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International Journal of Molecular... May 2024Cotton Verticillium wilt is mainly caused by the fungus , which threatens the production of cotton. Its pathogen can survive in the soil for several years in the form of...
Cotton Verticillium wilt is mainly caused by the fungus , which threatens the production of cotton. Its pathogen can survive in the soil for several years in the form of microsclerotia, making it a destructive soil-borne disease. The accurate, sensitive, and rapid detection of from complex soil samples is of great significance for the early warning and management of cotton Verticillium wilt. In this study, we combined the loop-mediated isothermal amplification (LAMP) with CRISPR/Cas12a technology to develop an accurate, sensitive, and rapid detection method for . Initially, LAMP primers and CRISPR RNA (crRNA) were designed based on a specific DNA sequence of , which was validated using several closely related spp. The lower detection limit of the LAMP-CRISPR/Cas12a combined with the fluorescent visualization detection system is approximately ~10 fg/μL genomic DNA per reaction. When combined with crude DNA-extraction methods, it is possible to detect as few as two microsclerotia per gram of soil, with the total detection process taking less than 90 min. Furthermore, to improve the method's user and field friendliness, the field detection results were visualized using lateral flow strips (LFS). The LAMP-CRISPR/Cas12a-LFS system has a lower detection limit of ~1 fg/μL genomic DNA of the , and when combined with the field crude DNA-extraction method, it can detect as few as six microsclerotia per gram of soil, with the total detection process taking less than 2 h. In summary, this study expands the application of LAMP-CRISPR/Cas12a nucleic acid detection in and will contribute to the development of field-deployable diagnostic productions.
Topics: CRISPR-Cas Systems; Nucleic Acid Amplification Techniques; Soil Microbiology; Plant Diseases; Ascomycota; Molecular Diagnostic Techniques; Gossypium; DNA, Fungal; Verticillium
PubMed: 38791224
DOI: 10.3390/ijms25105185 -
International Journal of Molecular... May 2024Next-generation sequencing has transformed the acquisition of vast amounts of genomic information, including the rapid identification of target gene sequences in...
Next-generation sequencing has transformed the acquisition of vast amounts of genomic information, including the rapid identification of target gene sequences in metagenomic databases. However, dominant species can sometimes hinder the detection of rare bacterial species. Therefore, a highly sensitive amplification technique that can selectively amplify bacterial genomes containing target genes of interest was developed in this study. The rolling circle amplification (RCA) method can initiate amplification from a single locus using a specific single primer to amplify a specific whole genome. A mixed cell suspension was prepared using ATCC17400 (targeting nonribosomal peptide synthetase [NRPS]) and (non-target), and a specific primer designed for the was used for the RCA reaction. The resulting RCA product (RCP) amplified only the genome. The was successfully amplified using RCP as a template from even five cells, indicating that the single-priming RCA technique can specifically enrich the target genome using gene-specific primers. Ultimately, this specific genome RCA technique was applied to metagenomes extracted from sponge-associated bacteria, and sequences were successfully obtained from an unknown sponge-associated bacterium. Therefore, this method could be effective for accessing species-specific sequences of in unknown bacteria, including viable but non-culturable bacteria.
Topics: Peptide Synthases; Nucleic Acid Amplification Techniques; Genome, Bacterial; High-Throughput Nucleotide Sequencing; Escherichia coli; Pseudomonas fluorescens; Sequence Analysis, DNA; Metagenome
PubMed: 38791129
DOI: 10.3390/ijms25105089 -
PloS One 2024This study investigates the impact of casein hydrolysates on the poultry ceca inoculated with Campylobacter focusing on microbial molecular preferences for different...
