This study investigated four different environmentally relevant microplastic (MP) pollutants which were derived from two facial cleansers, a plastic bag and polyethylene textile fleece. The mean size ...range of the particles (according to number distribution) was 20–250 μm when measured as a powder and 0.02–200 μm in suspension. In all MP exposures, plastic particles were found inside the guts of D. magna and A. franciscana, but only in the case of daphnids a clear exponential correlation between MP uptake in the gut and the size of the MP was identified. Exposure tests in which the majority of the MP particles were below 100 μm in size also had higher numbers of daphnids displaying evidence of MP ingestion. As the average MP particle size increased, the percentage of daphnids which had MP in their gut decreased. Using a number distribution value to measure particle size when in a suspension is more experimentally relevant as it provides a more realistic particle size than when samples are measured as a powder. Generally, artemias had fewer MP particles in the gut, than the daphnids, which could be explained by their different food size preferences. No acute effects on D. magna were found, but the growth of A. franciscana was affected. We conclude that zooplankton crustacean can ingest various MPs but none of the exposures tested were highly acutely hazardous to the test species. In addition, no delayed lethal effects in a 24 h post-exposure period were found.
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•All tested microplastics were found inside the guts of D. magna and A. franciscana.•The uptake of microplastics in daphnids is exponentially related to the size of particles.•A small mass of particles is sufficient to fill the gut of daphnids.•No acute mortality of daphnids and artemias was observed.•No delayed acute effects were found and microplastic was depurated from organisms.
Microplastic fibers (MP) from textile weathering and washing are increasingly being recognized as environmental pollutants. The majority of studies on the bioavailability and effects of microplastic ...focused on small polystyrene spherical plastic particles, while less data are available for fibers and for other materials besides polystyrene. We investigated the ingestion and effects of ground polyethylene terephthalate (PET) textile microfibers (length range: 62–1400 μm, width 31–528 μm, thickness 1–21.5 μm) on the freshwater zooplankton crustacean Daphnia magna after a 48 h exposure and subsequent 24 h of recovery in MP free medium and algae. The majority of ingested fibers by D. magna were around 300 μm, but also some very large twisted MP fibers around 1400 μm were found inside the gut. Exposure to these fibers results in increased mortality of daphnids after 48 h only in the case where daphnids were not pre-fed with algae prior to experiment, but no effect was found when daphnids were fed before the experiments. Regardless of the feeding regime, daphnids were not able to recover from MP exposure after additional 24 h incubation period in a MP free medium with algae. The uptake and effects of PET textile MP on D. magna are presented here for the first time.
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•D. magna ingests very long polyethylene terephthalate textile microfibers (even 1400 μm long).•Ingestion of fibres results in increased mortality.•Daphnia die even after 24 h of exposure in MP free medium following 48 h of MP exposure.
Daphnia magna ingest long textile microplastic fibers.
Genome editing techniques, such as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated systems (CRISPR/Cas9) are undoubtedly becoming an indispensable tool for improving food ...crops and tackling agricultural challenges. In the present study, key factors affecting transformation efficiency, such as PEG4000 concentration, incubation time, and plasmid amount were evaluated to achieve efficient delivery of CRISPR/Cas9 vector into cabbage protoplasts. Using amplicon sequencing, we confirmed a significant effect of PEG4000 concentration and incubation time on the induced target mutations. By optimizing the transformation protocol, editing efficiency of 26.4% was achieved with 40 µg of plasmid and 15 minutes incubation with 50% PEG4000. While these factors strongly affected the mutation rate, the viability of the transformed protoplasts remained high. Our findings would be useful for successful genome editing in cabbage and other brassicas, as well as in research areas such as gene function analysis and subcellular localization that rely on transient transformation methods in protoplasts.
Microspore embryogenesis (ME) is the most powerful tool for creating homozygous lines in plant breeding and molecular biology research. It is still based mainly on the reprogramming of microspores by ...temperature, osmotic and/or nutrient stress. New compounds are being sought that could increase the efficiency of microspore embryogenesis or even induce the formation of haploid embryos from recalcitrant genotypes. Among these, the mitogenic factor phytosulfokine alpha (PSK-α) is promising due to its broad spectrum of activity
and
. The aim of our study was to investigate the effect of PSK-α on haploid embryogenesis from microspores of oilseed rape (
L., DH4079), one of the most important oil crops and a model plant for studying the molecular mechanisms controlling embryo formation. We tested different concentrations (0, 0.01, 0.1 and 1 µM) of the peptide and evaluated its effect on microspore viability and embryo regeneration after four weeks of culture. Our results showed a positive correlation between addition of PSK-α and cultured microspore viability and a positive effect also on the number of developed embryos. The analysis of transcriptomes across three time points (day 0, 2 and 4) with or without PSK-α supplementation (15 RNA libraries in total) unveiled differentially expressed genes pivotal in cell division, microspore embryogenesis, and subsequent regeneration. PCA grouped transcriptomes by RNA sampling time, with the first two principal components explaining 56.8% variability. On day 2 with PSK, 45 genes (15 up- and 30 down-regulated) were differentially expressed when PSK-α was added and their number increased to 304 by day 4 (30 up- and 274 down-regulated).
,
, and
gene expression analysis revealed dynamic patterns, with
displaying the highest increase and overall expression during microspore culture at days 2 and 4. Despite some variations, only
showed significant differential expression upon PSK-α addition. Of 16 ME-related molecular markers, 3 and 15 exhibited significant differential expression in PSK-supplemented cultures at days 2 and 4, respectively. Embryo-specific markers predominantly expressed after 4 days of culture, with higher expression in medium without PSK, while on day 0, numerous sporophyte-specific markers were highly expressed.
