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Marine Environmental Research Jun 2024The assessment of fish diversity is crucial for effective conservation and management strategies, especially in ecologically sensitive regions such as marine protected...
The assessment of fish diversity is crucial for effective conservation and management strategies, especially in ecologically sensitive regions such as marine protected areas. This study contrasts the effectiveness of environmental DNA (eDNA) metabarcoding analysis employing Nanopore technology with compare beam trawl surveys at the Sylt Outer Reef, a Natura 2000 site in the North Sea, Germany. Out of the 17 fish species caught in a bottom trawl (using a 3m beam trawl), 14 were also identified through eDNA extracted from water samples. The three species not detected in the eDNA results were absent because they lacked representation in public DNA databases. The eDNA method detected twice as many fish species as the beam trawl, totalling 36 species, of which 14 were also detected by the trawl. Additionally, the selection of primers (Mifish) facilitated the identification of one marine mammal species, the harbour porpoise. In conclusion, the findings underscore the potential of eDNA coupled with MinION sequencing (Long read technology) as a robust tool for biodiversity assessment, surpassing traditional methods in detecting species richness.
PubMed: 38870557
DOI: 10.1016/j.marenvres.2024.106602 -
International Journal of... Apr 2024Torque teno virus (TTV) is a globally prevalent virus in humans, yet comprehensive knowledge about its prevalence, predominant transmission routes, and pathogenesis...
Torque teno virus (TTV) is a globally prevalent virus in humans, yet comprehensive knowledge about its prevalence, predominant transmission routes, and pathogenesis remains limited. This study aimed to assess the frequency of TTV infection among healthy blood donors in Yazd, Iran. A total of 236 healthy blood donors, devoid of HIV/HBV/HCV infection markers, participated in the study from 2015 to 2016. Nested Polymerase Chain Reaction (PCR) utilizing a set of oligo primers for the 5΄- UTR region was employed to detect TTV DNA in serum samples. The TTV genome was identified in 161 out of 236 (61.2%) healthy blood donors. The mean age for men and women was 43 and 57 years, respectively. Of the participants, 156 were male, and 107 were female. Donor age exhibited a significant association with virus presence (P=0.007); however, gender did not show a statistically significant association with the frequency of TTV infection in healthy blood donors (P=0.3). The study revealed a notably high frequency of the Torque teno virus in Yazd province, aligning with similar findings globally. Further investigations are warranted to elucidate the clinical implications of the virus in the healthy population.
PubMed: 38868806
DOI: 10.18502/ijhoscr.v18i2.15366 -
BMC Genomics Jun 2024Reverse transcription quantitative PCR (RT-qPCR) with intercalating dyes is one of the main techniques to assess gene expression levels used in basic and applied...
BACKGROUND
Reverse transcription quantitative PCR (RT-qPCR) with intercalating dyes is one of the main techniques to assess gene expression levels used in basic and applied research as well as in diagnostics. However, primer design for RT-qPCR can be complex due to the high demands on primer quality. Primers are best placed on exon junctions, should avoid polymorphic regions, be specific to the target transcripts and also prevent genomic amplification accurately, among others. Current software tools manage to meet all the necessary criteria only insufficiently. Here, we present ExonSurfer, a novel, user-friendly web-tool for qPCR primer design.
RESULTS
ExonSurfer combines the different steps of the primer design process, encompassing target selection, specificity and self-complementarity assessment, and the avoidance of issues arising from polymorphisms. Amplification of potentially contaminating genomic DNA is avoided by designing primers on exon-exon junctions, moreover, a genomic alignment is performed to filter the primers accordingly and inform the user of any predicted interaction. In order to test the whole performance of the application, we designed primer pairs for 26 targets and checked both primer efficiency, amplicon melting temperature and length and confirmed the targeted amplicon by Sanger sequencing. Most of the tested primers accurately and selectively amplified the corresponding targets.
CONCLUSION
ExonSurfer offers a comprehensive end-to-end primer design, guaranteeing transcript-specific amplification. The user interface is intuitive, providing essential specificity and amplicon details. The tool can also be used by command line and the source code is available. Overall, we expect ExonSurfer to facilitate RT-qPCR set-up for researchers in many fields.
