Basic Local Alignment Search Tool Research Articles

Molecular identification of the bacterium acute conjunctivitis by the method of sequensing gen 16S rRNA

Conjunctivitis is the most common eye disease, characterized by contextual inflammation, which can be caused by bacteria. The diagnosis of conjunctivitis is established based various factors, including the patients’s medical history, onset of eye symptoms, non-ocular symptoms, previous disease history, family medical history, allergies, and physical examination of the eyes, which may include assessments of visual acuity and vision field. Some cases of conjunctivitis require laboratory examinations to confirm the diagnosis. These may include cytological examination with Giemsa staining, Chlamydia Diagnostic Test, Nucleic acid amplification tests (NAATs) or Polymerase chain reaction (PCR), and microbiological tests. Microbiological examination helps identify the bacteria responsible for conjunctivitis and aids in treatment by prescribing antibiotics to suppress the growth of the infecting bacteria. While identification tests are not routinely performed, researchers often seek to determine the specific type of bacteria causing conjunctivitis infections, which may require several laboratory tests. Bacterial virulence plays a significant role, with genetic mutations potentially leading to severe infections of varying severity. Virulence genes encode proteins that express pathogenic properties. The species responsible for conjunctivitis can be definitively identified definitively through microbiological examination, utilizing methods such as the 16S Ribosomal RNA Sequencing (rRNA) technique, known for its accuracy and speed. This study aims to analyze the results of rRNA sequencing in cases of acute bacterial conjunctivitis caused by 16S rRNA genotyping. The research employed an exploratory metholodolot, with the results analyzed using the The Basic Local Alignment Search Tool (BLAST) tracking program database on the National Center for Biotechnology Information (NCBI) website. The findings revealed that Sphingomonas paucimobilis encoded the 16S rRNA using Universal Primary 27 F and 1492 R, obtained in a sequence size of 1351bp. The isolate demonstrated similarities to Sphingomonas paucimobilis.

[In silico analysis of Cit s 2: A highly conserved profilin].

Analyze phylogenetic relationships and molecular mimicry of Cit s 2 and other plant profilins. Online bioinformatics tools including Basic Local Alignment Search Tool (BLASTP), PRALINE and MEGA were used for multiple alignments and phylogenetic analysis. A 3D-homology model of Cit s 2 was predicted. Models were calculated with MODELLER. The best model was selected with the model scoring option of MAESTRO. Conserved regions between Cit s 2 and other profilins were located on the 3D model and antigenic regions were predicted by ElliPro server (3-5). Cit s 2 amino acid sequence (Uniprot code:P84177) was compared with other 30 profilins from different allergenic sources. The identity between Cit s 2 and other profilins ranged between 82 and 99%. The highest identity was observed with Cucumis melo (99%) followed by Prunus persica (98%) and Malus domestica (92%). High conserved antigenic regions were observed on the 3D predicted model. Seven lineal and six discontinuous epitopes were found in Cit s 2. High conserved antigenic regions were observed on the 3D predicted model of Cit s 2, which might involve potential cross-reactivity between Cit s 2 and other profilins. Future studies are needed to further analyze these results.

In silico and PCR Screening for a Live Attenuated Salmonella Typhimurium Vaccine Strain.

The application of live attenuated Salmonella Typhimurium vaccines has significantly helped control Salmonella in poultry products. Because the U.S. Department of Agriculture-Food Safety Inspection Service (USDA-FSIS) scores all Salmonella as positive, regardless of serovar, attenuated vaccine strains that are identified at processing contribute negatively toward Salmonella performance standards. This study was designed to determine the incidence of a live attenuated Salmonella serovar Typhimurium vaccine identified in broiler products by FSIS and to develop a PCR assay for screening of isolates. Salmonella Typhimurium short-read sequences from broiler samples uploaded to the National Center for Biotechnology Information (NCBI) Pathogen Detection database by the USDA-FSIS from 2016 to 2022 were downloaded and assembled. These were analyzed using the Basic Local Alignment Search Tool (BLAST) with a sequence unique to field strains, followed by a sequence unique to the vaccine strain. The PCR assays were developed against field and vaccine strains by targeting transposition events in the crp and cya genes and validated by screening Salmonella serovar Typhimurium isolates. Between 2016 and 2022, 1708 Salmonella Typhimurium isolates of chicken origin were found in the NCBI Pathogen Detection database, corresponding to 7.99% of all Salmonella identified. Of these, 104 (5.97%) were identified as the vaccine strain. The PCR assay differentiated field strains from the vaccine strain when applied to isolates and was also able to detect the vaccine strain from DNA isolated from mixed serovar overnight Salmonella enrichment cultures. Live attenuated Salmonella vaccines are a critical preharvest tool for Salmonella control and are widely used in industry. With forthcoming regulations that will likely focus on Salmonella Typhimurium, along with other serovars, there is a need to distinguish between isolates belonging to the vaccine strain and those that are responsible for causing human illness.

