Search Results (146)

The genera Pestalotiopsis, Neopestalotiopsis, and Pseudopestalotiopsis share highly similar morphological characteristics. Although species within these genera are recognized as plant pathogens, their pathogenicity can differ even on the same host plant, highlighting the importance of accurate genus-level identification for effective disease management. However, reliable discrimination among these genera based solely on morphology or internal transcribed spacer (ITS) amplicon length is difficult. Therefore, molecular approaches based on ITS sequence data are required for practical and reliable genus-level identification. This study aimed to develop genus-specific PCR primers through comparative genome analysis using genus-specific gene regions identified from available genomic data. The performance of these primers was evaluated using 49 isolates obtained from banana, Japanese andromeda, loquat, rubber, and tea. The primer sets achieved an overall identification accuracy of 97%. One strain could not be assigned to Pestalotiopsis, which exhibited morphological characteristics inconsistent with the genus and was positioned outside the main Pestalotiopsis clade in phylogenetic analyses, supporting the taxonomic validity of the primer-based identification. These results demonstrate that the developed primers provide a reliable and practical tool for genus-level identification and taxonomic assignment of these morphologically similar fungal pathogens, including direct detection from infected plant tissues. Full article

Background/Objectives: Strain-level detection of bacteria is essential for applications such as diagnostics, food safety, and microbial monitoring. While 16S rRNA gene sequencing provides genus- or species-level resolution, it cannot reliably discriminate closely related strains. Whole-genome sequencing (WGS) offers high-resolution strain differentiation but remains impractical for routine detection due to cost and analytical complexity. This study aims to enable the translation of WGS data into accurate and cost-effective strain-specific PCR assays. Methods: We developed Erimin, a modular, shell-based bioinformatics pipeline for the automated identification of strain-specific genomic regions from short-read WGS data. Erimin systematically analyzes all available reference genomes for a given bacterial species in combination with sequencing data from a target strain. The workflow integrates reference-based read alignment, extraction of unmapped reads, de novo assembly, contig filtering and validation, genome annotation, and in silico PCR primer design and specificity evaluation. Results: Erimin was applied to Lactiplantibacillus pentosus whole-genome sequencing data to identify genomic regions specific to strain L33 through comparative analysis against a comprehensive set of reference genome assemblies representing multiple Lactiplantibacillus species. These regions were used for in silico PCR primer design and computational specificity assessment against non-target bacterial genomes, supporting discrimination of closely related strains. Conclusions: Erimin provides a structured computational approach for identifying strain-specific genomic regions from WGS data and for supporting the in silico design of PCR primers. This framework facilitates strain-level discrimination using targeted molecular assays. Full article

Trypanosomiasis, caused by flagellated protozoa of the genus Trypanosoma, has recently emerged as a major threat to aquaculture in China, particularly in farmed large yellow croaker (Larimichthys crocea). Outbreaks lead to high mortality rates and severe economic losses. Conventional diagnostic tools, such as blood-smear microscopy and molecular assays including polymerase chain reaction or quantitative polymerase chain reaction (qPCR), are often limited by low sensitivity during early infection or by their dependence on sophisticated instruments and trained personnel, restricting their utility in field conditions. To address these challenges, a multienzyme isothermal rapid amplification (MIRA) assay coupled with a lateral-flow dipstick (LFD) was developed for the rapid detection of trypanosoma strains circulating in L. crocea targeting the 18S ribosomal ribonucleic acid gene. After optimizing primer-probe sets, the assay performance was evaluated using plasmid standards and a panel of common aquaculture pathogens. The MRA-LFD assay consistently detected plasmid DNA at concentrations as low as 0.01 fg/µL (≈2.1 copies/µL) and demonstrated no cross-reactivity with other pathogens. Using clinical DNA samples positive for Trypanosoma, the detection limit was 100 fg µL⁻¹. Validation with 150 tissue samples from fish with and without clinical symptoms demonstrated high diagnostic consistency (94%) with qPCR results, confirming the reliability of the assay. This MIRA-LFD platform provides a sensitive, specific and portable diagnostic tool for early detection of Trypanosoma infections in large yellow croaker, offering valuable support for surveillance and disease management in aquaculture. Full article

