High Degree Of Genetic Similarity Research Articles

Genetic analysis of drug resistance mechanisms and phylogenetic clustering in Candida auris isolates from Western India

Objectives: Candida auris is an emerging multidrug-resistant fungal pathogen that poses a significant threat to global health. Limited information is available from the Indian subcontinent regarding mutations associated with drug resistance and genetic variability among the isolates. In this study, we employed whole-genome sequencing (WGS) to investigate the genetic variations and drug resistance mechanisms within C. auris isolates from the western region of India. Materials and Methods: A total of twenty archived isolates were subjected to WGS on the Illumina NextSeq 2000 platform. A set of 18 genes was analyzed to check for the presence of drug-resistant mutations. Phylogenetic analysis was done using MEGA v6.06 software to identify the C. auris subgroup or clade and to check genetic relatedness among species. Statistical Analysis: The data related to drug resistance were presented in numbers and percentages. Results: Through manual analysis, drug-resistant mutations were detected in ERG11, CDR1, and TAC1b genes, which are known to be associated with reduced susceptibility to antifungal agents. Phylogenetic analysis revealed that all the isolates clustered within Clade I, indicating a high degree of genetic similarity among isolates. The absence of comprehensive antifungal mutation databases and automated tools for drug resistance detection necessitated the utilization of specialized computational skills of bioinformaticians for data analysis. Conclusions: The study provides valuable insights into the genetic diversity and drug resistance mechanisms of C. auris isolates in the western region of India and emphasizes the need for continued research and surveillance to combat this emerging pathogen. Our findings underscore the need for the development of user-friendly automated tools and comprehensive databases to facilitate rapid and accurate identification of drug resistance in C. auris.

Ultrastructural and molecular characterization of Glugea sp. (microsporidia), a parasite of the Red Sea fish Carangoides bajad (Carangidae).

Glugea sp. found infecting the liver of the teleost fish Carangoides bajad from the Red Sea, Egypt, is described based on light microscopy and ultrastructural characteristics combined with phylogenetic analyses. This microsporidium forms whitish xenomas up to ~4 mm in size. Xenomas display numerous parasitophorous vacuoles totally filled by mature spores, no other life cycle stages were observed. Mature spores ellipsoidal and measuring 6.3 × 4.0 μm in size. The polaroplast appears composed of two distinct regions: an electron-dense vesicular region and a densely packed lamellar region. The polar tubule forms approximately 24-27 coils arranged in three layers encircling the posterior vacuole. The small subunit (SSU) rRNA gene and its ITS region were sequenced and showed the highest similarity of 99.4% to other Glugea spp. Bayesian inference and maximum likelihood analyses place the novel isolate within the Glugea clade, more specifically within a subclade that predominantly grouped species described from fish inhabiting the Arabian Gulf or Red Sea. The results validate the parasite's classification in the Glugea genus. Nevertheless, until more detailed ultrastructural and molecular data are obtained, the identification of the current Glugea species is hampered by the absence of some developmental stages and the high degree of genetic similarity.

Effects of antimony on antioxidant system, damage indexes of blood-brain barrier and ultrastructure of zebrafish (Danio rerio)

Antimony (Sb) and its compounds can be harmful to people and are known to cause cancer, so they are a key pollutant to control. This study investigated the influence of antimony on non-enzymatic antioxidants and the blood-brain barrier (BBB) in zebrafish(Danio rerio), a model organism that shares a high degree of genetic similarity with humans. Zebrafish were exposed to different doses of antimony in water for 7, 18, and 30 days. The results indicated that antimony accumulated most in the liver, followed by the gills, flesh, and brain, with the accumulation increasing as the exposure duration extends. Additionally, under identical antimony concentrations, the buildup in the four tissues was positively correlated with the duration of exposure. After 18 days of exposure, the total antioxidant capacity (T-AOC) and endogenous non-enzymatic antioxidants vitamin C (VC) and vitamin E (VE) decreased as a result of antimony ingestion in zebrafish, although cysteine secretion was increased in the liver, gills, and brain. The structural integrity of the BBB was compromised by the elevation of ApoE4 and MMP-9 levels as a result of antimony exposure, which led to the breakdown of the basal lamina, tight junctions, and nerve fibers in the brain. At this injured region, 5-HT and MBP were also able to easily enter and leave the BBB, albeit at variable rates. Additionally, when the antimony exposure level reached 16.58 mg·L−1, antimony penetrated the BBB and bound to erythrocytes, causing their lysis.

