GenBank Database Research Articles

Borrelia theileri infections in Rhipicephalus annulatus ticks from the north of Iran.

Ticks serve as vectors and reservoirs of various Borrelia species, potentially causing diseases in humans and animals. Mazandaran, a fertile green land in northern Iran, provides ample grazing grounds for livestock and harbors at least 26 hard tick species. This study investigated Borrelia infection in hard ticks from forest areas in this region and compared their genetic identity with the species data in the GenBank database. A total of 2,049 ticks were collected manually from mammalian hosts or using dragging and flagging methods. These ticks were then grouped into 190 pools and 41 individuals based on host, species, developmental stage, and gender. A real-time PCR (qPCR) detected Borrelia DNA in 26 pools from female, male, and nymph of Rhipicephalus annulatus (n = 17) and Ixodes ricinus (n = 9) ticks and one individual female Haemaphysalis punctata tick. The generated partial flaB and glpQ sequences from qPCR-positive Rh. annulatus ticks exhibited the highest identities of 98.1-100% and 98.2% with Borrelia theileri and closely related undefined isolates. Additionally, in phylogenetic analysis, these sequences clustered within well-supported clades with B. theileri and the closely related undefined isolates from various geographic regions, confirming the presence of B. theileri in the north of Iran. Divergence in B. theileri flaB and glpQ sequences across various geographical areas suggests potential subspeciation driven by adaptations to different tick species. This divergence in our flaB sequences implies the possible introduction of B. theileri-infected ticks from different geographical origins into Iran.

Elucidating Mycotoxin-Producing Aspergillus Species in River Water: An Advanced Molecular Diagnostic Study for the Assessment of Ecological Health and Contamination Risk

The primary goal of this research is to isolate mycotoxin-producing fungus from the Nagavali River. Examining isolated fungi involved analyzing their mycelium growth on culture media and detailed microscopic inspection. We employed PCR analysis utilizing universal primers ITS1 and ITS4 to accurately identify the species. Furthermore, we sequenced the amplified ITS region and rigorously analyzed the sequences using NCBI-BLASTn and the ITS2 database. The analysis found a high 96.38% genetic similarity to the Aspergillus flavus strain, resulting in a 600-base pair fragment size. The sequence was given the accession number OR536222 in the NCBI GenBank database. Phylogenetic analysis was performed to ascertain the particular strain of A. flavus and its source. Remarkably, this analysis led to the identification of a single new strain gene, which represents a novel discovery in the field of fungal research. These results underscore the vital significance of molecular techniques in promptly and precisely identifying organisms. This research enhances our understanding of mycotoxin contamination in water, providing valuable insights to improve detection and prevention strategies. It accentuates the overarching importance of conserving our water resources and upholding ecological equilibrium, ultimately safeguarding the well-being of both humanity and the environment.

Isolation and Enzymatic Characterization of Fungal Strains from Grapevines with Grapevine Trunk Diseases Symptoms inCentral Mexico.

Grapevine production is economically indispensable for the global wine industry. Currently, Mexico cultivates grapevines across approximately 28 500 hectares, ranking as the 26th largest producer worldwide. Given its significance, early detection of plant diseases' causal agents is crucial for preventing outbreaks. Consequently, our study aimed to identify fungal strains in grapevines exhibiting trunk disease symptoms and assess their enzymatic capabilities as indicators of their phytopathogenic potential. We collected plant cultivars, including Malbec, Shiraz, and Tempranillo, from Querétaro, Mexico. In the laboratory, we superficially removed the plant bark to prevent external contamination. Subsequently, the sample was superficially disinfected, and sawdust was generated from the symptomatic tissue. Cultivable fungal strains were isolated using aseptic techniques from the recovered sawdust. Colonies were grown on PDA and identified through a combination of microscopy and DNA-sequencing of the ITS and LSU nrDNA regions, coupled with a BLASTn search in the GenBank database. We evaluated the strains' qualitative ability to degrade cellulose, starch, and lignin using specific media and stains. Using culture morphology and DNA-sequencing, 13 species in seven genera were determined: Acremonium, Aspergillus, Cladosporium, Dydimella, Fusarium, Sarocladium, and Quambalaria. Some isolated strains were able to degrade cellulose or lignin, or starch. These results constitute the first report of these species community in the Americas. Using culture-dependent and DNA-sequencing tools allows the detection of fungal strains to continue monitoring for early prevention of the GTD.

