Petri Dishes Research Articles

Low-adhesion culture selection for human iPS cell-derived cardiomyocytes

Despite progress in generating cardiomyocytes from pluripotent stem cells, these populations often include non-contractile cells, necessitating cardiomyocyte selection for experimental purpose. This study explores a novel cardiomyocyte enrichment mechanism: low-adhesion culture selection. The cardiac cells derived from human induced pluripotent stem cells were subjected to a coating-free low-adhesion culture using bovine serum albumin and high molecular weight dextran sulfate. This approach effectively increased the population of cardiac troponin T-positive cardiomyocytes. Similar results were obtained with commercially available low-adhesion culture dishes. Subsequently, we accessed the practicality of selection of cardiomyocytes using this phenomenon by comparing it with established methods such as glucose-free culture and selection based on puromycin resistance genes. The cardiomyocytes enriched through low-adhesion culture selection maintained autonomous pulsation and responsiveness to beta-stimuli. Moreover, no significant differences were observed in the expression of genes related to subtype commitment and maturation when compared to other selection methods. In conclusion, cardiomyocytes derived from pluripotent stem cells were more low-adhesion culture resistant than their accompanying non-contractile cells, and low-adhesion culture is an alternative method for selection of pluripotent stem cell-derived cardiomyocytes.

IMAC application of extracellular polymeric substances doped composite membranes for α-amylase immobilization and kinetic studies

Immobilized metal affinity chromatography (IMAC), extensively utilized technique in affinity chromatography, has proven to be highly effective in purifying α-amylase, a crucial enzyme in industrial applications. Here, Poly (2-hydroxyethyl methacrylate) (PHEMA) based composite membranes were fabricated using the radical copolymerization technique in a petri dish. Extracellular polymeric materials (EPS) as the metal binding agent. The attachment of Cu2+ ions to the membranes occurred through the utilization of EPSs (Cu2+-EPS). The membranes having Cu2+-EPS (Cu2+-EPS-PM) were subjected to various characterization techniques, including scanning electron microscopy (SEM), attenuated total reflectance-fourier transform infrared spectroscopy (ATR-FTIR), swelling tests, and surface area measurements. The investigation also encompassed the examination of various factors, including pH, initial α-amylase concentration, temperature, and ionic strength, that exert an influence on the adsorption of α-amylase from an aqueous medium. The Cu2+-EPS-PMs demonstrated maximum adsorption capacity with a value of 54.2 mg/g polymer (pH 5.0 sodium acetate buffer, temperature: 25°C, initial α-amylase concentration of 2 mg/mL). Additionally, it has been observed that the enzyme is able to be used in adsorption and desorption cycles on Cu2+-EPS-PM many times. The investigation also focused on examining the impact of pH, temperature, storage, and operational stability over the activities of both free and immobilized α-amylase. The outcomes showed that immobilization had positive effects on activity.

Allelopathic Effects of Corn Straw and Its Water Extracts on Four Weed Species and Foxtail Millet

Straw covering is a protective tillage measure in agricultural production, but there is relatively little research on the allelopathic effects of corn straw on weeds and foxtail millet. This experiment studied the allelopathic effects of corn straw on four weeds (Chenopodium album, Setaria viridis, Echinochloa crus-galli and Amaranthus retroflexus) in foxtail millet fields, and also measured the growth indicators of foxtail millet. The study consisted of Petri dish and field experiments. Five treatments were used in the Petri dish experiment: clear water as control (0 g/L, TCK) and four types of corn straw water extracts. They were, respectively, the stock solution (100 g/L, T1), 10 X dilution (10 g/L, T2), 50 X dilution (2 g/L, T3), and 100 X dilution (1 g/L, T4) of corn straw water extracts. Additionally, seven treatments were set up in the field experiment, consisting of three corn straw covering treatments, with covering amounts of 3000 (Z1), 6000 (Z2) and 12,000 kg/ha (Z3), and four control treatments—one treatment with no corn straw cover (CK) and three treatments involving the use of a black film to create the same shading area as the corn straw covered area, with black film coverage areas of 50% (PZ1), 70% (PZ2), and 100% (PZ3), respectively. The results showed that the corn straw water extract reduced the germination rate of the seeds of the four weeds. The T1 treatment resulted in the allelopathic promotion of C. album growth but the inhibition of S. viridis, E. crus-galli, and A. retroflexus growth. Treatments T2, T3, and T4 all induced the allelopathic promotion of the growth of the four weeds. The order of the effects of the corn straw water extracts on the comprehensive allelopathy index of the four weed seeds was as follows: C. album > S. viridis > A. retroflexus > E. crus-galli. With an increase in the corn straw mulching amount, the density and total coverage of the four weeds showed a gradual downward trend, whereas the plant control effect and fresh weight control effect showed a gradual upward trend. All indices showed the best results under 12,000 kg/ha of mulching and returning to the field. Overall, corn straw coverage significantly impacted the net photosynthetic rate and transpiration rate of foxtail millet and increased the yield of foxtail millet. Under coverages of 6000 and 12,000 kg/ha, the growth of foxtail millet is better. Based on our findings, we recommend a corn straw coverage of 12,000 kg/ha for the allelopathic control of weeds in foxtail millet fields.