Topics: Exons; Software; DNA Primers; Internet; Humans; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 38867172
DOI: 10.1186/s12864-024-10456-2 -
IMA Fungus Jun 2024Molecular studies of fungi within the order Laboulbeniales (Ascomycota, Pezizomycotina) have been hampered for years because of their minute size, inability to grow in...
Molecular studies of fungi within the order Laboulbeniales (Ascomycota, Pezizomycotina) have been hampered for years because of their minute size, inability to grow in axenic culture, and lack of reliable and cost-efficient DNA extraction protocols. In particular, the genus Laboulbenia is notorious for low success with DNA extraction and polymerase chain reaction (PCR) amplification. This is attributed to the presence of melanin, a molecule known to inhibit PCR, in the cells. We evaluated the efficacy of a standard single cell-based DNA extraction protocol by halving the recommended amount of reagents to reduce the cost per extraction and adding bovine serum albumin (BSA) during the multiple displacement amplification step to reverse the effect of melanin. A total of 196 extractions were made, 111 of which were successful. We found that halving the reagents used in the single cell-based extraction kit did not significantly affect the probability of successful DNA extraction. Using the halved protocol reduces cost and resource consumption. Moreover, there was no significant difference in the probability of successfully extracting DNA based on whether BSA was added or not, suggesting that the amount of melanin present in cells of the thallus has no major inhibitory effect on PCR. We generated 277 sequences from five loci, but amplification of the internal transcribed spacer region, the mitochondrial small subunit rDNA, and protein-coding genes remains challenging. The probability of successfully extracting DNA from Laboulbeniales was also impacted by specimen storage methods, with material preserved in > 95% ethanol yielding higher success rates compared to material stored in 70% ethanol and dried material. We emphasize the importance of proper preservation of material and propose the design of Laboulbeniales-specific primers to overcome the problems of primer mismatches and contaminants. Our new insights apply not only to the genus Laboulbenia; Laboulbeniales generally are understudied, and the vast majority of species remain unsequenced. New and approachable molecular developments will benefit the study of Laboulbeniales, helping to elucidate the true diversity and evolutionary relationships of these peculiar microfungi.
PubMed: 38863065
DOI: 10.1186/s43008-024-00146-9 -
Plant Disease Jun 2024Mung bean (Vigna radiata (L.) R. Wilczek) is a legume with high nutritional and economic value that is cultivated widely across Asia (Kang et al. 2014). In March 2022, a...
Mung bean (Vigna radiata (L.) R. Wilczek) is a legume with high nutritional and economic value that is cultivated widely across Asia (Kang et al. 2014). In March 2022, a leaf spot disease in mung bean was observed at the Gangneung-Wonju National University Experimental farm (Gangneung, South Korea, 37.77°N, 128.86°E). The affected plants had irregular brown-gray leaf spots, and the bottom of the leaves showed concentric brown-gray rings that eventually progressed to necrotic lesions. Regardless of the cultivar, approximately 30% of the plants in the field were infected. To isolate the pathogen, the affected leaves were surface-sterilized by washing with 70% ethanol for 1 min, followed by washing with 2% NaClO for 2 min, then rinsing with sterile distilled water. We placed 3-mm sized diseased lesions on potato-dextrose agar (PDA), then incubated them at 27 ± 1 °C in the dark for 7 days and we obtained 1 isolate (CC1). The fungus on PDA had white aerial mycelia that became gray toward the center. Single spore cultures were obtained from fungal isolate. Isolated conidia were single celled, hyaline, cylindrical, and measured between 20.75 to 22.07 μm × 5.85 to 6.92 μm (n = 20), similar to other reports of C. camelliae(Wang et al. 2016). Mycelium from the single spore isolate was used for DNA extraction using Exgene™ Plant SV / (GeneAll®, Cat.No. 117-152), and we amplified the ITS region with primers ITS1 + ITS2 and ITS3 + ITS4, a portion of the actin gene with primers ACT-512F + 738R, and a portion of the beta-tubulin gene with primers BT2aF + BT2bR (Carbone et al. 1999; Glass et al. 1995; White et al. 1990). The amplified regions were sequenced by by Macrogen® (Seoul, South Korea). Sequences were deposited under GenBank accession numbers OR523262 (ITS), OR582483 (Actin), and OR566953 (beta-tubulin). MegaBLAST