Morphological and Molecular Characterization of Spirogyra Species from Water Bodies in Chiang Rai Province, Thailand: Insights into Bioactivity and Antioxidant Potentialin

This study aimed to identify Spirogyra species collected from lentic (sampling site code CR01) and lotic (sampling site code CR02) water areas in Chiang Rai Province, Thailand. The diagnosis was proceeded by studying the morphological characteristics combined with molecular techniques with the chloroplast rbcL gene (ribulose bisphosphate carboxylase). Additionally, the bioactive properties of Spirogyra extracts were evaluated through antimicrobial and antioxidant assays. The extraction was performed with water and ethanol extraction. The results indicated that the morphological characteristics of Spirogyra CR01 resembled Spirogyra neglecta, but the data of rbcL gene for this species were not available in the database. Therefore, the Spirogyra CR01 sequence was added to the GenBank database, representing the report of rbcL gene of Spirogyra neglecta sequencing data. Spirogyra CR02 was robustly identified as S. fluviatilisthrough molecular analysis. The extracts from both Spirogyra samples showed that the ethanolic extract has more activity than the water extract. The antimicrobial ability of the ethanolic extract from Spirogyra CR02 was observed to inhibit the growth of Bacillus cereus and Staphylococcus aureus, while the ethanolic extract from Spirogyra CR01 only inhibited B. cereus. Moreover, the ethanolic extract of Spirogyra CR01 had statistically significant (p<0.05) antioxidant activity, with the highest value recorded at 114.92±2.38 mg GAE/g extract. Overall, this study provides new sequencing information on the S. neglecta and their potential for use Spirogyra in functional food, functional ingredients and cosmeceutical products.

Genetic variation and evolutionary characteristics of Echovirus 11: new variant within genotype D5 associated with neonatal death found in China

ABSTRACT Echovirus 11 (E11) has gained attention owing to its association with severe neonatal infections. From 2018 to 2023, a surge in severe neonatal cases and fatalities linked to a novel variant of genotype D5 was documented in China, France, and Italy. However, the prevention and control of E11 variants have been hampered by limited background data on the virus circulation and genetic variance. Therefore, the present study investigated the circulating dynamics of E11 and the genetic variation and molecular evolution of genotype D5 through the collection of strains from the national acute flaccid paralysis (AFP) and hand, foot, and mouth disease (HFMD) surveillance system in China during 2000–2022 and genetic sequences published in the GenBank database. The results of this study revealed a prevalent dynamic of E11 circulation, with D5 being the predominant genotype worldwide. Further phylogenetic analysis of genotype D5 indicated that it could be subdivided into three important geographic clusters (D5-CHN1: 2014–2019, D5-CHN2: 2016–2022, and D5-EUR: 2022–2023). Additionally, variant-specific (144) amino acid mutation sites and positive-selection pressure sites (132, 262) were identified in the VP1 region. Cluster-specific recombination patterns were also identified, with CVB5, E6, and CVB4 as the major recombinant viruses. These findings provide a preliminary landscape of E11 circulation worldwide and basic scientific data for further study of the pathogenicity of E11 variants.

The chloroplast genome inheritance pattern of the Deli-Nigerian prospection material (NPM) × Yangambi population of Elaeis guineensis Jacq.

The chloroplast genome has the potential to be genetically engineered to enhance the agronomic value of major crops. As a crop plant with major economic value, it is important to understand every aspect of the genetic inheritance pattern among Elaeis guineensis individuals to ensure the traceability of agronomic traits. Two parental E. guineensis individuals and 23 of their F1 progenies were collected and sequenced using the next-generation sequencing (NGS) technique on the Illumina platform. Chloroplast genomes were assembled de novo from the cleaned raw reads and aligned to check for variations. The sequences were compared and analyzed with programming language scripting and relevant bioinformatic softwares. Simple sequence repeat (SSR) loci were determined from the chloroplast genome. The chloroplast genome assembly resulted in 156,983 bp, 156,988 bp, 156,982 bp, and 156,984 bp. The gene content and arrangements were consistent with the reference genome published in the GenBank database. Seventy-eight SSRs were detected in the chloroplast genome, with most located in the intergenic spacer region.The chloroplast genomes of 17 F1 progenies were exact copies of the maternal parent, while six individuals showed a single variation in the sequence. Despite the significant variation displayed by the male parent, all the nucleotide variations were synonymous. This study show highly conserve gene content and sequence in Elaeis guineensis chloroplast genomes. Maternal inheritance of chloroplast genome among F1 progenies are robust with a low possibility of mutations over generations. The findings in this study can enlighten inheritance pattern of Elaeis guineensis chloroplast genome especially among crops' scientists who consider using chloroplast genome for agronomic trait modifications.