3D hydrogel microfibers promote the differentiation of encapsulated neural stem cells and facilitate neuron protection and axon regrowth after complete transactional spinal cord injury

Spinal cord injury (SCI) can cause permanent impairment to motor or sensory functions. Pre-cultured neural stem cell (NSC) hydrogel scaffolds have emerged as a promising approach to treat SCI by promoting anti-inflammatory effects, axon regrowth, and motor function restoration. Here, in this study, we performed a coaxial extrusion process to fabricate a core-shell hydrogel microfiber with high NSC density in the core portion. Oxidized hyaluronic acid, carboxymethyl chitosan, and matrigel blend were used as a matrix for NSC growth and to facilitate the fabrication process. During the in vitro differentiation culture, it was found that NSC microfibers could differentiate into neurons and astrocytes with higher efficiency compared to NSC cultured in petri dishes. Furthermore, during in vivo transplantation, NSC microfibers were coated with polylactic acid nanosheets by electrospinning for reinforcement. The coated NSC nanofibers exhibited higher anti-inflammatory effect and lesion cavity filling rate compared with the control group. Meanwhile, more neuron- and oligodendrocyte-like cells were visualized at the lesion epicenter. Finally, axon regrowth across the whole lesion site was observed, demonstrating that the microfiber could guide renascent axon regrowth. Experiment results indicate that the NSC microfiber is a promising bioactive treatment for complete SCI treatment with superior outcomes.

Exploiting non-permissive CHO cells as a rapid and efficient method for recombinant HSV-1 isolation

Using herpes simplex virus type 1 (HSV-1) as a therapeutic tool has recently emerged as a promising strategy for enhancing the treatment of various cancers, particularly those associated with the nervous system, which is the virus's natural site of infection. These viruses are specifically engineered to infect and eradicate tumor cells while leaving healthy cells unharmed. To introduce targeted mutations in specific viral genes, gene-modification techniques such as shuttle vector homologous recombination are commonly employed. Plaque purification is then utilized to select and purify the recombinant virus from the parental viruses. However, plaque purification becomes problematic when the insertion of the desired gene at the target site hampers progeny virus replication, resulting in a lower titer of cell-released virus than the parental virus. This necessitates a laborious initial screening process using approximately 10–15 tissue culture dishes (10 cm), making plaque purification time-consuming and demanding. Although the recently developed CRISPR-Cas9 system significantly enhances the efficiency of homologous integration and editing precision in viral genes, the purification of recombinant variants remains a tedious task. In this study, we propose a rapid and innovative method that employs non-permissive Chinese hamster ovary (CHO) cells, representing a remarkable improvement over the aforementioned arduous process. With this approach, only 1–2 rounds of plaque purification are required. Our proposed protocol demonstrates great potential as a viable alternative to current methods for isolating and purifying recombinant HSV-1 variants expressing fluorescent reporter genes using CHO cells and plaque assays.