Prevalence, antibiogram and molecular characterization of Listeria monocytogenes from ruminants and humans in New Valley and Beheira Governorates, Egypt

BackgroundListeriosis is a global health threat to both animals and humans, especially in developing countries. This study was designed to isolate Listeria monocytogenes from faeces; environmental samples; and cow, sheep and goat milk, as well as human stool, to study its molecular characteristics and antibiotic sensitivity in the New Valley and Beheira Governorates, Egypt. The isolation and identification of L. monocytogenes were carried out using traditional culture and biochemical methods, followed by antibiography, genus confirmation of some isolates and detection and sequencing of InlB genes via PCR.ResultsOut of 2097 examined samples, the prevalence of L. monocytogenes was 13.4% in animals; the prevalence was 9.2%, 2.4%, 25.4%, 4%, 42.4%, and 6.4% in cattle faeces, cattle milk, sheep faeces, sheep milk, goat faeces, and goat milk, respectively. However, the prevalence of L. monocytogenes was 8.3% in human samples. Both animal and human isolates showed 100% resistance to trimethoprim-sulfamethoxazole, and the isolates showed the highest sensitivity to flumequine (100%), amikacin (99.2%), gentamicin (97.6%), and levofloxacin (94.6%). Multidrug resistance (MDR) was detected in 86.9% of the tested isolates. The 16 S rRNA and inlB genes were detected in 100% of the randomly selected L. monocytogenes isolates. Phylogenetic analysis of three isolates based on the inlB gene showed 100% identity between faecal, milk and human stool isolates.ConclusionsFaeces and milk are major sources of listeriosis, and the high degree of genetic similarity between animal and human isolates suggests the possibility of zoonotic circulation. The high prevalence of MDR L. monocytogenes in both animal and human samples could negatively impact the success of prevention and treatments for animal and human diseases, thereby imposing serious risks to public health.

First Report of Botrytis cinerea Causing Stem Rot on Hydrangea macrophylla in China.