Occurrence of Praxelis clematidea Witches'-Broom Disease Association with 16SrI Group 'Candidatus Phytoplasma asteris' in Hainan Island of China.

Praxelis clematidea is an invasive herbaceous plant belonging to Asteraceae family. From August to November 2020, the plants showing severe witches'-broom symptoms were found in farms and roadsides from Ding'an of Hainan Province, a tropical island of China. The disease symptoms were suggestive of phytoplasma infection. For pathogen detection, P. clematidea samples consisting of six symptomatic and three asymptomatic plants were collected from the farms and roadsites of Ding'an with 40 % incidence by conducting surveys and statistics. Total nucleic acids were extracted using 0.10 g of fresh leaf tissues of the plant through CTAB DNA extraction method. Conserved gene sequences of 16S rRNA and secA genes from phytoplasma were amplified by direct PCR using primer pairs of R16mF2/R16mR1 and secAfor1/secArev3, respectively. R16mF2/R16mR1 PCR amplicons were obtained for all symptomatic samples but not from the symptomless plants. The amplicons were purified and sequenced by Biotechnology (Shanghai) Co., Ltd. (Guangzhou, China). Sequences of 16S rRNA gene (1323 bp) and secA (732 bp) were obtained and all the gene sequences were identical, designated as PcWB (Praxelis clematidea witches'-broom)-hnda. Representative sequencs were deposited in Genbank with accession numbers of PP098736 (16S rDNA) and PP072216 (secA). Nucleotide BLAST (Basic Local Alignment Search Tool) search based on 16S rRNA gene sequences indicated that PcWB-hnda had 100% sequence identity (1323/1323) with 'Candidatus Phytoplasma asteris'-related strains belonging to 16SrI group like Waltheria indica virescence phytoplasma (MW353909) and Capsicum annuum yellow crinkle phytoplasma (MT760793); had 99.62 % sequence identity (1321/1326) with the phytoplasma strains of 16SrI group such as Oenothera phytoplasma (M30790). RFLP (Restriction Fragment Length Polymorphism) pattern derived from 16Sr RNA gene sequences by iPhyClassifier showed identical (similarity coefficient=1.00) to the reference pattern of 16SrI-B subgroup (GenBank accession number: AP006628). The results obtained demonstrate that the phytoplasma strain PcWB-hnda under study is a member of 16SrI-B subgroup. A BLAST search based on secA gene sequences indicated that PcWB-hnda shares 100% sequence identity (732/732 bp) with Pericampylus glaucus witches'-broom phytoplasma (MT875200), 99% sequence identify (728/732 bp) with onion yellows phytoplasma OY-M(AP006628), and 99% sequence identify (729/732 bp) with rapeseed phyllody phytoplasma isolate RP166 (CP055264), among other phytoplasma strains that belong to 16SrI group. Previous studies demonstrated that P. clematidea can be infected by phytoplasmas affiliate to the 16SrII group (GenBank accession number: KY568717 and EF061924) in Hainan Island of China. To our knowledge, this is the first report of a natural infection of P. clematidea by a group 16SrI phytoplasma in the Island of China. 16SrI group can infect agronomic important species such as areca palm in the island and P. clematidea can be a reservoir of 16SrI phytoplasmas. Therefore, it is necessary to search of potential vectors of the pathogens, which would contribute to epidemiological monitoring and prevention of the related diseases.

Using nanopore sequencing to identify bacterial infection in joint replacements: a preliminary study.

This project investigates if third-generation genomic sequencing can be used to identify the species of bacteria causing prosthetic joint infections (PJIs) at the time of revision surgery. Samples of prosthetic fluid were taken during revision surgery from patients with known PJIs. Samples from revision surgeries from non-infected patients acted as negative controls. Genomic sequencing was performed using the MinION device and the rapid sequencing kit from Oxford Nanopore Technologies. Bioinformatic analysis pipelines to identify bacteria included Basic Local Alignment Search Tool, Kraken2 and MinION Detection Software, and the results were compared with standard of care microbiological cultures. Furthermore, there was an attempt to predict antibiotic resistance using computational tools including ResFinder, AMRFinderPlus and Comprehensive Antibiotic Resistance Database. Bacteria identified using microbiological cultures were successfully identified using bioinformatic analysis pipelines. Nanopore sequencing and genomic classification could be completed in the time it takes to perform joint revision surgery (2-3h). Genomic sequencing in this study was not able to predict antibiotic resistance in this time frame, this is thought to be due to a short-read length and low read depth. It can be concluded that genomic sequencing can be useful to identify bacterial species in infected joint replacements. However, further work is required to investigate if it can be used to predict antibiotic resistance within clinically relevant timeframes.