RNA interference is an evolutionary conserved mechanism by which organisms regulate the expression of genes in a sequence-specific manner to modulate defense responses against various abiotic or ...biotic stresses. Hops are grown for their use in brewing and, in recent years, for the pharmaceutical industry. Hop production is threatened by many phytopathogens, of which
, the causal agent of Verticillium wilt, is a major contributor to yield losses. In the present study, we performed identification, characterization, phylogenetic, and expression analyses of three Argonaute, two Dicer-like, and two RNA-dependent RNA polymerase genes in the susceptible hop cultivar Celeia and the resistant cultivar Wye Target after infection with
. Phylogeny results showed clustering of hop RNAi proteins with their orthologues from the closely related species
,
and
which form a common cluster with species of the Rosaceae family. Expression analysis revealed downregulation of argonaute 2 in both cultivars on the third day post-inoculation, which may result in reduced AGO2-siRNA-mediated posttranscriptional gene silencing. Both cultivars may also repress ta-siRNA biogenesis at different dpi, as we observed downregulation of argonaute 7 in the susceptible cultivar on day 1 and downregulation of RDR6 in the resistant cultivar on day 3 after inoculation.
(
) is one of the most problematic hop (
L.) pathogens, as the highly virulent fungal pathotypes cause severe annual yield losses due to infections of entire hop fields. In recent years, the RNA ...interference (RNAi) mechanism has become one of the main areas of focus in plant-fungal pathogen interaction studies and has been implicated as one of the major contributors to fungal pathogenicity. MicroRNA-like RNAs (milRNAs) have been identified in several important plant pathogenic fungi; however, to date, no milRNA has been reported in the
species. In the present study, using a high-throughput sequencing approach and extensive bioinformatics analysis, a total of 156 milRNA precursors were identified in the annotated
genome, and 27 of these milRNA precursors were selected as true milRNA candidates, with appropriate microRNA hairpin secondary structures. The stem-loop RT-qPCR assay was used for milRNA validation; a total of nine
milRNAs were detected, and their expression was confirmed. The milRNA expression patterns, determined by the absolute quantification approach, imply that milRNAs play an important role in the pathogenicity of highly virulent
pathotypes. Computational analysis predicted milRNA targets in the
genome and in the host hop transcriptome, and the activity of milRNA-mediated RNAi target cleavage was subsequently confirmed for two selected endogenous fungal target gene models using the 5' RLM-RACE approach.
Diseases caused by viruses and virus-like organisms are one of the major problems in viticulture and grapevine marketing worldwide. Therefore, rapid and accurate diagnosis and identification is ...crucial. In this study, we used HTS of virus- and viroid-derived small RNAs to determine the virome status of Slovenian preclonal candidates of autochthonous and local grapevine varieties (
L.). The method applied to the studied vines revealed the presence of nine viruses and two viroids. All viral entities were validated and more than 160 Sanger sequences were generated and deposited in NCBI. In addition, a complete description into the co-infections in each plant studied was obtained. No vine was found to be virus- and viroid-free, and no vine was found to be infected with only one virus or viroid, while the highest number of viral entities in a plant was eight.
The main challenge associated with genotyping based on conventional length polymorphisms is the cross-laboratory standardization of allele sizes. This step requires the inclusion of standards and ...manual sizing to avoid false results. Capillary electrophoresis (CE) approaches limit the information to the length polymorphism and do not allow the determination of a complete marker sequence. As an alternative, high-throughput sequencing (HTS) offers complete information regarding marker sequences and their flanking regions. In this work, we investigated the suitability of a semi-quantitative sequencing approach for microsatellite genotyping using Illumina paired-end technology. Twelve microsatellite loci that are well established for grapevine CE typing were analysed on 96 grapevine samples from six different countries. We redesigned primers to the length of the amplicon for short sequencing (~100 bp). The primer pair was flanked with a 10 bp overhang for the introduction of barcodes on both sides of the amplicon to enable high multiplexing. The highest data peaks were determined as simple sequence repeat (SSR) alleles and compared with the CE dataset based on 12 reference samples. The comparison showed that HTS SSR genotyping can successfully replace the CE system in further experiments. We believe that, with next-generation sequencing, genotyping can be improved in terms of its speed, accuracy, and price.
Phenolic compounds are involved in plant responses to various biotic and abiotic stress factors, with many studies suggesting their role in defense mechanisms against fungal pathogens. Soilborne ...vascular pathogen
causes severe wilting and consequent dieback in a wide range of economically important crops, including hops (
L.). In this study, we investigated the differential accumulation of phenolics in the susceptible "Celeia" and resistant "Wye Target" hop cultivars during the pathogenesis of
wilt. Quantitative polymerase chain reaction showed that colonization in the roots of both cultivars was intensive, but decreased continuously throughout the experiment in the resistant cultivar, while the relative fungal amount continuously increased in the stems of the susceptible cultivar. In response to colonization in the roots of the resistant cultivar, a significant increase in total flavanols was detected at three days postinoculation (dpi), suggesting a possible role in preventing fungus spread into the stems. The accumulation of phenolic compounds was less pronounced in the stems of the resistant cultivar since, compared to the latter, significant increases in flavonols at 3 and 15 dpi and hydroxycinnamic acids at 6 dpi were observed in the stems of the susceptible cultivar.
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