Lycium barbarum Is a New Host of Botryosphaeria dothidea Associated with Soft Rot Disease in China.

Goji berry (Lycium barbarum) is a plant of the Solanaceae family that is cultivated in the Chinese provinces of Xinjiang, Ningxia, Gansu, and Qinghai, and its fruit is used as a traditional Chinese medicine (Yossa Nzeuwa et al. 2019). In July 2019, fruit rot was observed at an incidence of 20 to 25% on the Goji berry at a fruit market in Yinchuan, Ningxia, China. The fruit symptoms began as slightly shriveled areas on fruit peel, with noticeable softening of the infested portion of the tissue, followed by rotting and a sour odor. To isolate the pathogen, ten symptomatic tissues were randomly collected from different boxes, surface-sterilized for 30 s with 75% ethanol, followed by 0.1% mercuric chloride, then rinsed in sterile distilled water three times and plated onto PDA. The plates were incubated at 25°C in the dark for 7 days. Five purified fungal isolates from different fruit were obtained and single-spores. Emergent fungal colonies were white with 1 to 3 mm white margins and abundant aerial hyphae, 1 to 6 mm high, that became dark gray after 4 to 5 days. Conidia were hyaline, unicellular, fusiform, and measured 19.3 to 28.2 μm× 3.8 to 6.4 μm (n=50). All the morphological characteristics were consistent with Botryosphaeria spp. (Slippers et al. 2004). Five representative isolates, BJN1-BJN5, were selected for molecular identification. Total genomic DNA of the isolates was extracted with a Plant/Fungi DNA Isolation Kit. Translation elongation factor 1-alpha (EF1) gene and internal transcribed spacer (ITS) regions were amplified with primers EF1-728F/986R (Carbone and Kohn 1999) and ITS1/ITS4, respectively. The sequencing results of the five isolates were consistent, and those of the isolate BJN1 we deposited in the NCBI GeneBank database for EF1 (MK733274) and ITS (MK359291). A BLAST search of the GenBank database indicated that the EF1 and ITS sequences had 100% and 99% similarity, respectively, to B. dothidea ex-type strain (AY236898 and KF766151). A phylogenetic tree was constructed using maximum parsimony methods in MEGA11 and BJN1 isolate clustered with the reference sequence of B. dothidea. Pathogenicity tests were performed, inoculating healthy fruit with both mycelial plugs (7 days old) and conidial suspension (1 × 106 conidia/ml), repeated three times. Mycelial plugs of five isolates (BJN1-BJN5) growing on PDA with a colony diameter of 4 mm were placed on the sterilized surface of 20 Goji berry fruit. Sterile PDA plugs were placed on 12 healthy fruit as a control. In a second test, conidial suspensions of five isolates were sprayed on the surface of 20 healthy fruit and sterilized distilled water was used as a control. The inoculated fruits were maintained in an artificial climate chamber at 25°C and 80% to 85% relative humidity with a 12-h photoperiod for 7 days. The development of soft rot, similar to that observed on the original samples, was observed on inoculated fruit while control fruits remained asymptomatic. The pathogen was reisolated from infected fruit and confirmed as B. dothidea based on morphological characteristics and molecular sequences. To our knowledge, this is the first report of B. dothidea causing postharvest fruit rot of Goji berry, and this pathogen has been reported to cause fruit rot in Kiwifruit (Li et al. 2016) and Yellowhorn (Liu et al. 2018). This study provides information on a new postharvest fruit rot of Goji berry in China that has the potential to cause economic losses.