In this study, we conducted a comprehensive characterization of avocado accessions from the Canary Islands, focusing on molecular, morphological, phenological, and agronomic traits. A total of 311 trees were initially prospected across Tenerife, La Palma, Gran Canaria, and La Gomera Islands. DNA was extracted from young leaves, and genetic diversity was assessed using 28 microsatelites primers, 14 of which were highly polymorphic, revealing 6937 amplified fragments, 137 of which were polymorphic. The average polymorphism percentage was 85.68%, with an expected heterozygosity of 0.68, indicating high genetic diversity. A dendrogram based on genetic data identified four main groups, two of which are closely related to the most widely cultivated avocado cultivars: ‘Hass’, ‘Fuerte’, and ‘Pinkerton’. Morphological analysis (over three years), using 91 descriptors, revealed a clear differentiation between accessions, with several groups corresponding to specific commercial cultivars. Morphotypes that deviated from the main groupings were specimens obtained through sexual propagation. Principal component analysis revealed that fruit characteristics such as length-to-diameter ratio, seed size, and fruit weight were the most discriminating traits, consistent with findings in the Persea genus. These results highlight the genetic and morphological diversity within the Canary Islands avocado germplasm, providing valuable insights for future breeding and conservation efforts. Full article

This study aimed to classify Escherichia coli phages using bioinformatics analysis systematically and to establish corresponding PCR and qPCR detection methods for rapid molecular typing and identification. Based on 419 complete E. coli phage genomes available in NCBI, phylogenetic and pan-genomic analyses were conducted to classify the phages at the family, subfamily, and genus levels and to identify highly conserved core genes. Specific primers targeting these core genes were designed, and their specificity, sensitivity, and reproducibility were verified using conventional PCR and dye-based qPCR. A total of 357 phages were successfully classified, encompassing 10 families, 20 subfamilies, and 67 genera. Pan-genomic analysis identified type-specific core genes within 16 taxa, including Ackermannviridae and Demerecviridae, for which 16 pairs of primers were designed. Validation using bacteriophages isolated from Xinjiang cattle farms showed distinct single PCR bands with high specificity, and the qPCR assay achieved a sensitivity of up to 10⁻⁵ µg/µL. This study established an efficient and broad-spectrum molecular typing and detection method for E. coli phages, providing a powerful preliminary screening tool for phage selection. Full article

Background: Kenaf (Hibiscus cannabinus L.) is an important fiber crop belonging to the genus Hibiscus in the Malvaceae family. Research on its chloroplast genome holds significant importance for deciphering the evolutionary relationships of the Hibiscus species, developing genetic markers, and promoting kenaf (H. cannabinus) genetic breeding. Methods: Based on high-throughput sequencing technology, this study completed the sequencing and assembly of the kenaf (H. cannabinus) chloroplast genome. Results: (1) The kenaf (H. cannabinus) chloroplast genome exhibits a typical circular quadripartite structure with a total length of 163,019 bp, including a large single-copy region (LSC) of 90,467 bp, a small single-copy region (SSC) of 19,486 bp, and a pair of inverted repeat regions (IRa/IRb) of 26,533 bp each. The total GC content is 36.62%, among which, the IR region has the highest GC content (42.61%) and the SSC region the lowest (30.87%). (2) A total of 131 genes were annotated, including 85 mRNAs, 37 tRNAs, 8 rRNAs, and 1 pseudogene. Their functions cover photosynthesis (e.g., pet and atp family genes), self-replication (e.g., rpl, rps, and rpo family genes), and genes with unknown functions (e.g., ycf1 and ycf2). A codon usage bias analysis revealed that the relative synonymous codon usage (RSCU) value of the stop codon UAA is the highest (1.6329), and codons ending with A/U are preferentially used (e.g., GCU for alanine with RSCU = 1.778). (3) A repeat sequence analysis identified various interspersed repeat sequences (predominantly 30~31 bp in length, with a relatively high proportion in the 30~40 bp range, including forward and palindromic types) and simple sequence repeats (cpSSRs). Among them, single-base repeat SSRs account for the highest proportion (e.g., (A)8 and (T)9), and specific SSR primers were designed. (4) A comparative evolutionary analysis indicated that the Ka/Ks ratios (nonsynonymous substitution rate/synonymous substitution rate) of core chloroplast genes (e.g., rps2 and rpoC2) in kenaf (H. cannabinus) are all less than 1 (0.145~0.415), suggesting that they are under purifying selection. The collinearity similarity of chloroplast genomes between kenaf (H. cannabinus) and its closely related species reaches over 99.97%, and the IR region boundaries are relatively conserved. The phylogenetic tree shows that kenaf (H. cannabinus) clusters with closely related Hibiscus species with a 100% bootstrap value, indicating a close genetic relationship. Conclusions: This study provides basic data for the functional analysis of the kenaf (H. cannabinus) chloroplast genome, the phylogeny of Hibiscus, and the utilization of genetic resources. Full article

