Suchergebnisse

Blueberry cultivation is becoming increasingly important in Spain. There is currently a tendency to reconvert soils previously used for strawberry cultivation to blueberries, which could produce the transmission of common pathogens such as Macrophomina phaseolina (Ijaz et al., 2013). The use of chemical antifungals has been the common method used to prevent this kind of infection but, due to their high environmental impact, legislation is increasingly restricting its use which makes it necessary to look for new alternatives such as biocontrol strategy.This project aim was to identify the main rhizospheric microorganisms implicated in blueberry plant survival or death and to analyse the repercussions of three treatments used against Macrophomina.Two of the treatments use a biocontrol strategy and a third one supplied the plant with nutrients inputs. The biocontrol treatments consisted of a mix composed of Pseudomonas aeruginosa and Bacillus velezensis strains with growth-promoting activities and antagonist effects against Macrophomina coming from the IFAPA-Las Torres collection, and a second one with Bacillus amyloliquefaciens. The third is a mix of Nitrogen, Phosphorus, Potassium, aminoacids and microorganisms . We characterized blueberry's rhizosphere from healthy and dead plants from the three treatments plus a control using metabarcoding, bacteria were identified via the 16S and fungi via the ITS region.All treatments modified the soil microbiome, especially the bacteria population. Although infection with M. phaseolina was the cause of plant death, the presence of Fusarium, another blueberry fungus pathogen, seemed to favour the infection and weaken the plant (Israel et al., 2005). On the other hand, the higher abundance of Penicillium or Gaiella in the rhizosphere of healthy plants could be related to increased resistance against Macrophomina (Altaf et al., 2018; Qi et al., 2019). From all the treatments tested, the one coming from the IFAPA-Las Torres collection was the best in modifying the rhizosphere most efficiently, favouring the proliferation of microorganisms with plant growth-promoting activities and pathogens antagonists.The different microorganisms detected between death and healthy plants open the possibility of studying new targets to act against and potential microorganisms to be used in biocontrol.

Metabarcoding extra‐organismal DNA from environmental samples is now a key technique in aquatic biomonitoring and ecosystem health assessment. Of critical consideration when designing experiments, and especially so when developing community standards and legislative frameworks, is the choice of genetic marker and primer set. Mitochondrial cytochrome c oxidase subunit I (COI), the standard DNA barcode marker for animals, with its extensive reference library, taxonomic discriminatory power and predictable sequence variation, is the natural choice for many metabarcoding applications. However, for targeting specific taxonomic groups in environmental samples, the utility of COI has yet to be fully scrutinized. Here, by using a case study of marine and freshwater fishes from the British Isles, we quantify the in silico performance of twelve primer pairs from four mitochondrial loci – COI, cytochrome b, 12S and 16S – in terms of reference library coverage, taxonomic discriminatory power and primer universality. We subsequently test in vitro four primer pairs – three COI and one 12S – for their specificity, reproducibility, and congruence with independent datasets derived from traditional survey methods at five estuarine and coastal sites around the English Channel and North Sea. Our results show that for aqueous extra‐organismal DNA at low template concentrations, both metazoan‐targeted and fish‐targeted COI primers perform poorly in comparison to 12S, exhibiting low levels of reproducibility due to non‐specific amplification of prokaryotic and non‐target eukaryotic DNAs. An ideal metabarcode would have an extensive reference library upon which custom primers could be designed, either for broad assessments of biodiversity, or taxon specific surveys. Such a database is available for COI, but low primer specificity hinders practical application, while conversely, 12S primers offer high specificity, but lack adequate references. The latter, however, can be mitigated by expanding the concept of DNA barcodes to include whole mitochondrial genomes generated by genome‐skimming existing tissue collections.