analysis of the ITS1, ITS2, ACT, and TUB regions showed 99% (216/217 bp) similarity with C. camelliae strain HNCS-26 (MK041440.1), 99% (303/305 bp) similarity with C. camelliae strain G3 (ON025203.1), 99% (242/244 bp) similarity with C. camelliae strain FWT41 (MN525820.1), and 99% (456/460 bp) with C. camelliae strain LF152 (KJ955239.1), respectively. To fulfill Koch's postulates, we conducted a pathogenicity teston the mung bean cultivar VC1973A (Seonhwanokdu) grown for four weeks at 25 °C with a 16-h day/8-h night cycle, simulating the field conditions when the symptoms were observed. We tested the pathogenicity on six plants , three control and three infected plants. Using three leaf replicates per plant resulting in total of nine leaves per group. Leaves were first injured using a sterile needle then either sterile 5 mm PDA plugs or plugs with C. camelliae were placed on the leaf for control and infected conditions, respectively. Irregular gray leaf spots were observed on the abaxial and adaxial of the infected leaf after 2 weeks, like the symptoms observed in the field. This was observed only on infected leaves and nowhere else on the plant and the control plants had no infection. We re-isolated the pathogen from diseased leaves and identified it as C. camelliae using the same molecular markers described previously, completing Koch's postulate. To the best of our knowledge, this is the first report of leaf spot caused by C. camelliae in mung bean plants in Korea, previously the fungi was reported to infect tea plants in Korea (Hassan et al. 2023). More studies are required to investigate potentially resistant mung bean varieties to minimize future damage caused by this fungus.
PubMed: 38861469
DOI: 10.1094/PDIS-02-24-0335-PDN -
Plant Disease Jun 2024In early August 2023, a disease outbreak on hot banana peppers (Capsicum annuum cv. Golden Dagger) was reported in Cattaraugus County, New York (NY). Disease incidence...
In early August 2023, a disease outbreak on hot banana peppers (Capsicum annuum cv. Golden Dagger) was reported in Cattaraugus County, New York (NY). Disease incidence was at least 60%. Affected developing and mature fruit had at least one tan, soft, sunken lesion with salmon-colored spore masses surrounded by brown, necrotic margins. Microscopic observation of the lesions identified acervuli and setae typical of Colletotrichum spp. Isolations were made from these lesions by spreading conidia from the acervuli on 2% water agar (WA) + 0.02% (w/v) ampicillin. Colonies were hyphal tipped and transferred onto clarified V8 juice agar (CV8) and incubated at 20°C. The isolation frequency was 100% and a total of six isolates were obtained: Coll23Pep001, Coll23Pep003, Coll23Pep005, Coll23Pep007, Coll23Pep008, and Coll23Pep010. After 10 days, colonies were subcultured to potato dextrose agar (PDA) and CV8. On PDA, colonies appeared off-white to dark gray with sparse aerial mycelia. On CV8, the colony was pale gray with acervuli and orange-colored spore masses in the center. Conidia were hyaline, smooth and fusiform to round, and tapered at both ends. Mean conidial dimensions (n = 20) were 20.2 (13.75 to 25) µm long × 4.7 (3 to 6.25) µm wide. To confirm the identity of the isolates, DNA was extracted, and PCR performed to amplify the internal transcriber spacer (ITS) region (primers ITS1/ITS4; White et al. 1990), and actin (ACT) (primers ACT-512F/ACT-783R; Carbone and Kohn 1999) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes (primers GDF1/GDR1; Guerber et al. 2003). Pairwise alignment of the sequences showed all isolates had 100% similarity to C. scovillei ex. holotype CBS 126529 (Damm et al. 2012). Sequences from all isolates were deposited in GenBank with accessions PP556967 to PP556972 (ITS), PP565766 to PP565771 (ACT), and PP565772 to PP565777 (GAPDH). For pathogenicity testing, all isolates were grown on CV8 at 20°C in the dark for 10 days. Conidia were harvested by flooding the plates of each isolate with sterile distilled water and filtering the suspension through a double layer of cheesecloth. The concentration of the conidial suspension was adjusted to 5 × 105 spores per ml. Pathogenicity of the six isolates was tested on banana pepper fruit by using a sterile toothpick to pierce the skin at the two opposite ends. A droplet (10 µl) of the conidial suspension was placed on each wound. The same number of fruit were inoculated without wounding, and non-inoculated control fruit received a droplet of sterile distilled water (either wounded or unwounded). The experiment was repeated twice. All fruit were placed in a humid box at room temperature for 7 days. All