Phytochemical and biological assessment of secondary metabolites isolated from a rhizosphere strain, Sphingomonas sanguinis DM of Datura metel

BackgroundThe plant roots excrete a large number of organic compounds into the soil. The rhizosphere, a thin soil zone around the roots, is a hotspot for microbial activity, making it a crucial component of the soil ecosystem. Secondary metabolites produced by rhizospheric Sphingomonas sanguinis DM have sparked significant curiosity in investigating their possible biological impacts.MethodsA bacterial strain has been isolated from the rhizosphere of Datura metel. The bacterium’s identification, fermentation, and working up have been outlined. The ethyl acetate fraction of the propagated culture media of Sphingomonas sanguinis DM was fractioned and purified using various chromatographic techniques. The characterization of the isolated compounds was accomplished through the utilization of various spectroscopic techniques, such as UV, MS, 1D, and 2D-NMR. Furthermore, the evaluation of their antimicrobial activity was conducted using the agar well diffusion method, while cytotoxicity was assessed using the MTT test.ResultsThe extract from Sphingomonas sanguinis DM provided two distinct compounds: n-dibutyl phthalic acid (1) and Bis (2-methyl heptyl) phthalate (2) within its ethyl acetate fraction. Furthermore, the 16S rRNA gene sequence of Sphingomonas sanguinis DM has been registered under the NCBI GenBank database with the accession number PP422198. The bacterial extract exhibited its effect against gram-positive bacteria, inhibiting Streptococcus mutans (12.6 ± 0.6 mm) and Staphylococcus aureus (10.6 ± 0.6 mm) compared to standard antibiotics. Conversely, compound 1 showed a considerable effect against phytopathogenic fungi such as Alternaria alternate (56.3 ± 10.6 mm) and Fusarium oxysporum (21.3 ± 1.5 mm) with a MIC value of 17.5 µg/mL. However, it was slightly active against Klebsiella pneumonia (11.0 ± 1.0 mm). Furthermore, compound 2 was the most active metabolite, having a significant antimicrobial efficacy against Rhizoctonia solani (63.6 ± 1.1 mm), Pseudomonas aeruginosa (16.7 ± 0.6 mm), and Alternaria alternate (20.3 ± 0.6 mm) with MIC value at 15 µg/mL. In addition, compound 2 exhibited the most potency against hepatocellular (HepG-2) and skin (A-431) carcinoma cell lines with IC50 values of 107.16 µg/mL and 111.36 µg/mL, respectively.ConclusionSphingomonas sanguinis DM, a rhizosphere bacterium of Datura metel, was studied for its phytochemical and biological characteristics, resulting in the identification of two compounds with moderate antimicrobial and cytotoxic activities.

Molecular analysis of olive (Olea europaea L.) in Iraq using the internal transcribed spacer (ITS) region

Olea is a genus of about 40 species in the Oleaceae family distributed in warm temperate and tropical areas of southern Europe, Africa, southern Asia, and Australia. The study aimed to use the variations in the Internal Transcribed Spacer 1 (ITS1) region to construct a phylogenetic tree for 12 Iraqi olive varieties with 15 ITS1 sequences downloaded from the NCBI database. Leaf samples were collected from 12 olive varieties grown in five geographical regions of the north of Iraq. Using the BLAST algorithm, 12 sequences were manually adjusted by chromatogram comparison and aligned with the NCBI GenBank database. Haplotype diversity ranged from zero in the Australian population to one in the Moroccan and Spanish populations. Nucleotide diversity ranged from 0.0 in the Australian population to 0.08 in the Algerian population and Tajima's D value was −0.940. Genetic differentiation values ranged from 0.0177 between Iraqi Moroccan and Iraqi Spanish populations to 0.6540 between Iraqi and Algerian populations. The average pairwise nucleotide diversity within populations was 0.9036 in Iraqi and Turkish olive populations to 0.8671 in Iraqi and Algerian populations. The phylogenetic tree formed 2 main clusters. The first cluster includes all Algerian varieties and the Iraqi varieties are grouped in second cluster. The negative Tajima test value in all populations indicates an excess of rare alleles. The high value of gene flow between the Iraqi and Australian populations may have been mediated by human transport and these varieties may have been originated from the same geographical region.