Molecular characterization and evaluation of phytochemical constituents of endophytic fungi derived from Mitracarpus scaber Zucc. (Rubiaceae)

In quest to ameliorate the issues of recurring incidences of multidrug-resistant organisms and over-exploration of plants, researchers have delved into researches involving endosymbiotic microorganisms, known as endophytes. These are microorganisms inhabiting the internal part of plants, which have been verified to possess great potentials of bioengineering novel products for therapeutic purposes. The aim of this study was to isolate and identify endophytic fungal species present in Mitracarpus scaber, molecularly characterize the pure isolates, and test for phytochemical compounds present. Freshly collected non-diseased leaves of M. scaber were subjected to a four-step surface sterilization. Thereafter, they were sliced into fragments of 1 cm-length, exposing the leaf blades and the midribs, and were aseptically inoculated on sterilized malt extract agar, containing chloramphenicol (500 mg/l), in Petri dishes. Hyphal tips of actively growing fungi from the plant material were harvested and further sub-cultured for purification and isolation. Segments were aseptically cut from the actively growing pure isolates, on malt extract agar and inoculated onto the fermentation medium (sterilized local rice), in 500 ml Erlenmeyer flasks, which were properly sealed with sterile cotton and kept at static condition at 25 oC for 21 days. Ethyl acetate was used to extract the metabolites from the end products of the fermentation processes. Four isolates obtained from the endophytic fungi (EDF), present in the leaf of M. scaber, were subjected to molecular identification. The DNA extraction was done using Zr fungal/bacterial DNA miniprep. The extracted DNA was amplified through PCR, and sequenced. The resultant sequences were compared with GenBank database, using Basic Local Alignment Search Tool. The result of phytochemical screening of the extracts revealed the presence of flavonoids, tannins and phenols, in large amounts; terpenoids, alkaloids and cardiac glycosides, in moderate amounts; steroids, hydrogen cyanide and saponins in very low quantities, while that of molecular characterization identified four organisms: Penicillium sclerotiorum, Clasporium cladosporiodies, Cryptococcus nemorosus, and Phyllosticta capitalensis.

Analysis of the Authenticity of Stone Medicinal Plants by DNA Barcoding

The genus Pyrrosia belongs to the family Polypodiaceae and are medium-sized epiphytic ferns, where the dried leaves of Pyrrosia lingua, Pyrrosia sheareri, Pyrrosia lanceolata, and Pyrrosia calvata are commonly used in medicinal practice. In this study, the authenticity of the collected medicinal plant samples of Shiwei was identified with the help of DNA barcoding technology using the internal transcribed spacer 2 (ITS2) as the identifying sequence. The experimental samples were analyzed using the basic local alignment search tool (BLAST) and the authenticity of the samples was further verified with the results of similarity comparison. The results proved that the sequences of the experimentally collected samples of Pyrrosia lingua, Pyrrosia sheareri, Pyrrosia lanceolata, and Pyrrosia calvata had a similarity of more than 97% when compared with the corresponding sequences that were uploaded on the Internet.

First Report of 'Candidatus Phytoplasma australasiaticum' Associated with Phyllody, Virescence, and Witches'-Broom Disease in Chrysanthemum morifolium in Taiwan.