The increasing prevalence of the mobile tigecycline resistance gene tet(X) poses a severe global health threat, and the genus Acinetobacter is a major reservoir. This study aimed to develop a rapid and specific multiplex PCR assay for detecting the predominant tet(X)-positive Acinetobacter species. Through pan-genome analyses of 390 tet(X)-positive Acinetobacter genomes, a total of 20 tet(X) variants were identified in 24 Acinetobacter species, including 17 published lineages and seven taxonomically unresolved Taxa. Acinetobacter indicus (30.8%), Acinetobacter amyesii (17.2%), and Acinetobacter towneri (16.1%) were the top three hosts of diverse tet(X) variants. Species-specific signature genes were identified and used for primer design, yielding amplicons of 267 bp (tet(X)), 424 bp (A. indicus), 690 bp (A. amyesii), and 990 bp (A. towneri). The assay was rigorously adjusted for an optimal annealing temperature of 52.8 °C and a primer ratio of 1:1:1:1, demonstrating high sensitivity with a detection limit of 0.3 ng/μL DNA and excellent stability under −20 °C, 4 °C, 20 °C storage conditions. Validation experiments on 151 bacterial strains showed high accuracy for DNA templates (≥97.8%) and bacterial suspensions (≥93.5%) within two hours. This cost-effective and highly accurate multiplex PCR provides a powerful tool for proactive surveillance and control of the critical Acinetobacter sp. pathogens. Full article

Background/Objectives: While morphological similarity and incomplete specimens pose a challenge to the precise identification of Coelogyne orchids, accurate species and genus assignment is essential for conservation and CITES enforcement. This study evaluated the efficacy of five DNA barcode regions—rbcL, matK, trnH-psbA, atpF-atpH, and ITS2—and their combinations for species- and genus-level discrimination within the genus Coelogyne, aiming to develop a rapid and simple diagnostic tool for use by customs officers and trade inspectors. This is the first comprehensive comparative analysis of these five barcode regions specifically within Coelogyne, a genus underrepresented in molecular identification studies, and the first to propose multi-locus combinations for potential practical use. This study identified DNA barcode regions with high resolution and reliability, providing a solid basis for practical identification kits. Such tools will enhance CITES enforcement by enabling rapid detection of Coelogyne species in trade, directly supporting their conservation and contributing to the reduction in illegal orchid trade. Methods: Using a CTAB protocol, genomic DNA was extracted from leaf samples belonging to 19 Coelogyne species. Sanger sequencing was performed after PCR amplification using published primer sets for every barcode region. Sequences were modified in BioEdit, and BLASTn (accessed 15 June 2025) was used to compare them to GenBank (NCBI Nucleotide). Amplification efficiency was calculated per locus. Species and genus identification success rates were determined by the congruence of top BLAST hits with morphologically pre-identified taxa. Multi-barcode combinations (matK + rbcL, ITS2 + matK, matK + trnH-psbA, rbcL + trnH-psbA, and matK + rbcL + trnH-psbA) were also assessed. Results: With rbcL, atpF-atpH, and ITS2 yielding ≤11%, the highest single-locus species identification rates were for trnH-psbA (21%) and matK (16%). Among single-locus barcodes, matK showed the highest performance, with 84% genus assignment. ITS2 reached 27%, but genus-level resolution remained limited for the rbcL, trnH-psbA and atpF-atpH barcodes. Multi-barcode approaches maintained species resolution: matK + rbcL + trnH-psbA, matK + rbcL, and matK + trnH-psbA correctly identified 16% of species and achieved 74–79% genus assignment. Conclusions: No single locus achieves robust species discrimination in Coelogyne, but trnH-psbA, matK and atpF-atpH provide the best single-marker performance. Using the matK locus alone, in combination with either trnH-psbA or rbcL, or all three together ensures consistent genus-level identification and significantly improves taxonomic resolution. This study introduces a novel multi-locus barcode strategy tailored to Coelogyne, offering a practical solution for identification and enforcement. While promising, this approach represents a potential application that requires further validation before routine implementation. Full article