wounded and inoculated fruit developed sunken lesions filled with salmon-colored conidial masses. Disease did not occur on the unwounded, inoculated fruit nor the non-inoculated controls. C. scovillei was recovered from all inoculated fruit by reisolating onto CV8 media and isolates had similar morphology and conidial dimensions to the original isolates. To the best of our knowledge, this is the first report of C. scovillei causing anthracnose on pepper in NY. C. scovillei has been reported in South Carolina (Toporek and Keinath 2021), Brazil (Caires et al. 2014), eastern Asia (de Silva et al. 2019), and Kosovo (Xhemali et al. 2023). The pathogen is particularly aggressive on pepper and poses substantial threats to pepper production around the world.
PubMed: 38861466
DOI: 10.1094/PDIS-04-24-0735-PDN -
Plant Disease Jun 2024Lonicera japonica Thunb. is a traditional Chinese medicinal plant, which widely cultivated in China, Japan and Korea. From August to October in 2021 and 2022, severe...
Lonicera japonica Thunb. is a traditional Chinese medicinal plant, which widely cultivated in China, Japan and Korea. From August to October in 2021 and 2022, severe leaf spots symptoms were observed on L. japonica in medicinal botanical garden of Shandong University of Traditional Chinese Medicine (36°55'89"N, 116°79'91"E), Jinan, Shandong Province, China. The disease incidence was above 80% in the 25 acre cultivation area. Early symptoms were small brown spots on the leaves. Then the number of small spots gradually increased and spread over the entire leaves. The small brown spots seldom merge together to form larger lesions. Leaves with typical symptoms were collected from twenty individual plants, and cut into small 5×5 mm fragments in the junction of infected and healthy tissues. The fragments were sterilized in 75% ethanol for 30 s and 1% NaClO for 60 s, rinsed three times in sterile water, and then placed on potato dextrose agar (PDA). After 3 days of incubation at 25°C, fungal plugs along the edge of the colony were cut and transferred to new PDA for purification. A total number of 23 colonies with similar morphological characteristics were obtained, and three representative strains (Lj14, Lj18 and Lj20) were selected for subsequent study. The colonies grew rapidly on PDA and covered the entire petri dish in 4 days. Colonies had abundant aerial hyphae, initially white, round, later turning gray and black. Conidia were oblate or nearly spherical, single-celled, black, and measured in size from 9.6 to 13.2 μm × 7.9 to 16.1 μm in diameter (n=150) (Figure S1). The observed characteristics were close to those of Nigrospora spp. ( Wang et al. 2017). The genomic DNA was extracted, and PCR amplification of the rDNA internal transcribed spacer (ITS), β-tubulin gene (TUB), and translation elongation factor 1-alpha gene (TEF1) were completed by primers ITS1/ITS4, Bt2a/Bt2b and EF1-728F/EF1-986R (Carbone and Kohn, 1999). Sequences were deposited in GenBank (accession nos. OR936661, OR936662, OR936671 for ITS, OR947626, OR947627, OR947628 for TUB, and OR947629, OR947630, OR947631 for TEF1 sequences, respectively). BLAST analyses of ITS (OR936661), TUB(OR947626) and TEF1 (OR947629) sequences exhibited 100% (487 bp out of 487 bp), 99.48% (380 bp out of 382 bp), and 99.6% (248 bp out of 249 bp) similarity to the sequences of N. oryzae strains KoLRI_053384 (MZ855426), LC2991 (KY019496) and LC7307 (KY019409), respectively. Lj14, Lj18 and Lj20 formed a clade with N. oryzae LC6763 and LC2991 in phylogenetic tree (Figure S2). Based on morphological and molecular evidence, the pathogen was identified as N. oryzae (Berk. &Broome) Petch. To fulfill Koch's postulates, the pathogenicity was tested in vivo experiments. Thirty non-wounded healthy leaves of ten intact plants were inoculated with 10 µl spore suspension (10 spores/ml) of three strains, respectively. As negative control, thirty leaves of ten healthy plants were inoculated with sterile water. The inoculated plants were placed at 28°C in the growth chamber with high relative humidity. The pathogenicity tests were repeated three times. Distinct symptoms similar to that of natural conditions were observed on the leaves of inoculated plants after 4 to 7 days. The strain was reisolated from the lesions and identified as N. oryzae by morphological features and ITS sequence. The pathogen has been reported to cause leaf spot disease on tobacco (Wang et al. 2022) and asiatic dayflower (Qiu et al. 2022). To our knowledge, this is the first report of leaf spot caused by N. oryzae on Lonicera japonica in China. The research will be helpful for leaf spot disease control.