Chrysanthemum morifolium (Asteraceae) is commonly grown as commercial cut flowers or pot mums worldwide. Common diseases of chrysanthemum include bacterial blight, fungal diseases, viruses, and phytoplasmas (Verma et al. 2003; Taloh et al. 2020). In June 2022, C. morifolium plants showing virescence, stunting, witches' broom, and phyllody symptoms were observed in 10 plants representing 10% of the estimated 100 plants in a field in Taichung City, Taiwan (Fig. S1). Three symptomatic samples along with three asymptomatic ones were collected for further study. Nested PCR was performed with two primer sets, P1/P7 (Deng and Hiruki 1991; Schneider et al. 1995) and R16F2n/R16R2 (Gundersen and Lee 1996) to amplify nearly full-length of 16S rDNA from the collected samples. The target 1.2-kb DNA band was only amplified from the symptomatic chrysanthemum plants. The amplicons were sequenced and a representative sequence deposited in GenBank under accession number OR501416. This sequence was used to search GenBank database by the Basic Local Alignment Search Tool (BLAST) program through the web service of National Center for Biotechnology Information (NCBI). In the 16S rDNA analyses, the three randomly picked amplicons from chrysanthemum phyllody phytoplasma (CPP) shared 100% identity with one another, and all shared 99.5% identity with the, 'Candidatus Phytoplasma australasiae' reference phytoplasma strain (Y10097). Further analysis using iPhyClassifier (Wei et al. 2007) revealed that CPP was most similar to the pattern of the peanut witches' broom phytoplasma in the 16SrII-A subgroup (GenBank Acc. No. L33765), with a pattern similarity coefficient of 1.0. For confirmation, the secY gene was amplified by secY-F/R primers (Li et al. 2014), the 1.2-kb band was sequenced and deposit in GenBank (Acc. No. OR508986). BLAST analysis showed that the secY sequence of CPP shared 99.93% of sequence identities to several 'Ca. P. australasiaticum' strains (MN543069, CP097312, CP120449, KC953013, MW085916, MW070030, CP040925). The phylogenetic tree analysis based on the secY gene by MEGA11 employing maximum-likelihood algorithm was performed and the bootstrap value was set as 1000 times for support of the stability for the clades. The result showed that CPP is closely related to other strains in 16SrII group (Fig. S2). Taken together, CPP is a 'Ca. P. australasiaticum' related-strain in 16SrII-A subgroup. This is the first report of chrysanthemum as a host of this phytoplasma in Taiwan, and might have an impact to the horticultural industry and the growers.

Potential of Viruses as Environmental Etiological Factors for Non-Syndromic Orofacial Clefts.

In this study, we analyzed the potential of viral infections in the species Homo sapiens as environmental causes of orofacial clefts (OFCs). A scoring system was adapted for qualitatively assessing the potential of viruses to cause cleft lip and/or palate (CL/P). This assessment considered factors such as information from the literature, nucleotide and amino acid similarities, and the presence of Endogenous Viral Elements (EVEs). The analysis involved various algorithm packages within Basic Local Alignment Search Tool 2.13.0 software and databases from the National Center for Biotechnology Information and the International Committee on Taxonomy of Viruses. Twenty significant viral species using different biosynthesis strategies were identified: Human coronavirus NL63, Rio Negro virus, Alphatorquevirus homin9, Brisavirus, Cosavirus B, Torque teno mini virus 4, Bocaparvovirus primate2, Human coronavirus HKU1, Monkeypox virus, Mammarenavirus machupoense, Volepox virus, Souris mammarenavirus, Gammapapillomavirus 7, Betainfluenzavirus influenzae, Lymphocytic choriomeningitis mammarenavirus, Ledantevirus kern, Gammainfluenzavirus influenzae, Betapolyomavirus hominis, Vesiculovirus perinet, and Cytomegalovirus humanbeta5. The evident viral etiological potential in relation to CL/P varies depending on the Baltimore class to which the viral species belongs. Given the multifactorial nature of CL/P, this relationship appears to be dynamic.

Development of a Multiplex PCR Assay for Rapid Differentiation of Fowlpox and Pigeonpox Viruses.

The aim of this study was to develop a multiplex PCR assay capable of rapidly differentiating two major Avipoxvirus (APV) species, Fowlpox virus (FWPV) and Pigeonpox virus (PGPV), which cause disease in bird species. Despite the importance of a rapid differentiation assay, no such assay exists that can differentiate the APV species without sequencing. To achieve this, species-specific target DNA fragments were selected from the fpv122 gene of FWPV and the HM89_gp120 gene of PGPV, which are unique to each genome. Nine samples collected from unvaccinated chickens, pigeons, and a turkey with typical pox lesions were genetically identified as FWPV and PGPV. The designed primers and target DNA fragments were validated using in silico analyses with the nucleotide Basic Local Alignment Search Tool. The multiplex PCR assay consisted of species-specific primers and previously described PanAPV primers (genus-specific) and was able to differentiate FWPV and PGPV, consistent with the phylogenetic outputs. This study represents the first successful differentiation of FWPV and PGPV genomes using a conventional multiplex PCR test. This assay has the potential to facilitate the rapid diagnosis and control of APV infections.

FGCD: a database of fungal gene clusters related to secondary metabolism.