Hydrangea (Hydrangea macrophylla), commonly referred to as big leaf hydrangea, is a species within the Hydrangeaceae family notable for its ornamental value. Characterized by its vividly colored sepals and lush, striking inflorescences, this species is globally esteemed as both a potted and landscape plant. Notably, in 2022, an alarming incidence of stem rot was observed in approximately 40% of H. macrophylla plants aged between six and twelve months within 16 greenhouses situated in Nanjing City (N 31°14', E 118°22'), Jiangsu Province, China. Initial symptoms of the disease manifested as wet gray-black spots at the base of the seedlings and stems, progressing to a necrotic gray-white discoloration in the stems and accompanied by the growth of gray mold on the affected parts. This infection ultimately led to the wilting of the leaves and the death of the seedlings. For pathogen identification, stem tissues at the interface of diseased and healthy sections were excised, surface-sterilized with 75 % ethanol for 30 s, followed by a 2 - 3 min treatment with 3% sodium hypochlorite, and subsequently rinsed three times with sterile water before air drying. Sections measuring 2 - 3 mm were then cultured on potato dextrose agar (PDA) medium, supplemented with 50 mg/mL rifampicin (RFP), and incubated at 25 ℃ for 3 - 5 d (Zhou et al. 2022). Upon 2 - 3 days of incubation, notable growth of fungal colonies was observed. Mycelial clusters from the periphery of these colonies were subsequently transferred to fresh PDA plates and incubated at 25 ℃ for an additional 5 - 7 d. A particular colony, designated JSNJ2022-2 and now preserved at the Jiangsu Academy of Agricultural Sciences, was selected for detailed examination. This colony exhibited a flocculent texture, with a coloration ranging from grey-white to light brown. It was characterized by the presence of irregularly formed, hard sclerotia within the hyphae. The conidiophores were observed to be slender and erect, featuring dendritic branches at their extremities. The conidia were clustered on the conidiophore like grapes. These conidia were generally colorless or grey, oval in shape, smooth and transparent, and measured between 6.4 - 12.2 × 7.3 - 18.2 μm (n = 50). For genetic analysis, genomic DNA (gDNA) was extracted using the DNA secure Plant Kit (Tiangen Biotech, Beijing, China). Polymerase chain reaction (PCR) amplification was performed using a set of universal primers of ITS1/ITS4 (White et al. 1990), primers corresponding to the specific sequences of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60), and DNA-dependent RNA polymerase subunit II (RPB2) (Yang et al. 2020). The resultant PCR products were sequenced, and the resulting sequences were submitted to the GenBank database, under the accession numbers OP131597, OP142320, OP142321, and OP142322, respectively. BLAST analysis of the sequences obtained from the isolate JSNJ2022-2 revealed a high degree of genetic similarity, ranging from 99 to 100%, with known sequences of Botrytis cinerea (accessions MK051124.1, MH796662.1, MH479931.1, and KU760986.1). To elucidate the phylogenetic position of the isolate, a phylogenetic tree was constructed using the maximum likelihood method, supported by 1,000 bootstrap replications, in the Mega7 software (Kumar et al. 2016). The results of this analysis confirmed that the strains under study clustered within the same branch as B. cinerea. To establish the pathogenicity of the isolate, Koch's postulates (Falkow 1988) were employed. Healthy potted H. macrophylla seedlings, approximately three months old, were wound inoculated at the base of the seedlings with a 6 mm diameter mycelium plug of JSNJ2002-2 cultivated on PDA for 3 days, which was subsequently covered with moistened degreasing cotton. Control plants were treated with moistened degreasing cloths minus the pathogen. Post-inoculation, these plants were placed in a growth chamber maintained at 25 ℃ with a relative humidity range of 60 - 80%. After a 3-d incubation period, the inoculated plants displayed symptoms identical to those initially observed in the greenhouse. The pathogen was successfully re-isolated from these inoculated plants and was morphologically re-confirmed as B. cinerea, thus satisfying the criteria of Koch's postulates. To our knowledge, this report represents the first documented incidence of B. cinerea causing stem rot in H. macrophylla in China.

Identification of the allelic state of the Rca2 gene for resistance to anthracnose and determination of genetic relatedness in garden strawberry genotypes

The studies were conducted between 2022-2023. The purpose of the work was to analyze the relationship of hybrids and varieties of strawberries resistant and susceptible to anthracnose and to determine the putative allelic status of the anthracnose resistance gene Rca2. Selection of ISSR primers for identifying polymorphism and constructing dendrites of genetic similarity of 19 garden strawberry genotypes from the collection of the Federal State Budgetary Scientific Institution Federal Scientifi c Center for Horticulture. Th e eff ectiveness of ISSR primers was assessed by the ISSR-PCR method, followed by gel electrophoresis of ISSR-PCR products in an agarose gel. To assess the allelic state of the Rca2 gene, the SCAR marker STS-Rca2_240 was used. The genetic distance of the analyzed strawberry samples was determined using the Jaccard coeffi cient. Cluster analysis was carried out using the STATISTICA program (StatSoft ). The putative allelic status of the anthracnose resistance gene Rca2 in the studied varieties and hybrids was established. Diagnostic fragment of the STS-Rca2_240 marker, 240 bp in size. was found in the Alpha variety. Based on an array of genetic similarity coefficients, dendrograms were constructed, during the analysis of which the relationship of the studied variety samples was established. Promising forms were identifi ed that had a high degree of genetic similarity to varieties resistant to anthracnose, and forms that were closely related to susceptible varieties.

Diving into the zebrafish brain: exploring neuroscience frontiers with genetic tools, imaging techniques, and behavioral insights.

