary several orders of magnitude in biomass. This can be problematic in DNA metabarcoding and metagenomic high-throughput sequencing approaches, as large specimens contribute disproportionately high amounts of DNA template. Thus, a few specimens of high biomass will dominate the dataset, potentially leading to smaller specimens remaining undetected. Sorting of samples by specimen size and balancing the amounts of tissue used per size fraction should improve detection rates, but this approach has not been systematically tested. Here we explored the effects of size sorting on taxa detection using two freshwater macroinvertebrate monitoring samples, collected from a low-mountain stream in Germany. Specimens were morphologically identified and sorted into three size classes (body size < 2.5x5, 5x10 and up to 10x20 mm). Tissue from each size category was extracted individually, and pooled to simulate samples that were not sorted by biomass ("Unsorted"). Additionally, size fractions were pooled so that each specimen contributed approximately equal amounts of biomass ("Sorted"). Mock samples were amplified using four different DNA metabarcoding primer sets targeting the Cytochrome c oxidase I (COI) gene. Sorting taxa by size and pooling them proportionately according to their abundance lead to a more equal amplification of taxa compared to the processing of complete samples without sorting. The sorted samples recovered 30% more taxa than the unsorted samples, at the same sequencing depth. Our results imply that sequencing depth can be decreased approximately five-fold when sorting the samples into three size classes and pooling by specimen abundance. Our study demonstrates that even a coarse size sorting can substantially improve taxa detection using DNA metabarcoding. While high throughput sequencing will become more accessible and cheaper within the next years, sorting bulk samples by specimen biomass is a simple yet efficient method to reduce current sequencing costs.

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Sorting things out - assessing effects of unequal specimen biomass on DNA metabarcoding

10.7287/peerj.preprints.2561v2 ◽

2017 ◽

Author(s):

Vasco Elbrecht ◽

Bianca Peinert ◽

Florian Leese

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Size Fraction ◽

Sequencing Depth ◽

West Germany ◽

Coi Gene ◽

Mountain Stream ◽

Size Classes ◽

Dna Metabarcoding ◽

Size Sorting

1) Environmental bulk samples often contain many taxa with biomass differences of several orders of magnitude. This can be problematic in DNA metabarcoding and metagenomic high throughput sequencing approaches, as large specimens contribute disproportionate amounts of DNA template. Thus a few specimens of high biomass will dominate the dataset, potentially leading to smaller specimens remaining undetected. Sorting of samples and balancing the amounts of tissue used per size fraction should improve detection rates, but this approach has not been systematically tested. 2) Here we tested the effects of size sorting on taxa detection using freshwater macroinvertebrates. Kick sampling was performed at two locations of a low-mountain stream in West Germany, specimens were morphologically identified and sorted into small, medium and large size classes (< 2.5x5, 5x10 and up to 10x20 mm). Tissue from the 3 size categories was extracted individually, and pooled to simulate samples that were not sorted by biomass and samples that were sorted and then pooled so that each specimen contributed approximately equal amounts of biomass. DNA from all five extractions of samples from both sites was amplified using four different DNA metabarcoding primer sets targeting the Cytochrome c oxidase I (COI) gene. The library was sequenced on a HiSeq Illumina sequencer. 3) Sorting taxa by size and pooling them proportionately according to their abundance lead to a more equal amplification compared to the processing of complete samples without sorting. The sorted samples recovered 30% more taxa than the unsorted samples, at the same sequencing depth. Our results imply that sequencing depth can be decreased approximately five-fold when sorting the samples into three size classes. 4) Our results demonstrate that even a coarse size sorting can substantially improve detection of taxa using DNA metabarcoding. While high throughput sequencing will become more accessible and cheaper within the next years, sorting bulk samples by specimen biomass is a simple yet efficient method to reduce current sequencing costs.

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