Effect of the Biostimulants of Microbiological Origin on the Entomopathogenic and Plant Parasitic Nematodes from Miscanthus × Giganteus Plantations

Abstract This study aimed to evaluate the effect of biostimulants derived from Streptomyces avermitilis metabolites on entomopathogenic nematodes: Steinernema carpocapsae, Steinernema feltiae, and Heterorhabditis bacteriophora, obtained from the soil of several Miscanthus × giganteus plantations in 2020–2023. The nematodes were isolated, identified, and cultured using live insects (the greater wax moth Galleria mellonella). Three preparations containing biostimulants – Charkor, Stimpo, and Regoplant, were tested for compatibility with entomopathogenic and plant parasitic nematodes. Their effect on nematode survival was evaluated using the Petri dish test. The study showed that the effect of biostimulants on the survival of nematodes depended on the concentration of aversectin contained in the evaluated preparations. Stimpo and Regoplant had an adverse effect on plant parasitic nematodes. The highest G. mellonella mortality was observed at the higher dose of Charkor (0.4%), and the lowest at the lower dose of Regoplant (1.22%). The study showed that the virulence of the nematodes decreased after 48 hours of incubation in Regoplant and Stimpo solutions containing aversectin. The degree of interaction between nematodes depended on the nematode species, trophic preferences, and the concentration of active ingredients in the preparations. This conclusion is crucial for the selection of appropriate types of entomopathogenic nematodes and the application rate of plant parasitic nematodes in the assessment of their short- and long-term spread, persistence, and recycling in field conditions.

The Response of Hormones, Reactive Oxygen Species and Nitric Oxide in the Polyethylene-Glycol-Promoted, Salt–Alkali-Stress-Induced Embryo Germination of Sorbus pohuashanensis

Polyethylene glycol can abrogate plant seed dormancy and alleviate salt–alkali stress damage to plants, but its role in embryonic dormancy abrogation and germination in Sorbus pohuashanensis is not yet clear. The mechanism by which polyethylene glycol promotes the release of embryonic dormancy may be related to the synthesis and metabolism of endogenous hormones, reactive oxygen species and reactive nitrogen. In this article, germination in indoor culture dishes was used, and the most suitable conditions for treating S. pohuashanensis embryos, with polyethylene glycol (PEG) and sodium carbonate (Na2CO3), were selected. Germination was observed and recorded, and related physiological indicators such as endogenous hormones, reactive oxygen species and reactive nitrogen were measured and analyzed to elucidate the mechanism of polyethylene glycol in alleviating salt–alkali stress in S. pohuashanensis embryos. The results showed that soaking seeds in 5% PEG for 5 days is the best condition to promote germination, which can increase the germination rate of embryos under salt–alkali stress by 1–2 times and improve indicators such as germination speed and the germination index. Polyethylene glycol led to an increase in gibberellin (GA), indole-3-acetic acid (IAA), ethylene (ETH), cytokinin (CTK), nitric oxide (NO), soluble protein and soluble sugar in the embryos under salt–alkali stress; increased activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), nitrate reductase (NR) and nitric oxide synthase (NOS) in the embryos; a reduction in the accumulation of abscisic acid (ABA), hydrogen peroxide (H2O2) and malondialdehyde (MDA). Therefore, it is suggested that the inhibitory effect of polyethylene glycol on the salt–alkali-stress-induced germination of S. pohuashanensis embryos is closely related to the response of endogenous hormones, reactive oxygen species and nitric oxide signalling.

First Report of Fusarium vanettenii Causing Fusarium Root Rot in Fatsia japonica in China

Fatsia japonica plays an important role as a commonly used plant in urban landscaping. From 2022 to 2023, a root rot infestation was observed that caused extensive wilting of Fatsia japonica without leaf shedding and eventual death of the plant, severely reducing the ornamental qualities of the plant as well as the vigor of its growth. Fusarium species were isolated from the roots of the affected plants, exhibiting abundant and dense yellow mycelial colonies that proliferated radially from the center of the Petri dishes. Morphological examinations revealed the presence of falciform macro- and microconidia consistent with Fusarium, as well as chlamydospores characterized by their thick walls. For further identification, the amplification and sequencing of the ITS, TEF1 alpha, and RPB2 alpha genes were performed. Finally, healthy Fatsia japonica plants were inoculated with a spore suspension of the pathogen, to confirm that the disease symptoms were compatible with naturally occurring infection. Fusarium vanettenii was identified as the causative agent of Fatsia japonica root rot. To the best of our knowledge, this is the first report of F. vanettenii causing root rot of Fatsia japonica in China.