This is the pre-peer reviewed version of the following article: Collins, R.A., Bakker, J., Wangensteen, O.S., Soto, A.Z., Corrigan, L., Sims, D.W. . Mariani, S. (2019). Non-specific amplification compromises environmental DNA metabarcoding with COI. Methods in Ecology and Evolution, 10 (11), 1985-2001, which has been published in final form at https://doi.org/10.1111/2041-210X.13276. . This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. ; Metabarcoding extra‐organismal DNA from environmental samples is now a key technique in aquatic biomonitoring and ecosystem health assessment. Of critical consideration when designing experiments, and especially so when developing community standards and legislative frameworks, is the choice of genetic marker and primer set. Mitochondrial cytochrome c oxidase subunit I (COI), the standard DNA barcode marker for animals, with its extensive reference library, taxonomic discriminatory power and predictable sequence variation, is the natural choice for many metabarcoding applications. However, for targeting specific taxonomic groups in environmental samples, the utility of COI has yet to be fully scrutinized. Here, by using a case study of marine and freshwater fishes from the British Isles, we quantify the in silico performance of twelve primer pairs from four mitochondrial loci – COI, cytochrome b, 12S and 16S – in terms of reference library coverage, taxonomic discriminatory power and primer universality. We subsequently test in vitro four primer pairs – three COI and one 12S – for their specificity, reproducibility, and congruence with independent datasets derived from traditional survey methods at five estuarine and coastal sites around the English Channel and North Sea. Our results show that for aqueous extra‐organismal DNA at low template concentrations, both metazoan‐targeted and fish‐targeted COI primers perform poorly in comparison to 12S, exhibiting low levels of reproducibility due to non‐specific amplification of prokaryotic and non‐target eukaryotic DNAs. An ideal metabarcode would have an extensive reference library upon which custom primers could be designed, either for broad assessments of biodiversity, or taxon specific surveys. Such a database is available for COI, but low primer specificity hinders practical application, while conversely, 12S primers offer high specificity, but lack adequate references. The latter, however, can be mitigated by expanding the concept of DNA barcodes to include whole mitochondrial genomes generated by genome‐skimming existing tissue collections.

The use of genomic approaches to assist with biodiversity estimations is an alternative to traditional biomonitoring, which is very time-consuming and costly. In response to the high demand for quick community descriptions, DNA metabarcoding can simultaneously assign taxonomy to hundreds of samples rapidly and at low cost. However, the technique has not routinely been incorporated into biomonitoring network programs yet. Here, we applied DNA metabarcoding methodologies at stations within the monitoring network of the Basque Water Agency, the competent authority for the application of the European Water Framework Directive in this region. We characterized the benthic macroinvertebrate communities from 18 estuarine and coastal sediment samples using morphology and metabarcoding-based taxonomic identification and evaluated the performance of several versions of the AZTI's Marine Biotic Index (AMBI). Although metabarcoding detected 112 taxa against the 206 taxa identified through morphology, we showed that metabarcoding leads to similar biomonitoring conclusions compared with traditional techniques. Using the abundance and biomass of those taxa detected from morphological methodologies, we found a significant positive correlation with the number of reads obtained with metabarcoding approaches. The metabarcoding-based index derived from read counts, gAMBI, and the morphology-based index derived from individuals' biomass, (B)AMBI, showed the best correlation and revealed excellent agreement at determining the ecological status of the stations analyzed. We calculated that, for the analysis of the 51 stations included in the Basque monitoring network, metabarcoding was 55% less costly and 72% less time consuming. The results of our study are relevant to policy makers and researchers in the field of ecological assessment and will contribute to the quick implementation of DNA metabarcoding to intensive monitoring programs. ; We thank Iñaki Mendibil and Craig T. Mitchel for assistance in the laboratory work, and Xabier Irigoien and John Pearman whose comments helped to improve the manuscript. The specimen taxonomic identification based on morphology was done by experts from the Cultural Society INSUB following the identification protocols accepted and applied by the scientific community. This manuscript is a result of the DEVOTES (DEVelopment Of innovative Tools for understanding marine biodiversity and assessing good Environmental Status – http://www.devotes-project.eu) project funded by the European Union (7th Framework Program 'The Ocean of Tomorrow' Theme, grant agreement no. 308392) and the Basque Water Agency (URA) through a Convention with AZTI to monitor the Basque estuaries and coasts. This paper is contribution number 868 from AZTI (Marine Research Division).

Abstract
Background
Phytoplankton diversity can be difficult to ascertain from morphological analyses, because of the existence of cryptic species and pico- and concealed phytoplankton. In-depth sequencing and metabarcoding can reveal microbial diversity, and identify novel diversity. However, there has been little comparison of metabarcoding and morphological datasets derived from the same samples, and metabarcoding studies covering total eukaryotic phytoplankton diversity are rare. In this study, the variable V7 region of the 18S rDNA gene was employed to explore eukaryotic phytoplankton diversity in 11 Chinese freshwater environments, and further compared with the dataset obtained through morphological identification.