Environmental and ancient DNA are mostly present in degraded forms in nature. Plant forensics is necessary for plants like Taxus (Taxaceae), which is a medicinal, as well as poisonous, endangered plant. We designed a study to develop high-efficiency PCR mini-barcoding primers for the identification of Taxus. We collected environmental materials, fresh and old Taxus specimens from natural habitats, herbaria, and ex situ propagation sites. Taxon-specific mini-barcoding primers were prepared through primer3. All the primers were amplified onto Taxus specimens and environmental samples having Taxus DNA, while no amplification on fresh and herbarium specimens other than Taxus was noted. DNA sequencing of amplified regions of matK, ITS, and rbcL yielded lengths of 117, 175, and 200 bp. Blast taxonomy showed 100% identification power at the genus level, while 75–93% at the species level, and identified a total of 30 taxa within the genus Taxus, comprising 16 species, 5 varieties, 2 hybrids, and 7 variants. ITS was the most specific for genus identification, followed by matK and rbcL. Environmental, trade, socio-economic, and toxicological crimes were also identified. Our high-efficiency PCR mini-barcoding method can be useful in the prevention of Taxus illegal trade and habitat degradation to mitigate climate change in the Himalayan region of Pakistan. Full article

Root-knot nematodes (RKNs) of the genus Meloidogyne spp., are among the most economically important groups of plant-parasitic nematodes worldwide, causing significant economic losses through yield reduction across a wide range of crops. In Croatia, although the presence of Meloidogyne spp. has been documented for decades, data at the species level was limited. As accurate identification is crucial for implementation of effective management strategies, we attempted to fill this gap. This study presents the results of a national survey of RKNs affecting potato crops as well as an early warning programme targeting vegetable crops, conducted across Croatia between 2022 and 2024. Nematodes were identified using morphological analyses (female perineal patterns and second-stage juveniles) and molecular methods (PCR with group-specific and species-specific primers, as well as DNA sequencing). Meloidogyne spp. were detected in 61 out of 210 samples, corresponding to an infestation rate of 29%. Four species were identified: M. incognita, M. hapla, M. arenaria, and M. javanica. Notably, M. incognita and M. javanica are reported here for the first time in Croatia. These results provide updated insights into the distribution and identity of RKNs in Croatia, thereby establishing a foundation for the implementation of sustainable management strategies. Full article