Lithium Toxicity in Lepidium sativum L. Seedlings: Exploring Li accumulation’s Impact on Germination, Root Growth, and DNA Integrity

The predicted increase in demand for minor metals for modern technologies raises major concerns regarding potential environmental concentration increases. Among the minor metals, lithium (Li) is particularly noteworthy due to growing demand for battery production. Concerns have been raised about the impact on biota of increasing Li concentrations in the environment. To expand the knowledge of the effects of Li on plants, garden cress (Lepidium sativum L.), a model plant for ecotoxicity assay, was tested in a 72 h test in Petri plates. The results showed a stimulation effect of Li at the lowest concentration (Li chloride 10 mg L−1) on seed germination and primary root elongation. Conversely, higher Li concentrations (50 and 150 mg L−1) caused a progressive impairment in both parameters. A genotoxic effect of Li on root cells, evaluated through the alkaline comet assay, was observed at each concentration tested, particularly at 150 mg L−1 Li chloride. Elemental analysis showed that Li accumulated in the seedlings in a dose–concentration relationship, confirming its ability to be readily absorbed and accumulated in plants. Given the likely increase in Li levels in the environment, further research is required to clarify the toxicity mechanisms induced by Li on growth and nucleic acids.

Assessing selection potential for Pacific oyster (Crassostrea gigas) to a North American OsHV-1 μvar: Comparing two experimental assay methods

Ostreid Herpesvirus - 1 is a global threat to Pacific oyster aquaculture as catastrophic farm losses have occurred where OsHV-1 microvariants (μvars) become endemic. Genetic selection has proven effective for increasing survival to OsHV-1 exposure in multiple Pacific oyster breeding programs, but a novel μvar detected in San Diego Bay California has the potential to severely impact commercial aquaculture along the North American Pacific coast as no OsHV-1 μvar selected population is available commercially. Using a Molluscan Broodstock Program population descending from imports originating in the Miyagi prefecture (Japan), we evaluated the potential for genetic selection to improve survival against the San Diego Bay OsHV-1 μvar using a laboratory trial. Two experimental methods were evaluated, a common garden method where all 15 full-sibling families were pooled, and a petri dish method, where each family was confounded within replicate. No striking differences were found between experimental methods, and both experiments displayed high heritability (0.69 and 0.64) of the tested trait, a broad range of estimated breeding values, and a moderate prediction accuracy for the survival trait. A significant genotype-by-environment interaction occurred, highlighting variability in laboratory-based disease challenges, but the two experiments yielded similar results with minor to moderate re-ranking of mean family survival. Overall, the Miyagi population of Pacific oysters curated by the Molluscan Broodstock Program is a promising population to use for artificial selection to increase survival against San Diego Bay OsHV-1 μvar in attempts to obviate farm losses if the virus expands its currently isolated range.

First Report of Kiwifruit Root Rot Caused by Globisporangium spinosum in China.