Results
Annotation by the evolutionary placement algorithm (EPA) rather than alignment with the SILVA database improved the taxonomic resolution, with 346 of 524 phytoplankton operational taxonomic units (OTUs) being assigned to the genus or species level. The number of unassigned OTUs was greatly reduced from 259 to 178 OTUs by using the EPA in place of the SILVA database. Metabarcoding detected 3.5 times more OTUs than the number of morphospecies revealed by morphological identification; furthermore, the number of species and the Shannon–Wiener index inferred from the two methods were correlated. A total of 34 genera were identified via both methods, while 31 and 123 genera were detected solely in the morphological or metabarcoding dataset, respectively.

Conclusion
The dbRDA plot showed distinct separation of the phytoplankton communities between lakes and reservoirs according to the metabarcoding dataset. The same pattern was obtained on the basis of 10 environmental variables in the PCO ordination plot, while the separation of the populations based on morphological data was poor. However, 30 morphospecies contributed 70% of the community difference between lakes and reservoirs in the morphological dataset, while 11 morphospecies were not found by metabarcoding. Considering the limitations of each of the two methods, their combination could substantially improve phytoplankton community assessment.

The Water Framework Directive (WFD) is the main political instrument for management of the waterbodies in Europe and includes the bioassessment of rivers and streams based on diatoms, through the calculation of an autoecological index, the Indice de Polluosensibilité Spécifique (IPS) officially adopted for Portugal. This index requires a high level of taxonomic expertise for morphological identification of individuals. Advances in genomics, such as the DNA metabarcoding combined with high-throughput sequencing (HTS) techniques offer a promising alternative to classical methods, limiting expertise requirement and therefore reducing time and costs. The aim of this study was to test the potential of DNA metabarcoding of diatoms in the bioassessment of Portuguese rivers by comparing the IPS classifications obtained with morphological and molecular approaches. A total of 88 samples from rivers in central Portugal were collected in the spring of 2017 following WFD standards. The morphological approach comprised taxonomic identification of at least 400 valves, under the light microscope. The molecular approach included eDNA extraction followed by DNA sequencing (Illumina MiSeq) using a 312bp rbcL DNA barcode. Sequences were analysed with Mothur software, producing Operational Taxonomic Units (OTUs) that were taxonomically assigned to the R-Syst::diatom reference library. It was also tested the effect of a correction factor (CF) for biovolume applied on molecular data. Inventories of diatom communities revealed a total number of 306, 125 and 111 species identified with morphology, molecular method without and with the CF, respectively. The total percentage of successfully assigned OTUs to the reference library was 32%, with an average of 47.5% per sample, while the average percentage of unassigned reads from the converted OTUs to taxa list was 52.5% and varied between 2 and 95%, among all samples. When comparing species' abundances, the results showed statistical differences in the number of species between molecular ...

1. Accurate quantification of biodiversity can be demanding and expensive. Although environmental DNA (eDNA) metabarcoding can facilitate biodiversity assessments through non-invasive, cost-efficient, and rapid surveys, the approach struggles to outperform traditional morphological approaches in providing reliable quantitative estimates for surveyed species (e.g., abundance and biomass). ; 2. We present an integrated methodology for improving biodiversity surveys that pairs eDNA metabarcoding with morphological data, following a series of taxonomic and geographic filters. We demonstrate its power by applying it to a new spatiotemporal dataset generated on an Iberian-wide distributed aquatic mesocosm infrastructure that spans a wide biogeographic gradient. ; 3. By building upon the strengths that these two approaches offer, our framework improved taxonomic resolution for 30% of the taxa and enabled species' traits (e.g., body-size) and abundance to be assigned to 85% of the taxa in hybrid datasets. ; 4. These results indicate that eDNA-based assessments can complement, but not always replace, conventional approaches. Integrating conventional and modern eDNA metabarcoding approaches, already available in the ecologist's toolbox, will greatly enhance biodiversity assessments. ; This work was supported by the Portuguese Science and Technology Foundation (FCT) through the scientific projects TrophicResponses - Trophic responses to macroecological gradients (PTDC/BIA-BIC/0352/2014), and StateShifts - Predicting state shifts in energetic food webs under climate change (PTDC/AAG-MAA/3764/2014). This work was also funded by FEDER funds through the COMPETE 2020 Programme and National Funds through FCT under the project number POCI-01-0145-FEDER-007688 and PTDC/CTA-AMB/30793/2017 (AdaptAlentejo – Predicting ecosystem-level responses to climate change). The Iberian Pond network has received funding from the European Union's Horizon 2020 research and innovation programme under grant Nos 731065 (AQUACOSM) and 871081 (AQUACOSM-plus). CLP acknowledges support by the Portuguese Science and Technology Foundation (FCT) through PhD studentship (SFRH/BD/102020/2014). ; We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). ; Peer reviewed