DNA Barcoding Revealed a High Percentage of Mislabeling in Commercial Fish Products: The First Empirical Survey in South Texas

Seafood mislabeling is a widespread problem that have produced a growing distrust of seafood industry. In this study, we examined the prevalence of mislabeling in fish samples from regional markets in the greater Houston area and close-by coastal communities. A total of 63 fish fillet samples were purchased, labeled, and stored at -20˚C in individual packages until DNA extraction. DNA fragments of roughly 700 base pairs encoding cytochrome oxidase I (COI) were PCR-amplified from each DNA sample. With 99.6–100% nucleotide identity in the GenBank and BOLD databases, all samples were correctly identified at the species level. The scientific names identified by DNA barcoding were matched with legally acceptable market names using the US Food and Drug Administration (FDA) Seafood List. Out of the 63 samples examined, 13 samples (20.6%) were mislabeled. Moreover, 24 samples (38.1%) did not use the acceptable market names, indicating that the FDA policy is poorly implemented in seafood industry. The first DNA barcoding survey this area warranted the need of continuous monitoring and the dissemination of the regulation combined with taxonomic knowledge.

Pseudoplectania mystica (Ascomycota, Pezizales), a new cup fungus with an endophytic habit of a broad range of host plants

A cup fungus producing ascomata on senescing to dead rhizomes of bamboo in East China is described as Pseudoplectania mystica sp. nov. (Ascomycota, Pezizales) based on morphological, ecological and phylogenetic evidence. Some sequences of unidentified endophytic fungi uploaded to the GenBank database by other researchers were also identified as P. mystica in our phylogeny. By checking the original literature of these sequences, P. mystica was further found to act as an endophyte in a broad range of host plants including Aegiceras, Cinnamomum, Dendrobium and Lindera, probably lurking in the tissues of the host plants as invisible mycelium in most cases and only producing visible ascomata following senescence of the host tissues. Such ecology and a combination of all its morphological characteristics make P. mystica recognizable from other known species of Pseudoplectania. This study contributes to the understanding of the species diversity and lifecycle of Pseudoplectania.

Two proteocephalid cestodes in the fish Malapterurus electricus and Heterobranchus bidorsalis from Lake Nasser, Egypt: a morphological, molecular, and histopathological study

Despite the importance of the electric catfish (Malapterurus electricus) and the African giant catfish (Heterobranchus bidorsalis) in the foodweb of Lake Nasser, Egypt, little is known about their diseases and parasitic fauna. This work describes, for the first time, cestodiasis in M. electricus and H. bidorsalis. Corallobothrium solidum and Proteocephalus sp. were identified morphologically and molecularly from M. electricus and H. bidorsalis, respectively. Using PCR, sequencing, and phylogenetic analysis, the two cestodes shared rRNA gene sequence similarities yet were unique and the two new sequences for the proteocephalid genera were submitted to the GenBank database. The prevalence of infection was 75% and 40% for the two fish species, respectively. Infections significantly increased in the summer and spring and were higher in female fish than in male fish. The intestine was the preferred site of the two adult cestodes. However, in the case of C. solidum some larval cestodes were found outside the intestine in between the skin and abdominal musculature, attached to the mesentery, and within intestinal tunica muscularis. Desquamation of the intestinal epithelium and inflammation at the site of infection in addition to congestion of the intestinal wall of the tapeworm infected fish were evident, indicating that C. solidum and Proteocephalus sp. impacted the infected fish. The larval stages of C. solidum attempted to penetrate the intestine and sometimes they were encircled within fibrous layers infiltrated with inflammatory cells. The infected fish’s musculature was free of cestode infections. Preventive measures should be implemented to prevent the spread of infections.

Genetic characterization of bovine coronavirus strain isolated in Inner Mongolia of China

BackgroundBovine coronavirus (BCoV) is implicated in severe diarrhea in calves and contributes to the bovine respiratory disease complex; it shares a close relationship with human coronavirus. Similar to other coronaviruses, remarkable variability was found in the genome and biology of the BCoV. In 2022, samples of feces were collected from a cattle farm. A virus was isolated from 7-day-old newborn calves. In this study, we present the genetic characteristics of a new BCoV isolate. The complete genomic, spike protein, and nucleocapsid protein gene sequences of the BCoV strain, along with those of other coronaviruses, were obtained from the GenBank database. Genetic analysis was conducted using MEGA7.0 and the Neighbor-Joining (NJ) method. The reference strains’ related genes were retrieved from GenBank for comparison and analysis using DNAMAN.ResultsThe phylogenetic tree and whole genome consistency analysis showed that it belonged to the GIIb subgroup, which is epidemic in Asia and America, and was quite similar to the Chinese strains in the same cluster. Significantly, the S gene was highly consistent with QH1 (MH810151.1) isolated from yak. This suggests that the strain may have originated from interspecies transmission involving mutations of wild strains. The N gene was conserved and showed high sequence identity with the epidemic strains in China and the USA.ConclusionsGenetic characterization suggests that the isolated strain could be a new mutant from a wild-type lineage, which is in the same cluster as most Chinese epidemic strains but on a new branch.