Fungal secondary metabolites are not necessary for growth, but they are important for fungal metabolism and ecology because they provide selective advantages for competition, survival and interactions with the environment. These various metabolites are widely used as medicinal precursors and insecticides. Secondary metabolism genes are commonly arranged in clusters along chromosomes, which allow for the coordinate control of complete pathways. In this study, we created the Fungal Gene Cluster Database to store, retrieve, and visualize secondary metabolite gene cluster information across fungal species. The database was created by merging data from RNA sequencing, Basic Local Alignment Search Tool, genome browser, enrichment analysis and the R Shiny web framework to visualize and query putative gene clusters. This database facilitated the rapid and thorough examination of significant gene clusters across fungal species by detecting, defining and graphically displaying the architecture, organization and expression patterns of secondary metabolite gene clusters. In general, this genomic resource makes use of the tremendous chemical variety of the products of these ecologically and biotechnologically significant gene clusters to our further understanding of fungal secondary metabolism. Database URL: https://www.hebaubioinformatics.cn/FungalGeneCluster/.

Molecular identification of endophytic bacterium DBA2 isolated from the leaf of binahong (Anredera cordifolia (Ten.) Steenis) and its antagonistic activity against bacteria associated with dental caries

Binahong plants (Anredera cordifolia (Ten.) Steenis) are known to contain secondary metabolites which have important biological properties. The current study focused on the endophytic bacteria which lives in the leaves of A. cordifolia (Ten.) Steenis. The aim of this study is to isolate and identify the endophytic bacteria which can produce antibacterial metabolites from the leaves of A. cordifolia (Ten.) Steenis. Two isolates, DBA1 and DBA2 were isolated and purified from the leaves of A. cordifolia. These isolates were subjected to the screening for their antagonistic activity against the bacteria associated with dental caries, which are Streptococcus mutans and Lactobacillus acidophilus using the disk-diffusion method. The strain of DBA2 exhibited the largest diameter of inhibition zone against both S. mutans (31,17 mm) and L. acidophilus (35,57 mm). While DBA1 exhibited the diameter of inhibition zone of 23,47 mm and 25,87 mm against S. mutans and L. acidophilus, as respectively. The strain of DBA2 was then subjected for molecular identification. The genomic DNA of DBA2 was extracted with the Geno Plus™ Genomic DNA Extraction Miniprep System and molecular identification was performed by PCR amplification and sequencing of the 16S rRNA gene. The amplicons were then purified and sequenced, before the 16S rRNA gene sequences were analysed by a Basic Local Alignment Search Tool (BLAST) search against the National Centre Biotechnology Information (NCBI) database. The endophytic bacterial strain DBA2 from the leaves of A. cordifolia was identified to be closely related to Bacillus sp., and the top match from the database search revealed a similarity value of 100% with the reference Bacillus sp. strain x20. Future studies are required to analyse the bioactive compounds of strain DBA2, which can be considered as a potential source for the new antibacterial drugs for the dental caries treatment.

Molecular approach for identification and validation of Eurycoma longifolia, Labisia pumila, and Orthosiphon stamineus in herbal products

BackgroundEurycoma longifolia (E. longifolia), Labisia pumila (L. pumila), and Orthosiphon stamineus (O. stamineus) are popular species known for their therapeutic properties. An increase in local demand for herbal products makes them susceptible to adulteration, which poses a risk to their safety and efficacy. Current identification methods, such as organoleptic, microscopic, and macroscopic analysis, need to be revised to identify plant species in highly processed herbal products due to their limited ability to detect morphological features and provide comprehensive plant taxonomy information.MethodsThis research objective was to develop a simple, reliable, and accurate DNA molecular identification method based on polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) for E. longifolia, L. pumila, and O. stamineus, used to validate the species identification for herbal products. PCR–RFLP was developed for rapid identification using restriction enzymes TaqI, BamH I, HinfI, EcoRI, EcoRV, Mbol, and Mspl.ResultsThe nuclear DNA internal transcribed spacer 2 (ITS2) sequences were identified and compared between plant specimens of E. longifolia, L. pumila, and O. stamineus and 101 samples of commercial herbal products. Plant specimens of E. longifolia, L. pumila, and O. stamineus were successfully identified with high similarity of 100%, 100%, and 99.33%, respectively, based on National Center for Biotechnology Information (NCBI) GenBank. The recovery of DNA sequences from the herbal products was 60.4%, of which 81.97% were identified, and 18.03% showed no sequence through Basic Local Alignment Search Tool (BLAST) identification.ConclusionA reliable approach for identifying and validating plant species in herbal products has been created using restriction enzymes. This simple and accurate PCR–RFLP approach efficiently identifies E. longifolia, L. pumila, and O. stamineus by analysing ITS2 sequences, assuring consumer health and safety.