Diets during critical brooding and winter periods likely influence the growth of Lesser Prairie-Chicken (Tympanuchus pallidicinctus) populations. During the brooding period, rapidly growing Lesser Prairie-Chicken chicks have high calorie demands and are restricted to foods within immediate surroundings. For adults and juveniles during cold winters, meeting thermoregulatory demands with available food items of limited nutrient content may be challenging. Our objective was to determine the primary animal and plant components of Lesser Prairie-Chicken diets among native prairie, cropland, and Conservation Reserve Program (CRP) fields in Kansas and Colorado, USA, during brooding and winter using a DNA metabarcoding approach. Lesser Prairie-Chicken fecal samples (n = 314) were collected during summer 2014 and winter 2014-2015, DNA was extracted, amplified, and sequenced. A region of the cytochrome oxidase I (COI) gene was sequenced to determine the arthropod component of the diet, and a portion of the trnL intron region was used to determine the plant component. Relying on fecal DNA to quantify dietary composition, as opposed to traditional visual identification of gut contents, revealed a greater proportion of soft-bodied arthropods than previously recorded. Among 80 fecal samples for which threshold arthropod DNA reads were obtained, 35% of the sequences were most likely from Lepidoptera, 26% from Orthoptera, 14% from Araneae, 13% from Hemiptera, and 12% from other orders. Plant sequences from 137 fecal samples were composed of species similar to Ambrosia (27%), followed by species similar to Lactuca or Taraxacum (10%), Medicago (6%), and Triticum (5%). Forbs were the predominant (>50% of reads) plant food consumed during both brood rearing and winter. The importance both of native forbs and of a broad array of arthropods that rely on forbs suggests that disturbance regimes that promote forbs may be crucial in providing food for Lesser Prairie-Chickens in the northern portion of their distribution. ; Kansas Wildlife, Parks, and Tourism [KS W-73-R-3]; USDA Farm Services CRP Monitoring, Assessment, and Evaluation [12-IA-MRE CRP TA#7, KSCFWRU RWO 62]; USDA Natural Resources Conservation Service, Lesser Prairie-Chicken Initiative ; Funding for the project was provided by Kansas Wildlife, Parks, and Tourism (Federal Assistance Grant KS W-73-R-3); USDA Farm Services CRP Monitoring, Assessment, and Evaluation (12-IA-MRE CRP TA#7, KSCFWRU RWO 62); and USDA Natural Resources Conservation Service, Lesser Prairie-Chicken Initiative. ; Public domain authored by a U.S. government employee

Abstract Transfer of organisms with ships' ballast water is recognized as a major pathway of non-indigenous species introduction and addressed in a few recent legislative initiatives. Among other they imply scientific and technical research and monitoring to be conducted in a efficient and reliable way. The recent development of DNA barcoding and metabarcoding technologies opens new opportunities for biodiversity and biosecurity surveillance. In the current study, the performance of metabarcoding approach was assessed in comparison to the conventional (visual) observations, during the en route experimental ballast water survey. Opportunities and limitations of the molecular method were identified from taxonomical datasets rendered by two molecular markers of different degree of universality – the universal cytochrome oxydase sub-unit I gene and a fragment of RuBisCO gene. The cost-efficacy and possible improvements of these methods are discussed for the further successful development and implementation of the approach in ballast water control and NIS surveillance.