The zebrafish (Danio rerio) is increasingly used in neuroscience research. Zebrafish are relatively easy to maintain, and their high fecundity makes them suitable for high-throughput experiments. Their small, transparent embryos and larvae allow for easy microscopic imaging of the developing brain. Zebrafish also share a high degree of genetic similarity with humans, and are amenable to genetic manipulation techniques, such as gene knockdown, knockout, or knock-in, which allows researchers to study the role of specific genes relevant to human brain development, function, and disease. Zebrafish can also serve as a model for behavioral studies, including locomotion, learning, and social interactions. In this review, we present state-of-the-art methods to study the brain function in zebrafish, including genetic tools for labeling single neurons and neuronal circuits, live imaging of neural activity, synaptic dynamics and protein interactions in the zebrafish brain, optogenetic manipulation, and the use of virtual reality technology for behavioral testing. We highlight the potential of zebrafish for neuroscience research, especially regarding brain development, neuronal circuits, and genetic-based disorders and discuss its certain limitations as a model.

Epidemiological Characterisation of blaNDM-Positive Enterobacterales from Food-Producing Animal Farms in Southwest China.

Carbapenems are atypical β-lactam antibiotics with a broade antibacterial spectrum and strong antibacterial activity; however, the emergence and spread of carbapenemases have led to a decline in their effectiveness. New Delhi metallo-β-lactamase (NDM) is an important carbapenemase that has attracted widespread attention and poses a major threat to public health. To investigate the epidemiological characteristics of blaNDM in swine and chicken farms in southwestern China, we isolated 102 blaNDM-positive Enterobacterales strains from 18 farms in Sichuan and Yunnan provinces in 2021, with Escherichia coli and Klebsiella spp. being the main reservoirs of blaNDM, variant blaNDM-5 being the most prevalent, and all strains being multi-drug resistant. Whole-genome sequencing analysis of 102 blaNDM-positive Enterobacterales strains revealed that blaNDM had spread primarily through its carriers on the same farm and among the 18 farms in this study. A high degree of genetic similarity between animal-derived blaNDM-positive Escherichia coli strains and human-derived strains was also identified, suggesting a potential mutual transmission between them. Nanopore sequencing results indicated that blaNDM is predominantly present on the IncX3 plasmid, that an insertion sequence might be important for recombination in the blaNDM genetic environment, and that most of the plasmids carrying blaNDM are transferable. Collectively, our results enrich the current epidemiological information regarding blaNDM in pig and chicken farms in Southwest China, revealing its transmission pattern, as well as the potential risk of transmission to humans, which could help to better understand and control the spread of blaNDM.

Biological Features of Atlantic Cod <i>Gadus Morhua</i> L., 1758 (Gadiformes: Gadidae) of the Murmansk Coast: Race Composition and Fishing

The biological features of Atlantic cod in the coastal zones of the Murman for 1999–2006 were studied. The age composition, growth rate and maturation, feeding habits and structure of otoliths were analyzed. Histological and genetic studies were carried out. It was shown that young cod mainly lives and spawns in Western Murman bays. Older cod leaves the bays due to insufficient food supply. The growth rate of young coastal cod is comparable to that of cod in the open part of the Barents Sea. With age, the cod of open sea areas noticeably overtakes the cod that lives in the coastal zone in growth. It was noted that cod spawns in the bays with both coastal and Atlantic types of otoliths. Studies of genetic polymorphism indicate a high degree of genetic si-milarity between cod groups in the Norwegian and Barents Seas, which is evidenced under the influence of currents (branches of the Norwegian Atlantic Current, the Murmansk Coastal Current) and against the background of migrations at different stages of ontogeny of cod individuals from coastal and open water areas.

Isolation, characterization, and pathogenicity assessment of Corynebacterium pseudotuberculosis biovar equi strains from alpacas (Vicugna pacos) in China.