PubMed: 38861465
DOI: 10.1094/PDIS-01-24-0190-PDN -
Plant Disease Jun 2024Nanhaia speciosa, commonly known as Niudali, is a medicinal woody vine belonging to the Leguminosae family. Valued for its culinary and medicinal properties, it is...
Nanhaia speciosa, commonly known as Niudali, is a medicinal woody vine belonging to the Leguminosae family. Valued for its culinary and medicinal properties, it is extensively cultivated, covering approximately 5,973 hm2 in the Guangxi Zhuang Autonomous Region of China. The edible tubers of this plant are reported to possess antibacterial and antioxidant effects (Luo et al., 2023; Shu et al., 2020). In July 2021, a Niudali plantation in Yulin, Guangxi, China (22°64'N; 110°29'E) exhibited leaf spot symptoms, with an incidence rate exceeding 40% across a 46,690 m2 area. Initially, small circular, pale yellow spots appeared on the leaves, which subsequently evolved into dark brown lesions surrounded by yellow halos, ultimately leading to foliage wilting. Leaves exhibiting typical symptoms were collected for pathogen investigation. The leaves were thoroughly washed with sterile water and small tissue fragments (5×5 mm) were excised from the lesion periphery. These fragments were surface-sterilized with 75% ethanol and 1% NaClO, rinsed three times with sterile water, and subsequently cultured on potato dextrose agar (PDA) at 28 °C in darkness for 7 days. Through single-spore isolation, seven isolates with similar morphological traits were obtained. After 7 days of incubation on PDA at 28 °C in dark, the colonies exhibited a white to grey coloration on the upper surface with abundant aerial hyphae, while the underside appeared dark black. The conidia, cylindrical or obclavate in shape, were straight, pale brown, and measured 30.1-128.9 μm × 4.8-15.0 μm (n=50). The morphological characteristics matched those of Corynespora sp.(Wang et al. 2021). For molecular identification, the isolate N5-2 underwent DNA sequence analysis using genomic DNA and primers ITS1/ITS4 and EF1-688F/EF1-1251R. The sequences (ITS: OP550425; TEF1-α: OQ117118) were deposited in GenBank, exhibiting 98% identity to C. cassiicola (OP981637) for TEF1-α and 99% homology to C. cassiicola (OP957070) for ITS. Based on the concatenated ITS and TEF1-α, a maximum likelihood phylogenetic analyses using MEGA7.0 clustered the isolate with C. cassiicola. Consequently, the fungus was identified as C. cassiicola based on its morphological and molecular features. In the pathogenicity test on 1-year-old Nanhaia speciosa seedlings, leaves were gently scratched and inoculated with mycelial plugs (5 mm). Control seedlings received PDA plugs. Five leaves per plant and five plants per treatment were selected for assessment. All seedling were maintained in a greenhouse (12/12h light/dark cycle, 25 ± 2°C, 90% humidity). After a 7-day incubation period, all leaves subjected to fungal inoculation exhibited symptoms consistent with those observed in the field, while control plants remained symptom-free. The fungus was successfully reisolated from the infected leaves in three successive trials, fulfilling Koch's postulates. While C. cassiicola is well-documented for inducing leaf spots on various plant species, including Jasminum nudiflorum, Strobilanthes cusia, Acanthus ilicifolius, Syringa species (Hu et al., 2023; Liu et al., 2023; Xie et al., 2021; Wang et al., 2021), this study represents the first report of C. cassiicola causing leaf spots on Nanhaia speciosa in China. The identification of this pathogen in Nanhaia speciosa has significant implications for future epidemiological investigations and serves as a valuable reference for controlling leaf spot disease in Nanhaia speciosa.