Kiwifruit is widely cultivated for its high vitamin C content and nutritional value. In January 2022, root rot symptoms were found in about 30% of Actinidia chinensis cv. Jinyan plants grafted on A. deliciosa rootstocks in an orchard located in Sanming (26.32°N, 117.23°E), Fujian Province of China. The affected plants appeared stunted, with brown and decaying roots, some of which were covered with white hyphae. To isolate the pathogen, the surfaces of typical symptomatic roots were sterilized for 30 s using 75% ethanol, followed by four rinses in sterile water, placing on potato dextrose agar (PDA), and incubating away from light at 25°C for 7 days. 16 Globisporangium-like isolates were obtained through hyphal tip isolation, displaying a milky-white appearance with irregular protuberances on the surface, and yellow-white backs with radial fold lines. The isolates were then cultured on corn meal agar for 5 days at 25°C in dark for morphological characteristics. Under microscope, the hyphae appeared as long strips without septa and 4.1 to 8.2 µm wide (average 6.7 µm), containing irregularly sized spherical droplets. Both terminal and intercalary hyphae swellings were observed; these appeared either spherical or subspherical, with some having projections. Their dimensions were 12.3 to 27.6 µm (average 17.3 µm). The oospores were mostly spherical, either plerotic or aplerotic, 11.8 to 22.3 µm wide (average 18.9 µm), with occasional projections. The antheridia were rod-shaped and curved, with one end attached to the oogonia. Amplification of the sequences of internal transcribed spacer (ITS) regions and cytochrome c oxidase subunit I (COI) were conducted using the primers ITS1/ITS4 (White et al. 1990) and OomCoxI-Levlo/OomCoxI-Levup (Robideau et al. 2011), respectively. The sequencing results revealed identical ITS and COI sequences in all 16 isolates. BLASTn analysis of the 969-bp ITS sequence ON202808 showed 99.38-99.59% similarity (965/971bp, 967/971bp) with the KJ162353 and AY598701 sequences from Globisporangium spinosum isolates, while the 700-bp COI sequence ON075783 showed 100% and 99.41% identity (680/680bp, 676/680bp) with the GenBank sequences HQ708835 and HQ708832, respectively, from G. spinosum. Phylogenetic analysis also showed that the obtained isolate (termed MA16) clustered with isolates from G. spinosum on the same evolutionary branch. For pathogenicity testing, four-month-old healthy Jinyan (A. chinensis) plants grown in sterilized media were transferred to sterile petri dishes covered with wet filter paper, and their roots were inoculated with a 5-mm-wide disk of MA16 when cultivated on PDA medium for 5 days. Miliang-1 (A. deliciosa) and Hongyang (A. chinensis) plants were treated similarly. The control groups each included three plants that were inoculated with non-colonized PDA. The plants were kept at 25 °C with a 12-/12-h light/dark cycle for 10 days when the inoculated plants exhibited root rot symptoms similar to those seen in the field, together with rotting and browning of the leaves. The control plants appeared healthy with no symptoms. After re-isolated from infected tissues, the pathogen was verified to be G. spinosum according to its ITS sequence, thus fulfilling the Koch's postulates. Recently, Pythium spinosum has been classified as G. spinosum according to whole-genome sequencing and phylogenomic analysis (Nguyen et al. 2022). Based on the morphological features and pathogenicity results, MA16 was identified as G. spinosum (van der Plaats-Niterink 1981; Huo et al. 2023). This report appears to be the first description of kiwifruit root rots caused by G. spinosum in China, and its identification will assist the development of strategies to counteract the disease.

Identification of Urinary Tract Infection Causing Bacteria from Microbial Colony Images using Deep Learning

Abstract: Diagnosis of microbiological and bacterial infections involves extensive procedures ofsample culture and microscopic examination. The diagnosis process begins with the identification of symptoms followed by collection of test samples in the form of blood,scraps of skin lesions, mucus, sputum, urine etc. The entire process of bacteria routine culture and bacteriological examination may take as long as 10 to 11 days. Due to the long time required for the standard process of species identification, high costs and need of human expertise it is beneficial to use methods that do not rely on conventional methods. In this work we propose a comprehensive Deep Learning basedapproach to identify urinary Tract Infection (UTI) causing bacteria from microbial colony images of bacteria cultured on agar plates. The approach explains the use of VGG-19 and Inception-v3 deep learning architectures, in bacteria identification evaluated on The Annotated Germs for Automated Recognition (AGAR) dataset, an image dataset of microbial colonies cultured on agar plates. The findings affirmed the significant potential of employing deep learning techniques for the identification of microbial colonies and their classification using Petri dish images.

Morphological and Physiological Responses of Different Cotton Genotypes Primed with Salicylic Acid Under Salinity Conditions

This study was conducted as both petri dishes and pot experiments on four different salt-sensitive cotton genotypes (Laser, May 505, May 455 and Selin) in order to investigate the role of exogenous salicylic acid applications in reducing the effects of salt stress. Six saline treatments; 0, 30, 60, 90, 120 mM NaCl were used. Each group divided into three sub-groups (hydo-primed control, 0.5 mM and 1.0 mM SA) on the basis of seed priming treatments. They were applied in three replications according to the randomized block design. In all genotypes, 90 mM and 120 mM salt stress negatively affected germination and seedling development. In salt stress up to 60 mM, it was recommended to May 505 and Selin genotypes with 0.5 mM salicylic acid pre-application to the seeds.

Superabsorbent Seed Coating and Its Impact on Fungicide Efficacy in a Combined Treatment of Barley Seeds