Corynebacterium pseudotuberculosis is a zoonotic pathogen that causes lymphadenitis in humans, livestock, and wildlife. In this study, C. pseudotuberculosis biovar equi strains were isolated from three alpacas. Antibiotic susceptibility tests and pathogenicity tests were also conducted. Moreover, one strain was sequenced using DNBSEQ and Oxford Nanopore technology. The three strains exhibited resistance to aztreonam, fosfomycin, and nitrofurantoin. The median lethal doses (LD50) of strains G1, S2 and BA3 in experimentally infected mice was 1.66 × 105 CFU, 3.78 × 105 CFU and 3.78 × 105 CFU, respectively. The sequencing of strain G1 resulted in the assembly of a chromosomal scaffold comprising 2,379,166 bp with a G + C content of 52.06%. Genome analysis of strain G1 revealed the presence of 48 virulence genes and 5 antibiotic resistance genes (ARGs). Comparative genomic analysis demonstrates a high degree of genetic similarity among C. pseudotuberculosis strains, in contrast to other Corynebacterium species, with a clear delineation between strains belonging to the two biovars (ovis and equi). The data of the present study contribute to a better understanding of the properties of C. pseudotuberculosis biovar equi strains and the potential risk they pose to alpacas and other livestock, as well as the necessity of ongoing surveillance and monitoring of infectious diseases in animals.

Global distribution and genomic characteristics of tet(X)-positive Escherichia coli among humans, animals, and the environment

In recent years, the plasmid-mediated transmission of the tigecycline resistance gene tet(X) in Escherichia coli has received considerable attention. However, studies on the global distribution of tet(X)-positive E. coli remain scarce. Herein, we performed a systematic genomic analysis of 864 tet(X)-positive E. coli isolates from humans, animals and the environment around the world. These isolates were reported in 25 countries and isolated from 13 different hosts. China reported the most tet(X)-positive isolates (71.76 %), followed by Thailand (8.45 %) and Pakistan (5.9 %). Pigs (53.93 %), humans (17.41 %), and chickens (17.41 %) were determined to be important reservoirs of these isolates. The sequence types (STs) of E. coli were highly diverse, with the ST10 clone complex (Cplx) being the most prevalent clone. Correlation analysis revealed a positive association between the antibiotic resistance genes (ARGs) in ST10 E. coli and the presence of insertion sequences and plasmid replicons; however, we found no significant correlation between ARGs and virulence genes. Furthermore, the ST10 tet(X)-positive isolates from multiple sources displayed a high degree of genetic similarity (<200 single-nucleotide polymorphisms [SNPs]) to the mcr-1-positive but tet(X)-negative human-derived isolates, suggesting clonal transmission. The most prevalent tet(X) variant in the E. coli isolates was tet(X4), followed by tet(X6)-v. Genome-wide association study (GWAS) indicated that compared to tet(X4), tet(X6)-v harbored more significantly different resistance genes. Notably, certain tet(X)-positive E. coli isolates from different geographical locations or hosts shared a few SNPs (<200 SNPs), indicating cross-contamination. Therefore, continuous global surveillance of tet(X)-positive E. coli is imperative in the future.

The Western Lake Erie culture collection: A promising resource for evaluating the physiological and genetic diversity of Microcystis and its associated microbiome

Cyanobacterial harmful algal blooms (cyanoHABs) dominated by Microcystis spp. have significant public health and economic implications in freshwater bodies around the world. These blooms are capable of producing a variety of cyanotoxins, including microcystins, that affect fishing and tourism industries, human and environmental health, and access to drinking water. In this study, we isolated and sequenced the genomes of 21 primarily unialgal Microcystis cultures collected from western Lake Erie between 2017 and 2019. While some cultures isolated in different years have a high degree of genetic similarity (genomic Average Nucleotide Identity >99%), genomic data show that these cultures also represent much of the breadth of known Microcystis diversity in natural populations. Only five isolates contained all the genes required for microcystin biosynthesis while two isolates contained a previously described partial mcy operon. Microcystin production within cultures was also assessed using Enzyme-Linked Immunosorbent Assay (ELISA) and supported genomic results with high concentrations (up to 900 μg L⁻¹) in cultures with complete mcy operons and no or low toxin detected otherwise. These xenic cultures also contained a substantial diversity of bacteria associated with Microcystis, which has become increasingly recognized as an essential component of cyanoHAB community dynamics. These results highlight the genomic diversity among Microcystis strains and associated bacteria in Lake Erie, and their potential impacts on bloom development, toxin production, and toxin degradation. This culture collection significantly increases the availability of environmentally relevant Microcystis strains from temperate North America.