This study investigates the impact of casein hydrolysates on the poultry ceca inoculated with Campylobacter focusing on microbial molecular preferences for different protein sources in the presence of Campylobacter jejuni. Three casein sources (intact casein (IN), casein enzyme hydrolysate (EH), and casein acid hydrolysate (AH)) were introduced to cecal contents in combination with inoculated C. jejuni in an in vitro model system incubated for 48 h at 42°C under microaerophilic conditions. Samples were collected at 0, 24, and 48 h. Genomic DNA was extracted and amplified using custom dual-indexed primers, followed by sequencing on an Illumina MiSeq platform. The obtained sequencing data were then analyzed via QIIME2-2021.11. Metabolite extracts were analyzed with ultra-high-performance liquid orbitrap chromatography-mass spectrometry (UHPLC-MS). Statistical analysis of metabolites was conducted using MetaboAnalyst 5.0, while functional analysis was performed using Mummichog 2.0 with a significance threshold set at P < 0.00001. DNA sequencing and metabolomic analyses revealed that C. jejuni was most abundant in the EH group. Microbial diversity and richness improved in casein supplemented groups, with core microbial differences observed, compared to non-supplemented groups. Vitamin B-associated metabolites significantly increased in the supplemented groups, displaying distinct patterns in vitamin B6 and B9 metabolism between EH and AH groups (P < 0.05). Faecalibacterium and Phascolarctobacterium were associated with AH and EH groups, respectively. These findings suggest microbial interactions in the presence of C. jejuni and casein supplementation are influenced by microbial community preferences for casein hydrolysates impacting B vitamin production and shaping competitive dynamics within the cecal microbial community. These findings underscore the potential of nutritional interventions to modulate the poultry GIT microbiota for improved health outcomes.
Topics: Campylobacter jejuni; Animals; Cecum; Caseins; Metabolome; Chickens; Gastrointestinal Microbiome; Poultry
PubMed: 38787822
DOI: 10.1371/journal.pone.0303856 -
Insects Apr 2024spp. (Oleaceae) have become invasive species in the US and negatively affect native plant diversity and richness in forests. (Coleoptera: Curculionidae) is considered...
spp. (Oleaceae) have become invasive species in the US and negatively affect native plant diversity and richness in forests. (Coleoptera: Curculionidae) is considered a potential biological control agent in the US because adults feed on the foliage and larvae are seed-feeders of spp. To discover the relationships between . and spp., fruit dissections or rearing and field observations are required. In the current research project, novel PCR primers were developed to rapidly detect the DNA of . in molecular analyses without rearing and observation. The developed PCR primers worked even with 0.01 ng of DNA and did not amplify the DNA of the other five curculionid species tested. When the novel primers were tested with three spp. species common in the southeastern US, the DNA of . was detected from all three species. We expect that the novel primers will be utilized to find out the presence and impact of . on spp rapidly and accurately.
PubMed: 38786876
DOI: 10.3390/insects15050320 -
Gels (Basel, Switzerland) May 2024is a complex of anaerobic bacteria responsible for the epidemics of post-antibiotic diarrhea as one of the examples of CDI ( infection). As many as 70% of cases concern...
is a complex of anaerobic bacteria responsible for the epidemics of post-antibiotic diarrhea as one of the examples of CDI ( infection). As many as 70% of cases concern hospitalized patients, particularly those in intensive care units. Ribotyping is one of the most common methods for differentiating bacterial strains. The purpose of this work was to show the effectiveness of the gel electrophoresis-based PCR ribotyping method and the Webribo database for typing isolates, including the hypervirulent 027 ribotype. DNA samples extracted from 69 strains with previously marked genotypes were included in this study. PCR was performed using 16S-23S primers, and capillary gel electrophoresis was performed on the Applied Biosystem 3130xl Genetic Analyzer. The Webribo database was applied for ribotype assignment. Out of 69 samples, 48 belonged to already known ribotypes, 13 represented new ribotypes and 8 was indicated as similar to the existing ones, having some differences. Capillary gel electrophoresis-based PCR is an effective method for the differentiation of ribotypes and can be recognized as a very useful tool in epidemiological studies, while the Webribo database is a useful and an accessible database for a quick analysis of ribotypes.
PubMed: 38786259
DOI: 10.3390/gels10050343 -
RNA Biology Jan 2024The RNA world hypothesis confers a central role to RNA molecules in information encoding and catalysis. Even though evidence in support of this hypothesis has...
The RNA world hypothesis confers a central role to RNA molecules in information encoding and catalysis. Even though evidence in support of this hypothesis has accumulated from both experiments and computational modelling, the transition from an RNA world to a world where heritable genetic information is encoded in DNA remains an open question. Recent experiments show that both RNA and DNA templates can extend complementary primers using free RNA/DNA nucleotides, either non-enzymatically or in the presence of a replicase ribozyme. Guided by these experiments, we analyse protocellular evolution with an expanded set of reaction pathways made possible through the presence of DNA nucleotides. By encapsulating these reactions inside three different types of protocellular compartments, each subject to distinct modes of selection, we show how protocells containing DNA-encoded replicases in low copy numbers and replicases in high copy numbers can dominate the population. This is facilitated by a reaction that leads to auto-catalytic synthesis of replicase ribozymes from DNA templates encoding the replicase after the chance emergence of a replicase through non-enzymatic reactions. Our work unveils a pathway for the transition from an RNA world to a mixed RNA-DNA world characterized by Darwinian evolution, where DNA sequences encode heritable phenotypes.