Extensive Countrywide Molecular Identification and High Genetic Diversity of Haemonchus spp. in Domestic Ruminants in Greece.

The gastrointestinal nematode parasite Haemonchus spp. is one of the most pathogenic parasites of ruminants, due to its blood-sucking activity, which causes large economic losses in the ruminant industry. The latest epizootiological data recorded an increase in the infection, not only in Greece but also in other countries, mainly attributed to climatic changes. The study of the population structure and the investigation of the phylogenetic relationships of Haemonchus spp. are essential for the understanding of its biology and epizootiology to implement appropriate control and prevention strategies. In addition, the molecular approach allows the determination of evolutionary relationships between different species of this parasite, the diverse hosts they infect, as well as the different geographic compartments from which they originate. Therefore, the aim of the present study was to identify the species of the sympatric populations of the genus Haemonchus, a nematode parasite infecting ruminants (sheep, goats, cattle, and buffaloes) from different regions of Greece (continental and insular) using molecular methods. At the same time, an attempt was made to identify the possible subpopulations of Haemonchus spp. in Greece, to investigate their phylogenetic relationships, as well as to determine the genetic diversity of each population. A total of 288 worms of the genus Haemonchus were processed using molecular methods; of these, 96 were collected from sheep, 96 from goats, 48 from cattle, and finally, 48 from buffaloes. A fragment of 321 base pairs of the second internal transcribed spacer (ITS2) sequence of nuclear DNA was amplified for species identification, and, after basic local alignment search tool (Blast) analysis, it was revealed that they belonged to H. contortus. A fragment of 820 base pairs of subunit 4 of the nicotinamide dehydrogenase (ND4) gene of mitochondrial DNA was amplified for genetic diversity analysis. The Greek mitochondrial ND4 sequences of H. contortus were classified into 140 haplotypes, and the values of the average nucleotide and haplotype diversity were lower compared to the respective values derived from Italy, Malaysia, the USA, and China. The phylogenetic analysis of the ND4 gene revealed a clear grouping of the Greek haplotypes when compared with Asian ones, and, at the same time, there was no profound grouping of the same haplotypes with regard to their different hosts and geographical origin within different regions of Greece. The aforementioned findings confirmed that H. contortus prevails in our country and can infect all species of ruminants, without geographical boundaries, when the right conditions (i.e., common grazing) are created.

DNA BARCODING OF Caranx senegallus FROM THREE WATER BODIES ADJOINING THE GULF OF GUINEA, NIGERIA

Fish species show astonishing phenotypic and genetic diversity in populations. The delimitation and recognition of fish species are not only of interest to taxonomists but also a requirement in studies of ecology. In this study, the genetic structure of Caranx senegallus collected from three water bodies adjoining the Gulf of Guinea, Nigeria was characterized using the cytochrome c oxidase I (COI) barcode gene region. Fish samples collected from Qua Iboe estuary, Escravos estuary, and Lagos lagoon were identified morphologically, and muscle tissues were used for DNA barcoding using Fish1 and Fish2 primers. Evolutionary analysis showed the formation of diverging clades in the neighbor-joining tree and indicated the presence of multiple species. The result also revealed that morphological identification was not entirely successful, as the Basic Local Alignment Search Tool (BLAST) prediction confirmed the presence of three specimens initially identified as C. senegallus but genetically matching C. fischeri and Trachinotus goreensis. This observation further emphasized the need for an integrative method for species identification.

Unraveling Proto-Oncogene (ErbB2) Expression in Patients With Carcinoma Head of Pancreas and Chronic Pancreatitis Patients: A Case-Control Study.