Detection of Virulence and β-lactamase resistance genes of non-typhoidal Salmonella isolates from human and animal origin in Egypt "one health concern"

BackgroundNon-typhoidal Salmonella (NTS) is a major foodborne zoonotic pathogen worldwide. In the current study, Various NTS strains were isolated from (cows, milk and dairy products in addition to humans) in New Valley and Assiut Governorate, Egypt. NTS were firstly serotyped and tested by antibiotic sensitivity test. Secondly, some virulence genes and Antibiotic resistance genes have been identified by using PCR. Finally, Phylogenesis was performed depending on the invA gene, for two S. typhimurium isolates (one of animal origin and the other of human origin for evaluating zoonotic potential).ResultsOut of 800 examined samples, the total number of isolates was 87 (10.88%), which were classified into 13 serotypes, with the most prevalent being S. Typhimurium and S. enteritidis. Both bovine and human isolates showed the highest resistance to clindamycin and streptomycin, with 90.80% of the tested isolates exhibiting MDR. The occurrence of the invA gene was 100%, while 72.22%, 30.56%, and 94.44% of the examined strains were positive for stn, spvC, and hilA genes, respectively. Additionally, blaOXA-2 was detected in 16.67% (6/ 36) of the tested isolates, while blaCMY-1 was detected in 30.56% (11of 36) of the tested isolates. Phylogenesis revealed a high degree of similarity between the two isolates.ConclusionsThe high occurrence of MDR strains of NTS in both human and animal samples with high degree of genetic similarity, shows that cows, milk and milk product may be a valuable source of human infection with NTS and interfere with treatment procedures.

GENETIC RELATIONSHIP BETWEEN SOME BARLEY GENOTYPES (HORDEUM VULGARE L.) USING SSR TECHNIQUE (MICROSATELLITE)

The study was carried out in order to determine the degree of genetic kinship among fifteen barley genotypes using SSR technique with 14 pairs of primers. The study showed that, all the primers gave amplification products and showed polymorphism between the studied genotypes. The total number of alleles was 19, with an average of 1.35, and the number of polymorphic alleles was 19, the percentage of polymorphism was 100%. The number of total and polymorphic alleles ranged between one allele as the lowest value for primers (Bmac0209, Bmac0067, Bmag0225, Bmag012, Bmag0394, Bmag0353, Bmag0206, Bmac0031, GBM1362, EBmac0603) and three alleles as the highest value for the primer Bmag0006. The polymorphism information content (PIC) for each primer ranged from 0.32 (Bmac0209, Bmag0225) as the lowest value, to 0.721 (Bmag0385) as the highest value, and average PIC value was 0.446. The highest value of the percent agreement values (PAV) matrix was about 0.909, between Furat 7 and Mohsen White cultivars, which indicate the present of a high degree of genetic kinship, followed by the Furat 5 and Asowad Arabic (0.863), while the lowest value was 0.318 between H10 and Furat 4, indicating a great genetic variation between them. The previous results were in agreement with the genetic kinship analysis and cluster analysis which showed a high degree of genetic similarity between Furat 7 and Mohsen White, followed by Furat 5 and Asowad Arabic.

Cadmium-resistant Chryseobacterium sp. DEMBc1 strain: characteristics and potential to assist phytoremediation and promote plant growth.

The effectiveness of phytoremediation is closely related to the various interactions between pollutants, soil particles, rhizosphere microorganisms, and plants. Therefore, the object of current study was a cadmium-tolerant bacterium isolated from the rye rhizosphere, with a high degree of genetic similarity to the genus Chryseobacterium. Chryseobacterium sp. DEMBc1 was able to grow with 36 different BiologGN2 carbon sources and show the adaptation to stress factors such as Cd (100 μg ml-1), low temperature (8 °C), and salinity (2% NaCl). Furthermore, it was shown that DEMBc1 had the characteristics of plant growth-promoting microorganisms: it was able to produce ammonia, indole acetic acid, 1-aminocyclopropane-1-carboxylic acid deaminase, and siderophores, as well as solubilize Ca3(PO4)3. After inoculation with DEMBc1, a significant decrease in the concentration of Cd was observed in the roots of Festuca ovina grown in Cd-polluted soil, compared to the non-inoculated Cd-polluted soil. It was also noticed that DEMBc1 produced a large amount of extracellular polymeric substances that were significantly higher than the cellular biomass. These polymers can form a barrier to reduce the translocation of Cd from the growth medium to the plant roots. According to the current study, DEMBc1 has a stabilizing potential and can decrease the mobility of Cd in the F. ovina rhizosphere, bioaccumulate metals in plant tissues, and effectively improve the bioavailability of nutrients, especially Fe, N, and P in a polluted environments.