Topics: DNA; RNA; RNA, Catalytic; Evolution, Molecular; RNA-Dependent RNA Polymerase; Artificial Cells
PubMed: 38785360
DOI: 10.1080/15476286.2024.2355391 -
Open Research Africa 2024( ) is a common sexually transmitted infection (STI). In 2019, the World Health Organization reported about 131 million infections. The majority of infected patients...
( ) is a common sexually transmitted infection (STI). In 2019, the World Health Organization reported about 131 million infections. The majority of infected patients are asymptomatic with cases remaining undetected. It is likely that missed infections contribute to preventable adverse health outcomes in women and children. Consequently, there is an urgent need of developing efficient diagnostic methods. In this study, genome-mining approaches to identify identical multi-repeat sequences (IMRS) distributed throughout the genome were used to design a primer pair that would target regions in the genome. Genomic DNA was 10-fold serially diluted (100pg/μL to 1×10 pg/μL) and used as DNA template for PCR reactions. The gold standard PCR using 16S rRNA primers was also run as a comparative test, and products were resolved on agarose gel. The novel assay, IMRS-PCR, had an analytical sensitivity of 4.31 pg/µL, representing better sensitivity compared with 16S rRNA PCR (9.5 fg/µL). Our experimental data demonstrate the successful development of lateral flow and isothermal assays for detecting DNA with potential use in field settings. There is a potential to implement this concept in miniaturized, isothermal, microfluidic platforms, and laboratory-on-a-chip diagnostic devices for reliable point-of-care testing.
PubMed: 38783971
DOI: 10.12688/openresafrica.14316.2 -
Sheng Wu Gong Cheng Xue Bao = Chinese... May 2024To develop an accurate and efficient protocol for multi-fragment assembly and multi-site mutagenesis, we integrated and optimized the common multi-fragment assembly...
To develop an accurate and efficient protocol for multi-fragment assembly and multi-site mutagenesis, we integrated and optimized the common multi-fragment assembly methods and validated the established method by using fructose-1,6-diphosphatase 1 (FBP1) with 4 mutant sites. The fragments containing mutations were assembled by introducing mutant sites and I recognition sequences. After digestion/ligation, the ligated fragment was amplified with the primers containing overlap region to the linearized vector. The amplified fragment was ligated to the linearized vector and the ligation product was transformed into . After screening and sequencing, the recombinant plasmid with 4 mutant sites was obtained. This protocol overcame the major defects of Gibson assembly and Golden Gate assembly, serving as an efficient solution for multi-fragment assembly and multi-site mutagenesis.
Topics: Escherichia coli; Fructose-Bisphosphatase; Homologous Recombination; Plasmids; Genetic Vectors; DNA; Mutation; Mutagenesis, Site-Directed; Cloning, Molecular
PubMed: 38783816
DOI: 10.13345/j.cjb.230793 -
Plant Disease May 2024The fungal genus includes numerous important plant pathogenic species, some of which causes fruit bitter rot as well as leaf lesions (leaf black spot) on major crops,...