Background The pre-malignant tendency of the normal, non-affected portion of the pancreas is not as well explored as the multicentricity documented in pancreatic cancer cases. In order to ascertain the expression of inflammatory markers and Erythroblastic Oncogene B (ErbB2) in the non-affected pancreas in patients with pancreatic cancer, a case-control study was carried out. Materials and methods In patients who underwent pancreatoduodenectomy for pancreatic cancer (PC), pro-inflammatory genes and a tumor marker, erythroblastic oncogene 2 (ErbB2) in the epidermal growth factor receptor family were analyzed in the pancreatic tissue at the cut surface of the normal pancreas using qRT-PCR. Twenty patients diagnosed with Chronic pancreatitis (CP) after Frey's surgical procedure were selected, and their pancreatic tissues were analyzed as controls. The HPLC-purified primers were designed using National Center for Biotechnology Information (NCBI) software. The primer's specificity was verified for gene expression analysis using the Basic Local Alignment Search Tool (BLAST). The genes under study were normalized using β-actin as the housekeeping gene, and the 2-ddct method was used to compute the fold change compared to the control sample. Results Patients with margin-positive were not included. Pro-inflammatory genes (TNF-α, NF-kβ,andCOX-2) had significantly lower foldchange in PC patients compared to the CP group. The CP control group had higher levels ofIL-6gene expression than the PC group. Patients with pancreatic cancer had a considerably higher expression of theErbB2gene than patients with CP. Conclusion The upregulatedErbB2gene in the unaffected pancreatic tissue of pancreatic cancer patients, when compared to controls, indicates that the remaining pancreas may have the capacity to cause cancer. Proto-oncogene may play a role in the pathophysiologic process in patients with pancreatic cancer.

Cryptic diversity of mudskipper genus Boleophthalmus (Gobiiformes: Oxudercidae) from the north coast of East Java, Indonesia

Abstract. Shabrina FN, Wibowo K, Arisuryanti T. 2024. Cryptic diversity of mudskipper genus Boleophthalmus (Gobiiformes: Oxudercidae) from the north coast of East Java, Indonesia. Biodiversitas 25: 412-420. Mudskippers of genus Boleophthalmus are commonly found in the Indo-Pacific area. This study aimed to examine the diversity of the genus Boleophthalmus from the north coast of East Java Province, Indonesia by employing the mitochondrial COI gene for DNA barcoding and morphological characterization. Ten individuals of this genus were analyzed. According to the similarity percentage evaluated using Basic Local Alignment Search Tool (BLAST) and Barcode of Life Data (BOLD) identification engines, all the individuals were identified as Boleophthalmus boddarti Pallas, 1770. However, phylogenetic analysis revealed that three of the individuals were identified as B. boddarti, and seven were identified as Boleophthalmus pectinirostris Linnaeus, 1758 originated from East Java, which is separated from the clade of B. pectinirostris from East Asia population. These results were supported by the genetic distance of 8.93%-13.81% and the morphological characteristics determined by principal component analysis (PCA). PCA revealed that B. boddarti and B. pectinirostris are primarily distinguished by the number of (longitudinal) scales (69-75 versus 113-123), length of second dorsal fin base (42.3-43.9 versus 38.1-49.0), and caudal fin length (18.1-22.4 versus 17.5-21.6). Further detailed studies are needed to clarify whether the seven individuals belonging to B. pectinirostris complex should be regarded as a new species or a new subspecies.

Management of Fusarium fruit rot of Luffa cylindrical caused by Fusarium incarnatum and Fusarium chlamydosporum

AbstractFusarium rot of Luffa cylindrical was detected at Kafr-El-Dawar province, Beheira governorate, North Egypt during the growing season 2021. Surveyed disease incidence at Beheira, Kafr El-Shekh and Sharkia governorate showed fruit rot incidences of 18.33%, 16.12%, and 12.42%, respectively. Two fungal isolates of Fusarium spp. were tested for their pathogenic ability to induce Fusarium rot disease and typical disease symptoms. The online Basic Local Alignment Search Tool for Nucleotide Sequences (BLASTN) was used for identification and confirmation of identity performance of Internal Transcribed Spacer (ITS) and DNA sequencing, then compared with sequences available in GenBank. The two fungal isolates were identified as Fusarium incarnatum and Fusarium chlamydosporum and registered under accession number MN480497 and MN480498, respectively. Under laboratory conditions, organic acids, essential oils and organic salts significantly inhibited the growth of the two fungal isolates. Complete inhibition of fungal growth (100%) was recorded at 0.75 g/l of Topsin-M70 (Thiophanate-methyl) and 0.5% of salicylic acid. Meanwhile, a moderate growth reduction was observed using benzoic acid, coumarine, and cinnamon, thyme oil and garlic oil treatments. Under field conditions for two successive seasons, similar trends were observed. Foliar sprays with the fungicide Topsin-M70 showed the largest reduction in disease expression. The highest fruit rot disease reduction was recorded for salicylic acid and coumarin treatments followed by thyme oil, benzoic acid and potassium carbonate, garlic oil, boric acid and sodium bicarbonate. We concluded that the treatments are effective, safe and cost-effective methods for the control of this pre-harvest fruit disease under field conditions.