Omsk hemorrhagic fever is an acute zoonotic disease caused by Omsk hemorrhagic fever virus, a member of the genus Flavivirus (family Flaviviridae), with a reported case-fatality rate of approximately 3%. Historically confined to southwestern Siberia, ecological changes raise concerns about possible spread to non-endemic regions. Although no Omsk hemorrhagic fever cases have been reported in the Republic of Korea, the risk of accidental importation highlights the importance of establishing a reliable diagnostic protocol. We established and validated an institutionally developed diagnostic protocol employing real-time reverse transcription polymerase chain reaction targeting the NS2A and C genes of Omsk hemorrhagic fever virus. Primers and probes were designed from all available genomes to ensure broad strain coverage. Human ribonuclease P was used as an internal control to verify nucleic acid extraction and amplification. Using synthetic deoxyribonucleic acid fragments and in vitro-transcribed ribonucleic acid, assay performance was optimized, and analytical sensitivity was determined using probit analysis. The limits of detection were 74.50 copies/µL (threshold cycle 32.99) for NS2A and 70.41 copies/µL (threshold cycle 35.38) for C. Specificity testing using representative flaviviruses (West Nile virus, Yellow fever virus, Zika virus, St. Louis encephalitis virus, and Tick-borne encephalitis virus) and an alphavirus (Venezuelan equine encephalitis virus) demonstrated no cross-reactivity. The assay demonstrated high sensitivity, specificity, and reproducibility, supporting its potential application in national and international Omsk hemorrhagic fever virus surveillance systems. Full article

The increased presence of harmful algal blooms (HABs) is a concern for many aquatic environments, especially with the increasing effects of climate change. Members of the dinoflagellate genus Dinophysis have been shown to produce toxins that can cause Diarrheic Shellfish Poisoning (DSP) in humans who consume infected shellfish. The advancing oyster aquaculture industry in Delaware will require the development of management practices and monitoring HAB species to protect environmental and human health. Temperature, nutrients, and prey abundance can be drivers of Dinophysis blooms. D. acuminata has been historically identified at high concentrations (>200,000 cells L⁻¹) in water samples from Rehoboth Bay, DE, USA. However, the reach of spring blooms and how far they extend to aquaculture sites have not been determined. This study monitored an emergent HABs threat of a toxin-producing dinoflagellate, Dinophysis acuminata, by assessing a transect during the first recorded winter bloom in Torquay Canal and analyzing concentrations of chemical nutrients of combined nitrate and nitrite, and orthophosphate. Pearson correlation coefficient analysis between cell density (cells L⁻¹) and environmental variables across all sites was conducted to determine significant relationships between water temperature, Chl-a concentration, conductivity, dissolved oxygen (DO), combined nitrate and nitrite concentrations (NO_x), and orthophosphate concentrations (PO₄³⁻). Genetic techniques and PCR were utilized to determine the presence of Dinophysis using genus-specific primers to monitor cell density or abundance within the sediments during winter months. There were no significant correlations between environmental variables, and nutrient concentrations did not exceed EPA regulations. Molecular analyses of benthic sediments detected Dinophysis spp., offering insight into potential bloom origins. Overall, there is limited ecological data on Dinophysis acuminata in Rehoboth Bay, DE, USA. The results of this study will help strengthen resources for monitoring HAB species and understanding potential risks to oyster aquaculture in Delaware. Full article

Accurate normalization is crucial for reliable gene expression quantification and depends on stably expressed housekeeping genes (HKGs) as internal controls. However, HKGs expression varies with developmental stage, tissue type, and treatments, potentially introducing bias and compromising data accuracy. Thus, validating candidate reference genes under defined conditions is essential. Reynoutria, also known as giant Asian knotweeds, is a Polygonaceae family genus of several medicinal plants producing a diverse array of specialized metabolites of pharmacological interest. Outside their native range, these plants are also noxious invasive weeds, causing significant environmental and economic threats. Research on stable reference genes in these species is limited, with a primary focus on R. japonica. To enable accurate gene expression analysis related to specialized metabolism and natural product biosynthesis, we aimed to identify the most stable reference genes across the most common species: R. japonica Houtt., R. sachalinensis (F. Schmidt) Nakai, and their hybrid—R. × bohemica Chrtek & Chrtková. In this study, we evaluated twelve candidate HKGs (ACT, TUA, TUB, GAPDH, EF-1γ, UBQ, UBC, 60SrRNA, eIF6A, SKD1, YLS8, and NDUFA13) across three tissue types (rhizomes, leaves, and flowers) from three Reynoutria species sampled at peak flowering. Primer specificity and amplification efficiency were confirmed through standard-curve analysis. We assessed expression stability using ΔCt, geNorm, NormFinder, and BestKeeper, and generated comprehensive rankings with RefFinder. Our integrated analysis revealed organ- and species-dependent stability differences, yet identified up to three reference genes suitable for interspecific normalization in Reynoutria. This represents the first systematic, comparative validation of HKGs across closely related knotweed species, providing a robust foundation for future transcriptomic and functional studies of their specialized metabolism and other biological processes. Full article