The fungal genus includes numerous important plant pathogenic species, some of which causes fruit bitter rot as well as leaf lesions (leaf black spot) on major crops, leading to yield losses (Fu et al. 2019; Talhinhas & Baroncelli, 2023). was reported associated with black spot on fallen, immature fruit of pear () in New Zealand (Damm et al. 2012); however, to our knowledge, this species has not been reported in Italy or nowhere else. In 2022, a significant increase of anthracnose symptoms was observed on pears in Emilia-Romagna region, Italy. Symptoms, such as round brown lesions of 1 to 4 cm, appeared on more than 50% of refrigerated stored fruit. These symptoms were undetectable at the end of September 2022 and appeared after a five-month period of storage (February 2023) at 4°C (e-Xtra 1A and B). Fungal isolates were obtained from symptomatic pears after surfaces sterilization with 96% ethanol by culturing necrotic tissue pieces on Potato Dextrose Agar at 25°C in the dark (e-Xtra 1C and D). Cultures were shades of coral color, from opalescent to sunkist coral, with slight aerial mycelium becoming grey and darker with age. When observed from the reverse side, the color was pink and, with age, became coral orange to dark amaranth. Conidia observed with a light microscope appeared hyaline and fusiform, 8 to 16 × 2.5 to 4 μm, with two pointed ends or one rounded end. (e-Xtra 1E) One reference isolate, named L51, was used for molecular characterization. Total genomic DNA was extracted, and the ITS region of rDNA amplified using the universal primers ITS1 and ITS4, then sequenced. The resulting sequences were 100% identical to those of (NR_144794.1: strain CBS 112996 ITS region; from TYPE material). Based on Damm et al. (2012), partial ACT, GAPDH, CHS and TUB2 gene sequences were also amplified and sequenced (GenBank Accession numbers: ITS: OR882016, ACT: OR882013, GAPDH: OR882011, CHS: OR882012, TUB2: OR882010), to characterize the isolates. Additionally, the multilocus phylogenetic analysis carried out with the obtained and reference sequences (Damm et al. 2012) revealed the species of analyzed isolates and confirmed the BLAST results, identifying the strain as (e-Xtra 1F). Koch's postulates were performed on 10 'Kaiser' pears. Surfaces sterilized fruits with 96% ethanol were subjected to wound inoculation with a conidial suspension (106 conidia ml-1) while 10 fruit were used as negative control and inoculated with sterile water. Following an incubation period of 8-14 days at 15-20°C, symptoms around the inoculation site resembled those initially observed, while the negative control showed no symptoms. Fungal colonies re-isolated from the lesions exhibited the same morphological characteristics as the original isolates. To our knowledge, this is the first report of pear bitter rot caused by in Italy and in Europe (Talhinhas & Baroncelli, 2023). Yet, bitter rot had not been recognized as a notable issue in pear cultivation. Nevertheless, given that pears rank as the 8th most cultivated fruit globally and economically very significant for the Emilia Romagna region in Italy the emergence of pear bitter rot caused by species has the potential to evolve into a significant worldwide problem, warranting further investigation.
PubMed: 38783583
DOI: 10.1094/PDIS-12-23-2598-PDN -
Plant Disease May 2024is a genus of trees and shrubs in the Rosaceae commonly known as rowan and mountain-ash. They are usually found in temperate regions of the Northern Hemisphere and...
is a genus of trees and shrubs in the Rosaceae commonly known as rowan and mountain-ash. They are usually found in temperate regions of the Northern Hemisphere and cultivated as ornamental trees for parks and gardens. In September 2023, infection by a rust was observed on a single tree in Sólbrekkuskógur, Reykjanesbær (64.046645, -22.707276; ~13 m) in Iceland. Infected leaves were collected from this single cultivated tree at an outdoor recreation area in a natural wooded location, with a 2% disease severity. Sori were infrequent, scattered, embedded within circular yellow lesions on leaf margins. On average, one sorus was observed per leaf and only 2% of leaves were infected. Spermogonia epiphyllous, punctate and aggregated, pale yellow to black. Hypophyllous aecia roestelioid with cornute peridium rupturing at apex with peridial cells rhomboidal, aeciospores yellowish brown globoid 17.67-25.17 x 17.20-21.94 µm, walls 1.22-2.28 µm thick (n = 20). The features of this rust and dimensions of spores are consistent with descriptions of (Arthur 1909, Kern 1911). To confirm the identity (specimen MCA9732), a ~620 bp region of the 28S subunit of the ribosomal DNA repeat was sequenced using primers Rust2inv and LR6 following published protocols (Aime 2006). The sequence (GenBank PP413765) shared 100% (649/649 bp) identity with a sequence deposited as (KY764066, BPI910184; J. E. Demers, M. K. Romberg, and L. A. Castlebury, unpublished data) from and 100% (620/620 bp) identity with (PURN11049) on sp. from Canada when blasted against the RustHUBB database (Kaishian et al. 2024). The specimen has been deposited in the Arthur Fungarium at Purdue University as PURN24233. Disease on sp. caused by has been reported in various countries in Africa, Asia, North America, and Europe (Kern 1911). To our knowledge, this is the first report of the occurrence of this genus in Iceland from any host. alternates on species. In Iceland, (sect. Oxycedrus) seems to be the only naturally occurring species but it is an alternate host for G. cornutum. The presence of the primary and alternative hosts in Iceland and the ability of spp. to produce systemic infections in spp., represents the potential for reinfection of every year, resulting in potential impacts on both host species. With being the only spp. in natural habitats in Iceland, the presence of this rust represents a potential ecological disruption, as repeated infections may reduce host vitality and predispose the host to winter injury and attack from opportunistic pathogens or insects.
PubMed: 38783582
DOI: 10.1094/PDIS-03-24-0674-PDN