An Improved Agrobacterium-Mediated Transformation and Genome-Editing Method for Maize Inbred B104 Using a Ternary Vector System and Immature Embryos.

For maize genome-editing and bioengineering, genetic transformation of inbred genotypes is most desired due to the uniformity of genetic background in their progenies. However, most maize inbred lines are recalcitrant to tissue culture and transformation. A public, transformable maize inbred B104 has been widely used for genome editing in recent years. This is primarily due to its high degree of genetic similarity shared with B73, an inbred of the reference genome and parent of many breeding populations. Conventional B104 maize transformation protocol requires 16–22 weeks to produce rooted transgenic plants with an average of 4% transformation frequency (number of T0 plants per 100 infected embryos). In this Method paper, we describe an advanced B104 transformation protocol that requires only 7–10 weeks to generate transgenic plants with an average of 6.4% transformation frequency. Over 66% of transgenic plants carried CRISPR/Cas9-induced indel mutations on the target gene, demonstrating that this protocol can be used for genome editing applications. Following the detailed and stepwise procedure described here, this quick and simplified method using the Agrobacterium ternary vector system consisting of a T-DNA binary vector and a compatible helper plasmid can be readily transferable to interested researchers.

Cell Wall Properties Determine Genotype-Specific Response to Cold in Miscanthus × giganteus Plants.

The cell wall plays a crucial role in plant growth and development, including in response to environmental factors, mainly through significant biochemical and biomechanical plasticity. The involvement of the cell wall in C4 plants’ response to cold is, however, still poorly understood. Miscanthus × giganteus, a perennial grass, is generally considered cold tolerant and, in contrast to other thermophilic species such as maize or sorgo, can maintain a relatively high level of photosynthesis efficiency at low ambient temperatures. This unusual response to chilling among C4 plants makes Miscanthus an interesting study object in cold acclimation mechanism research. Using the results obtained from employing a diverse range of techniques, including analysis of plasmodesmata ultrastructure by means of transmission electron microscopy (TEM), infrared spectroscopy (FTIR), and biomechanical tests coupled with photosynthetic parameters measurements, we present evidence for the implication of the cell wall in genotype-specific responses to cold in this species. The observed reduction in the assimilation rate and disturbance of chlorophyll fluorescence parameters in the susceptible M3 genotype under cold conditions were associated with changes in the ultrastructure of the plasmodesmata, i.e., a constriction of the cytoplasmic sleeve in the central region of the microchannel at the mesophyll–bundle sheath interface. Moreover, this cold susceptible genotype was characterized by enhanced tensile stiffness, strength of leaf wall material, and a less altered biochemical profile of the cell wall, revealed by FTIR spectroscopy, compared to cold tolerant genotypes. These changes indicate that a decline in photosynthetic activity may result from a decrease in leaf CO2 conductance due to the formation of more compact and thicker cell walls and that an enhanced tolerance to cold requires biochemical wall remodelling. Thus, the well-established trade-off between photosynthetic capacity and leaf biomechanics found across multiple species in ecological research may also be a relevant factor in Miscanthus’ tolerance to cold. In this paper, we demonstrate that M. giganteus genotypes showing a high degree of genetic similarity may respond differently to cold stress if exposed at earlier growing seasons to various temperature regimes, which has implications for the cell wall modifications patterns.