Detection and genetic analysis of bovine rhinitis B virus in Japan.

Bovine rhinitis B virus (BRBV) (genus Aphthovirus, family Picornaviridae) is a significant etiological agent of the bovine respiratory disease complex. Despite global reports on BRBV, genomic data for Japanese strains are not available. In this study, we aimed to obtain genomic information on BRBV in Japan and analyze its genetic characteristics. In nasal swabs from 66 cattle, BRBV was detected in 6 out of 10 symptomatic and 4 out of 56 asymptomatic cattle. Using metagenomic sequencing and Sanger sequencing, the nearly complete genome sequences of two Japanese BRBV strains, IBA/2211/2 and LAV/238002, from symptomatic and asymptomatic cattle, respectively, were determined. These viruses shared significant genetic similarity with known BRBV strains and exhibited unique mutations and recombination events, indicating dynamic evolution, influenced by regional environmental and biological factors. Notably, the leader gene was only approximately 80% and 90% identical in its nucleotide and amino acid sequence, respectively, to all of the BRBV strains with sequences in the GenBank database, indicating significant genetic divergence in the Japanese BRBV leader gene. These findings provide insights into the genetic makeup of Japanese BRBV strains, enriching our understanding of their genetic diversity and evolutionary mechanisms.

Isolation and Identification of Morganella morganii from Rhesus Monkey (Macaca mulatta) in China

A bacterium was isolated and identified from the secretion of a rhesus monkey with endometritis. The morphological results showed that the strain exhibited round, convex, gray-white colonies with smooth surfaces and diameters ranging from 1 to 2 mm when cultured on Columbia blood agar at 37 °C for 24 h; on salmonella–shigella agar (S.S.) at 37 °C for 24 h, the colonies appeared round, flat, and translucent. Gram staining showed negative results with blunt ends and non-spore-forming characteristics. Molecular biology results showed that the 16S rRNA sequence of the strain revealed over 96.9% similarity with published sequences of M. morganii from different sources in the NCBI GenBank database. Morphological and molecular biology analysis confirmed that the strain (RM2023) isolated from cervical secretions of rhesus monkey was M. morganii. Drug sensitivity testing demonstrated that the isolated strain (RM2023) was sensitive to ceftriaxone, amikacin, gentamicin, cefazolin, cefuroxime, ceftazidime, levofloxacin, cotrimoxazole, norfloxacin, and tetracycline; moderately sensitive to ampicillin; and resistant to penicillin, vancomycin, ciprofloxacin, and clindamycin. The research findings provide valuable insights for disease prevention in rhesus monkeys and contribute to molecular epidemiological studies.

Speciation patterns of Aedes mosquitoes in the Scutellaris Group: a mitochondrial perspective

The Scutellaris Group of Aedes comprises 47 mosquito species, including Aedes albopictus. While Ae. albopictus is widely distributed, the other species are mostly found in the Asia–Pacific region. Evolutionary history researches of Aedes species within the Scutellaris Group have mainly focused on Ae. albopictus, a species that raises significant public health concerns, neglecting the other species. In this study, we aimed to assess genetic diversity and estimate speciation times of several species within the Scutellaris Group. Mosquitoes were therefore collected from various Asia–Pacific countries. Their mitochondrial cytochrome c oxidase subunit 1 (cox1) and subunit 3 (cox3) sequences were analyzed alongside those of other Scutellaris Group species available in the GenBank database. To estimate the divergence time, we analyzed 1849 cox1 gene sequences from 21 species, using three species (Aedes aegypti, Aedes notoscriptus and Aedes vigilax) as outgroups. We found that most of the speciation dates occurred during the Paleogene and the Neogene periods. A separation between the Scutellaris Subgroup and the Albopictus Subgroup occurred approximately 64–61 million years ago (MYA). We also identified a split between species found in Asia/Micronesia and those collected in Melanesia/Polynesia approximately 36–35 MYA. Our findings suggest that the speciation of Aedes species within the Scutellaris Group may be driven by diversity in mammalian hosts, climate and environmental changes, and geological dynamics rather than human migration.