PubMed: 38853332
DOI: 10.1094/PDIS-03-24-0562-PDN -
Heliyon Jun 2024Molecular techniques of nucleic acid testing recommended by the World Health Organization (WHO) for the sis (MTB) detection were considered to have the potential access...
Molecular techniques of nucleic acid testing recommended by the World Health Organization (WHO) for the sis (MTB) detection were considered to have the potential access to the accurate tuberculosis (TB) notifications. In this study, a new method, which coupled real-time (rt) fluorescence technique with multiple cross displacement amplification (MCDA), was developed for the rapid, sensitive and specific detection of MTB (termed MTB-rt-MCDA). According to the principle of the rt-MCDA test, a set of ten primers were designed for the MCDA reaction, of which one was engineered with a restrictive endonuclease recognition site, a fluorophore and a quencher for achieving the real-time fluorescence detection. MTB-rt-MCDA test was conducted under the optimized conditions (67 °C, 40 min) on the real-time fluorescence platform. The MTB-rt-MCDA assay accurately identified the MTB strains with no cross reaction with other bacteria. The lowest detectable genomic DNA concentration of the MTB-rt-MCDA assay was 25 fg/μl. We employed the genomic DNA templates extracted from sputum of clinical cases for validating the practical applicability of this assay, and the detection power of the MTB-rt-MCDA assay was comparable to that of the Xpert method and MCDA-based biosensor detection and superior to smear microscope method. The complete process of the MTB-rt-MCDA assay, including rapid extraction of DNA and rt-MCDA test, takes less than 1 h. In conclusion, the presented MTB-rt-MCDA assay provided an effective and simple option for the rapid screening of MTB infection.
PubMed: 38845879
DOI: 10.1016/j.heliyon.2024.e31901 -
Veterinary Pathology Jun 2024Fixation and demineralization protocols for bone marrow (BM) across diagnostic laboratories are not standardized. How different protocols affect histomorphology and DNA...
Fixation and demineralization protocols for bone marrow (BM) across diagnostic laboratories are not standardized. How different protocols affect histomorphology and DNA amplification is incompletely understood. In this study, 2 fixatives and 3 demineralization methods were tested on canine BM samples. Twenty replicate sternal samples obtained within 24 hours of death were fixed overnight in either acetic acid-zinc-formalin (AZF) or 10% neutral-buffered formalin (NBF) and demineralized with formic acid for 12 hours. Another 53 samples were fixed in AZF and demineralized with hydrochloric acid for 1-hour, formic acid for 12 hours, or ethylenediamine tetraacetic acid (EDTA) for 24 hours. Histologic sections were scored by 4 raters as of insufficient, marginal, good, or excellent quality. In addition, DNA samples extracted from sections treated with the different fixation and demineralization methods were amplified with 3 sets of primers to conserved regions of and genes. Amplification efficiency was graded based on review of capillary electrophoretograms. There was no significant difference in the histomorphology scores of sections fixed in AZF or NBF. However, EDTA-based demineralization yielded higher histomorphology scores than demineralization with hydrochloric or formic acid, whereas formic acid resulted in higher scores than hydrochloric acid. Demineralization with EDTA yielded DNA amplification in 29 of 36 (81%) samples, whereas demineralization with either acid yielded amplification in only 2 of 72 (3%) samples. Although slightly more time-consuming and labor-intensive, tissue demineralization with EDTA results in superior morphology and is critical for polymerase chain reaction (PCR) amplification with the DNA extraction method described in this article.
PubMed: 38842072
DOI: 10.1177/03009858241257920