Sequential infections by 32 isolates of

Context Studies of Phoma black stem and leaf spot disease (caused by Phoma medicaginis) in annual medics (Medicago spp.) normally involve a ‘once-only’ inoculation not reflecting multiple pathogen infection and phytoestrogen production cycles in the field. Phytoestrogen production by plants can result in lower ovulation rates in grazing animals. Aims We aimed to determine whether sequential infections by P. medicaginis increase production of phytoestrogens in annual medics, and to measure the genetic diversity of isolates. Methods In a greenhouse experiment, pathogenicity and virulence were investigated across 32 isolates of P. medicaginis following one, two or three rounds of inoculation of M. polymorpha var. brevispina. Production of the phytoestrogens coumestrol and 4′-O-methyl coumestrol was measured, and correlation with disease parameters assessed. DNA sequencing using ITS, β-tubulin, calmodulin and P. medicaginis-specific EFNI-1α was applied for phylogenetic analysis of isolates from Western Australia and elsewhere. Key results Across isolates, highest leaf disease incidence was 76%, petiole disease incidence 61%, leaf disease severity 52% and petiole disease severity 53%. Stem coumestrol content range was 45–1247 mg kg−1, and 4′-O-methyl coumestrol 0–344 mg kg−1. All measures were highest after three rounds of inoculation. Overall, there was a positive correlation of leaf disease incidence with coumestrol content (P &lt; 0.05) and of both leaf and petiole disease incidence with 4′-O-methylcoumestrol content (P &lt; 0.01, P &lt; 0.05, respectively). Phylogenetic analysis revealed a high degree of genetic similarity among Western Australian isolates, generally grouping into a single separate cluster across the four markers, and genetically distinct from isolates sourced outside Australia. Conclusions Leaf disease incidence was the best discriminating disease parameter for coumestrol and 4′-O-methylcoumestrol content. Western Australian isolates of P. medicaginis were genetically similar and unique, possibly due to geographic separation. Implications The study emphasised the importance of sequential inoculations when screening annual Medicago genotypes towards developing cultivars with superior disease resistance and enhanced animal reproductive outcomes.

First identification and genomic characterization of equine hepacivirus subtype 2 in China.

Equine hepacivirus (EqHV) is a newly discovered hepatitis C virus-like virus that can infect equines. EqHV strains circulating worldwide have been classified into subtypes 1-3. In previous studies, we detected the presence of EqHV strains of subtype 1 and 3 in China. To determine whether EqHV strains of subtype 2 are prevalent in China, serum samples were collected from 133 racehorses in Guangdong province in 2021 and were tested for EqHV RNA by RT-PCR, and the positive rate was 9% (12/133). Sequencing of the NS3 gene revealed that one field strain (GD2021) had a high degree of genetic similarity to EqHV strains of subtype 2. Subsequent genome sequencing and analysis demonstrated that strain GD2021 belongs to subtype 2. The present study enriches our knowledge about the genetic diversity of EqHV in China.

Ophiocordyceps salganeicola, a parasite of social cockroaches in Japan and insights into the evolution of other closely-related Blattodea-associated lineages

The entomopathogenic genus Ophiocordyceps includes a highly diverse group of fungal species, predominantly parasitizing insects in the orders Coleoptera, Hemiptera, Hymenoptera and Lepidoptera. However, other insect orders are also parasitized by these fungi, for example the Blattodea (termites and cockroaches). Despite their ubiquity in nearly all environments insects occur, blattodeans are rarely found infected by filamentous fungi and thus, their ecology and evolutionary history remain obscure. In this study, we propose a new species of Ophiocordyceps infecting the social cockroaches Salganea esakii and S. taiwanensis, based on 16 years of collections and field observations in Japan, especially in the Ryukyu Archipelago. We found a high degree of genetic similarity between specimens from different islands, infecting these two Salganea species and that this relationship is ancient, likely not originating from a recent host jump. Furthermore, we found that Ophiocordyceps lineages infecting cockroaches evolved around the same time, at least twice, one from beetles and the other from termites. We have also investigated the evolutionary relationships between Ophiocordyceps and termites and present the phylogenetic placement of O. cf. blattae. Our analyses also show that O. sinensis could have originated from an ancestor infecting termite, instead of beetle larvae as previously proposed.