Lytic Capsule-Specific Acinetobacter Bacteriophages Encoding Polysaccharide-Degrading Enzymes

The genus Acinetobacter comprises both environmental and clinically relevant species associated with hospital-acquired infections. Among them, Acinetobacter baumannii is a critical priority bacterial pathogen, for which the research and development of new strategies for antimicrobial treatment are urgently needed. Acinetobacter spp. produce a variety of structurally diverse capsular polysaccharides (CPSs), which surround the bacterial cells with a thick protective layer. These surface structures are primary receptors for capsule-specific bacteriophages, that is, phages carrying tailspikes with CPS-depolymerizing/modifying activities. Phage tailspike proteins (TSPs) exhibit hydrolase, lyase, or esterase activities toward the corresponding CPSs of a certain structure. In this study, the data on all lytic capsule-specific phages infecting Acinetobacter spp. with genomes deposited in the NCBI GenBank database by January 2024 were summarized. Among the 149 identified TSPs encoded in the genomes of 143 phages, the capsular specificity (K specificity) of 46 proteins has been experimentally determined or predicted previously. The specificity of 63 TSPs toward CPSs, produced by various Acinetobacter K types, was predicted in this study using a bioinformatic analysis. A comprehensive phylogenetic analysis confirmed the prediction and revealed the possibility of the genetic exchange of gene regions corresponding to the CPS-recognizing/degrading parts of different TSPs between morphologically and taxonomically distant groups of capsule-specific Acinetobacter phages.

Polymorphism of spore germination receptor genes in Bacillus anthracis strains of major genetic lineages

Introduction. The genetic structure of the global population of Bacillus anthracis characterized by an unequal distribution of isolates of the main genetic lineages A, B and C, the reason for which is unknown. Determining the characteristics of genes encoding factors that determine the existence of this pathogen at the intra- and extra organismal stages of the life cycle, which can influence the prevalence of strains, is relevant. Aim — сharacterization of the genes and proteins of spore germination in strains of the anthrax pathogen of different genetic lineages. Materials and methods. Whole genome sequences of 46 B. anthracis strains and the CI strain of B. cereus biovar anthracis from the NCBI GenBank database studied. In silico analysis carried out using the programs “BLASTn”, “MEGA X”, “Tandem Repeat Finder”. Results. The number of SNPs, indels and pseudogenes in B. anthracis strains of line B was 2,7–25,6 times higher, in line C 2–23,5 times, and in the B. cereus biovar anthracis strain was 20–2841 times higher than in strains of line A. Significant substitutions in genes leading to changes in the amino acid composition of 10 germination receptor proteins were also significantly more common in B. anthracis strains of lines B, C and the B. cereus biovar anthracis strain. Undescribed VNTRs within the gerHA gene with repeat units of 78 and 117 bp identified, varying between and within isolates of different genetic lineages. Six germination receptor genes have been shown to have rare starting codons. Conclusion. A larger number of non-synonymous SNPs in the genes of spore germination receptors with changes in the amino acid composition of proteins in B. anthracis strains of the main genetic lines B, C and B. cereus biovar anthracis than in strains of line A suggests their limited adaptive capabilities and may be one of the explanations for the lower prevalence compared to line A. Differences in the gerHA and gerM genes make it possible to differentiate the major B and C genetic lineages from A.

Assessment of Durian Diversity and Its Wild Relatives (Durio spp.) Based on Leaf Morphology and Molecular Marker

Durian (Durio spp.) is native to Southeast Asia and has potential for development. However, some species are threatened due to deforestation and extensive land conversion. This study aimed to determine the genetic diversity and relationships of durian and wild relatives (Durio spp.) on the Indonesia Island Borneo using a leaf morphology and DNA barcoding (matK) marker. In this study, 15 durian samples from this region were used, excluding ‘Monthong’ (Durio zibethinus) and ‘Bengang’ (Neesia strigosa) as the outgroups from the GenBank database. The leaf morphology was analyzed descriptively, whereas the genetic diversity was by the nucleotide diversity index (π%). The relationship of durians was revealed by the maximum likelihood (ML) method and examined with the bootstrap statistics for 1000 replicates, also confirmed by the PCA (principal component analysis). Based on the leaf morphology, the durians are divided into five forms, i.e., obovate-lanceolate, elliptic, ovate, oblong, and linear-oblong. ‘Pampaken’ and ‘Pampaken Burung Kecil’ indicated the earliest form (obovate-lanceolate), whereas the linear-oblong was by ‘Kamundai.’ Following the molecular marker, it was seen that the durians have low genetic diversity (π%) with only 0.015. However, phylogenetically, the durians were separated into four similar clades or groups for ML and PCA. In this instance, it has appeared that most of the durians evaluated in the current study have close relationships, except for the taxa with the farthest relationship. The results provide valuable information for the local and global durian conservation mission, including future breeding programs.