Listeria monocytogenes, Staphylococcus aureus, Salmonella enterica, and Escherichia coli O157:H7 are four major foodborne pathogenic bacteria found in meat and meat products, which pose significant threats to human health. In this study, we developed specific loop-mediated isothermal amplification (LAMP) primers targeting these four pathogenic bacteria. Following the optimization of system components and reaction parameters, four rapid and simplified LAMP-based detection assays were established, which enabled the visual detection of these four pathogenic bacteria within 40–50 min. The three established LAMP assays targeting L. monocytogenes, S. aureus, and E. coli O157:H7 achieved species-level discrimination, whereas the LAMP method for Salmonella exhibited genus-level specificity. The detection limits of the LAMP assays were determined as follows: 1.8 × 10¹ colony forming units (CFU)/mL for L. monocytogenes, 5.1 × 10¹ CFU/mL for S. aureus, 1.2 × 10¹ CFU/mL for S. enterica, and 3.3 × 10³ CFU/mL for E. coli O157:H7, with sensitivity improved by 10–1000-fold compared to conventional PCR. The developed LAMP assays were used to analyze 52 meat and meat product samples, and 7 samples were positive, which was consistent with the results of the conventional PCR and culture-based methods, demonstrating an accuracy rate of 100% for the LAMP methods. In conclusion, the established LAMP assays exhibit high specificity, enhanced sensitivity, and result visualization, making them suitable for on-site rapid detection in food safety monitoring. Full article

Tusavirus 1 of species Protoparvovirus incertum 1 (family Parvoviridae) was first identified in humans and later in small ruminants (caprine and ovine). This study reports the full-length coding sequences (~4400–4600 nt) of three novel tusavirus-related protoparvoviruses from ovine (“misavirus”, PV540792), for the first time bovine (“sisavirus”, PV540793) and subsequently from caprine (“gisavirus” PV540850/51) fecal samples, using next-generation sequencing (NGS) and PCR techniques. Their NS1, VP1 and VP2 proteins shared 61–63% amino acid identities with each other and with tusaviruses, suggesting these three viruses belong to three novel species in the genus Protoparvovirus. Phylogenetic analyses placed them with tusaviruses on a separate main branch, implying a shared origin among these most likely ruminant protoparvoviruses. A small-scale epidemiological investigation on 318 ruminant enteric samples using novel generic NS1 primers found misavirus in 14/51 (27.5%) ovine and sisavirus in 19/203 (9.4%) bovine samples from multiple Hungarian farms. Tusavirus was present in 5/51 (9.8%) ovine and 15/62 (24.2%) caprine samples, all from one farm. The highest prevalences for all three viruses were found in animals aged 2–12 months, though sporadic cases were also found in other age groups. Partial NS and VP sequence-based phylogenetic trees showed virus-specific lineages for misa-, sisa-, gisa- and tusaviruses, with various strains forming sub-lineages. These findings suggest the presence of multiple genotypes and/or members of additional species, which was supported by a VP sequence-based hierarchical cluster analysis. The study’s viruses were mostly phylogenetically separated by host; however, two bovine sisavirus strains with diverse phylogenetic localizations in the NS (belonging to bovine sisaviruses) and VP1 trees (distantly related to ovine misaviruses) could indicate previous (interspecies?) recombination events. Full article