Phylogenetic Relationships of Isognomon (Lightfoot, 1786) Oysters from North Sulawesi, Indonesia

The Isognomon (Lightfoot, 1786) is a genus of oysters found in various coastal ecosystems throughout the world. Along with other bivalves, it performs significant ecological functions in marine ecosystems by providing food and habitat for fish and invertebrate habitats, filtering water, and protecting shorelines. Taxonomic classification of the Isognomon oyster can be challenging due to the varied or cryptic phenotypic characters, particularly shell characters.  In this study, two specimens with different shell characters of Isognomon oyster were collected from mangrove waters in Likupang, North Sulawesi, Indonesia, and subjected to molecular analysis to determine their identity.  The mitochondrial cytochrome C oxidase subunit I (COI) gene was utilized as a primer for this purpose, and the genetic distance and phylogenetic position of the two specimens were determined by comparing them with the GenBank database. The Basic Local Alignment Search Tool (BLAST) revealed that the two specimens were of belonged to Isognomon ephippium, with a similarity of 99.84%. The genetic distance between the two specimens was calculated using the Tamura Nei model and found to be 0.00, while the genetic distance between I. ephippium and other species in the Isognomon genus ranged from 0.00 to 0.14. The results of the Neighbor Joining (NJ) tree analyses showed that the two specimens clustered together with I. ephippium, which was divided into two distinct clades with a strong bootstrap value of 100 at the node

First Report of Charcoal Rot Caused by Macrophomina phaseolina on Dry Bean (Phaseolus vulgaris L.) in Western Canada.

In 2021, several dry bean (Phaseolus vulgaris L.) plants at the mid-seed-fill growth stage displaying wilting, chlorosis of the leaves and reduced vigor were collected near the Pembina - Emerson Border of Manitoba, Canada and North Dakota, USA. When symptomatic plants were examined, gray to dark brown discoloration was observed on the lower stem and the roots. Afterwards, brown to black discoloration was noticed on stem and root sections. Root and lower stem pieces (1 to 2 cm) from affected plants were surface sterilized with 70 % ethanol, followed by 1% NaOCl, rinsed twice in sterilized water, air dried on sterilized filter papers, and placed on potato dextrose agar (PDA) amended with 1 mg/mL of streptomycin sulfate. The PDA plates were incubated at 28°C with 12 h light/12 h dark for 10 days. The growing hyphae were transferred using the hyphal tip method to new PDA plates. Growing cultures were initially hyaline and turned from light gray to dark brown or black with age. Abundant dark and spherical to oblong shaped sclerotia with an average diameter of 97.9 µm (range: 66.8 to 143.5 µm, n =30) formed on the pure cultures 7 days after incubation. Additional pure culture was obtained through an isolation of a single microsclerotium followed by a single hyphal tip transfer. One isolate was identified as Macrophomina phaseolina based on morphological characteristics (Smith and Wyllie 1999). The morphological identity was confirmed by sequencing the rDNA internal transcribed spacer (ITS) region with universal primers ITS1/ITS4 (White et al. 1990) and calmodulin (CAL) and translation elongation factor-1 alpha (TEF-1α) genes with MpTefF/MpTefR, and MpCalF/MpCalR primer sets (Santos et al. 2020), respectively. The online resource Basic Local Alignment Search Tool (BLAST; https://www.ncbi.nlm.nih.gov/BLAST) confirmed the fungus identity as 100% M. phaseolina. The sequences of the original isolate BF21-25 were deposited in GenBank with accession numbers OQ615297 (ITS), OR357630 (CAL), and OR363106 (TEF-1α). To confirm pathogenicity, bioassays were conducted under controlled conditions. Four seeds of cultivar 'Etna' were sown per pot, and five pots were used for inoculated (approx. 4 × 105 microsclerotia/pot) and control (mock-inoculated with sterile PDA medium) treatments. For the inoculum, 20 g of macerated 10 to 14-day old M. phaseolina culture grown on PDA medium was applied to each pot using an inoculum layering technique. Pots were kept in the greenhouse with 28/17°C day/night, 13 h light/11 h dark cycle, and 70% relative humidity and watered weekly. Disease symptoms similar to those observed in the field were visible on all inoculated plants at the mid-seed-fill growth stage. Mock-inoculated control plants didn't show any symptoms. The experiment was repeated twice with similar results. The pathogen was re-isolated from infected plants to confirm Koch's postulates and identified as M. phaseolina based on the morphology and sequences of ITS, CAL and TEF-1α regions. To our knowledge, this is the first report of charcoal rot caused by M. phaseolina on dry bean in Western Canada.