First record of Diplodia gallae causing branch cankers on declining scarlet oak (Quercus coccinea) in Virginia.

Members of Botryosphaeria s.l. have an extensive history as cankering pathogens of stressed and declining oak trees in the eastern United States (Ferreira et al. 2021). The host range, distribution, and virulence among two closely related species, Diplodia corticola and D. gallae, remains unclear (Brazee et al. 2023). On 15 August 2023, a survey was conducted at a declining natural hardwood site in Shenandoah County, Virginia (GPS coordinates 38.922089, -78.606125). One mature Quercus coccinea tree that displayed scorched leaf margins and branch dieback was felled and a cankered branch from the crown was sampled (Fig. 1A and B). A 4-mm piece of necrotic tissue was selected from the margin of the canker, disinfected with 2.5% NaOCl, again with 70% ethanol, and air-dried before being placed on half-strength acidified PDA medium (pH 4.8) and incubated in the dark at 22 ± 2°C. After 5 days, four colonies were transferred to full-strength PDA medium and incubated in the dark at 22 ± 2°C. After 10 days, all four colonies displayed thick, gray, floccose mycelium and pigmented hyphae (Fig. 1C). Mycelia was harvested from 10-day-old colonies with a sterile pin and DNA was extracted using a Qiagen DNeasy Plant Pro Kit (Germantown, MD) according to the manufacturer's instructions. A fragment of the internal transcribed spacer (ITS) and translation elongation factor 1-α (tef1) loci were amplified using ITS4/ITS5 (White et al. 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999) primer sets, respectively. The PCR amplicons were purified with ExoSap-IT (Affymetrix, Santa Clara, CA) and sequenced at Eurofins (Louisville, KY).&xa0; The raw nucleotide sequences were analyzed using Geneious 11.1.5 software (Biomatters, Auckland, NZ). All four colonies had identical ITS sequences. A 523 and 276-bp fragment of the ITS and tef1 loci, respectively, from isolate R1.2 was deposited into the GenBank database (accessions OR934498 and OR961039). A dataset of 43 strains consisting of 38,658 characters was aligned using MAFFT v7.49 (Katoh et al. 2013), and a concatenated ITS + tef1 maximum likelihood phylogenetic tree (1000 bootstraps) was built with PhyML 3.0 (Guindon et al. 2010) using the GTR substitution model. Isolate R1.2 was grouped with isolates of D. gallae although the species failed to form a well-supported clade (BS = 67) due to intraspecific variation (Fig. 1D). Koch's postulates were fulfilled by inoculating five healthy, containerized Q. coccinea trees (average stem caliper 5.3 cm) with isolate R1.2, with five plants as controls. After disinfecting the bark with 70% ethanol, a 0.5 mm section of the bark was removed 13 cm above the soil line with a sterile scalpel, and a 0.5 mm agar plug taken from the edge of a 10-day-old PDA culture was placed in the wound with the mycelium facing the cambial tissue, sealed with Parafilm, and maintained at 22 ± 4°C. The same procedure was performed on the control plants using sterile PDA plugs. After five weeks the bark was removed, and all five stems treated with R1.2 had necrotic lesions with a mean linear growth ([length+width]/2) of 9.2 ± 2.72 mm from the edge of the wound, which was significantly larger (P = 0.003) than the controls (1 ± 0.66 mm; Fig. 1E - L). Necrotic stem tissue was sampled as previously described, and the isolate recovered was confirmed as D. gallae based on morphology and 100% ITS sequence homology to isolate R1.2. D. gallae was not recovered from the control plants. In the United States, D. gallae has been isolated from Q. rubra and Q. velutina twig cankers in Maine, Massachusetts, New Hampshire, New York, and Vermont (Brazee et al. 2023). This is the first report of the species in Virginia causing branch cankers on Q. coccinea.