The work is part of the EU ERA-Net program BiodivERsA, under the project BioMarKs (Biodiversity of Marine euKaryotes).-- 14 pages, 8 figures, supplementary material https://doi.org/10.1038/s41598-018-27509-8 ; Although animals are among the best studied organisms, we still lack a full description of their diversity, especially for microscopic taxa. This is partly due to the time-consuming and costly nature of surveying animal diversity through morphological and molecular studies of individual taxa. A powerful alternative is the use of high-throughput environmental sequencing, providing molecular data from all organisms sampled. We here address the unknown diversity of animal phyla in marine environments using an extensive dataset designed to assess eukaryotic ribosomal diversity among European coastal locations. A multi-phylum assessment of marine animal diversity that includes water column and sediments, oxic and anoxic environments, and both DNA and RNA templates, revealed a high percentage of novel 18S rRNA sequences in most phyla, suggesting that marine environments have not yet been fully sampled at a molecular level. This novelty is especially high among Platyhelminthes, Acoelomorpha, and Nematoda, which are well studied from a morphological perspective and abundant in benthic environments. We also identified, based on molecular data, a potentially novel group of widespread tunicates. Moreover, we recovered a high number of reads for Ctenophora and Cnidaria in the smaller fractions suggesting their gametes might play a greater ecological role than previously suspected ; This work was supported by an Institució Catalana de Recerca i Estudis Avançats contract, two grants (BFU-2011-23434 and BFU2014-57779-P) from the Ministerio de Economia y Competitividad (MINECO), one of which (BFU2014-57779-P) was co-funded by the European Regional Development Fund (FEDER), and a European Research Council Consolidator Grant (ERC-2012-Co -616960) to I.R.-T. We also acknowledge financial support from the Secretaria d'Universitats i Recerca del Departament d'Economia i Coneixement de la Generalitat de Catalunya (Project 2014 SGR 619). J.d.C. was supported by a Marie Curie International Outgoing Fellowship grant (FP7-PEOPLE-2012-IOF - 331450 CAARL). J.P. acknowledges support from the European Research Council under the European Union's Seventh Framework Program (FP7/2007–2013)/ERC grant [268513] ; Peer Reviewed

Although animals are among the best studied organisms, we still lack a full description of their diversity, especially for microscopic taxa. This is partly due to the time-consuming and costly nature of surveying animal diversity through morphological and molecular studies of individual taxa. A powerful alternative is the use of high-throughput environmental sequencing, providing molecular data from all organisms sampled. We here address the unknown diversity of animal phyla in marine environments using an extensive dataset designed to assess eukaryotic ribosomal diversity among European coastal locations. A multi-phylum assessment of marine animal diversity that includes water column and sediments, oxic and anoxic environments, and both DNA and RNA templates, revealed a high percentage of novel 18S rRNA sequences in most phyla, suggesting that marine environments have not yet been fully sampled at a molecular level. This novelty is especially high among Platyhelminthes, Acoelomorpha, and Nematoda, which are well studied from a morphological perspective and abundant in benthic environments. We also identified, based on molecular data, a potentially novel group of widespread tunicates. Moreover, we recovered a high number of reads for Ctenophora and Cnidaria in the smaller fractions suggesting their gametes might play a greater ecological role than previously suspected. ; This work was supported by an Institució Catalana de Recerca i Estudis Avançats contract, two grants (BFU-2011-23434 and BFU2014-57779-P) from the Ministerio de Economia y Competitividad (MINECO), one of which (BFU2014-57779-P) was co-funded by the European Regional Development Fund (FEDER), and a European Research Council Consolidator Grant (ERC-2012-Co -616960) to I.R.-T. We also acknowledge financial support from the Secretaria d'Universitats i Recerca del Departament d'Economia i Coneixement de la Generalitat de Catalunya (Project 2014 SGR 619). J.d.C. was supported by a Marie Curie International Outgoing Fellowship grant (FP7-PEOPLE-2012-IOF - 331450 CAARL). J.P. acknowledges support from the European Research Council under the European Union's Seventh Framework Program (FP7/2007–2013)/ERC grant [268513]. The work is part of the EU ERA-Net program BiodivERsA, under the project BioMarKs (Biodiversity of Marine euKaryotes)

This study was funded from the European Union's Horizon 2020 Research and Innovation program under Grant Agreement No 689682, Adaptive Management of Barriers in European Rivers (AMBER) project