The technology of seed coating with superabsorbent polymer (SAP) has the potential to mitigate the negative impact of drought on seed germination and crop establishment. However, their application on the seed surface can affect the effectiveness of pesticides used for seed treatment in the protection against phytopathogens. In our work, the influence of the Aquaholder®Seed polymer coating on the effectiveness of fungicides in the protection of germinating seeds of spring barley cv. Bojos and Laudis against the fungal pathogen Bipolaris sorokiniana was studied. One-half of the seeds were first treated with fungicides, and then a polymer was applied. Fungicide efficacy was evaluated in a Petri dish test and pot test under the pathogen attack. Seed coating with SAP did not negatively affect fungicide efficacy. The percentage of germinated seeds, seedling emergence, plant height, and symptoms of the disease in the fungicide-treated variants were not significantly changed by the SAP application. Moreover, in cv. Laudis, the application of SAP alone partially protected germinating seeds against pathogen attack. The amount of pathogen DNA in plant tissues of cv. Laudis was not significantly different among seed treatments, while in cv. Bojos, the pathogen DNA increased in seeds coated with SAP alone but decreased in combined treatment with fungicides. These results demonstrated that SAP seed coating does not negatively affect the efficacy of fungicides used for seed protection against fungal pathogens.

Drosophila hydei as a potential vector of Ceratocystis fimbriata, the causal agent of sweetpotato black rot, in storage facilities.

Ceratocystis fimbriata, the causal agent of sweetpotato black rot, is a pathogen capable of developing and spreading within postharvest settings. A survey of North Carolina sweetpotato storage facilities was conducted to determine the arthropods present and identify potential vectors of C. fimbriata. Sixteen taxonomic categories were recovered and the genus Drosophila (Diptera: Drosophilidae) accounted for 79% of individuals sampled with Drosophila hydei (Sturtevant) being the most abundant species. Behavioral assays were conducted to determine if D. hydei is attracted to C. fimbriata inoculated roots and if the pathogen could be recovered from external or internal surfaces of the insect. Flies were released in insect trapping pitchers containing either C. fimbriata inoculated or non-inoculated roots or Petri dishes. No significant differences in fly number were detected in sweetpotato-baited pitchers; however, significant differences were found in the pitcher baited with a mature C. fimbriata culture. Flies were subjected to washes to determine if viable C. fimbriata was present (internally or externally); washes were plated onto carrot agar plates and observed for the presence of C. fimbriata colonies. Both external and internal washes had viable C. fimbriata inoculum with no significant differences, and inoculated sweetpotatoes had a significantly higher number of flies carrying C. fimbriata. This study suggests that D. hydei can carry C. fimbriata from infected sweetpotatoes and move viable C. fimbriata inoculum both externally and internally, making this the first report of any Drosophila spp. serving as a potential vector for the Ceratocystis genus.

First Report of Alternaria alternata Causing Leaf Spot on Smooth Bromegrass (Bromus inermis Leyss.) in China.

Smooth bromegrass (Bromus inermis Leyss.) is an important forage crop in northern China. In July 2021, leaf spot symptoms were observed on smooth bromegrass in Ewenki Banner, Hulunbuir, Inner Mongolia. In an area of approximately 0.12 hectares, 95% disease incidence was observed. Ten diseased plants were collected for pathogen isolation. Leaf tissues near the lesions were cut into 5 × 5 mm pieces, surface-disinfested in 75% ethanol for 3 min, and rinsed with sterile distilled water. The pieces were placed on water agar in petri plates and incubated at 25℃ for three days. The resulting colonies were flushed with sterile water and a spore suspension was serially diluted and plated on potato dextrose agar (PDA). A single-spore colony was obtained. Ten isolates were obtained and designated HE1 to HE10. The colony morphology was identical for all isolates, grayish white in color on the upper surface and light black on the underside. The mycelia were light gray and velvety. Conidia were light brown to brown in color and oblate, oblong or oval. The conidial dimensions were typically between 15 to 43 μm by 8 to 9 μm in size. The conidia possessed one to six transverse septa, with slight to distinct constrictions at each division, and zero to two longitudinal septa. These morphological characteristics resembled Alternaria alternata (Fr.) Keissl.. DNA was extracted from three isolates, HE3, HE4 and HE5, using the CTAB method. Polymerase chain reaction (PCR) was performed on the extracted DNA with a set of primers ITS1/ITS4, H31a/H31b, gpd1/gpd2, TEF1-728F/TEF1-986R, and RPB2-5F2/fRPB2-7cR. The amplicon sequences from the three isolates were analyzed using the BLAST in GenBank (https://www.ncbi.nlm.nih.gov/). The results showed a high sequence identity, ranging from 99 to 100%, with the A. alternata strain YTMZ-20-2 across all the genetic markers tested. The strong match reinforced the identification of the strains as A. alternata. The sequences were deposited in GenBank (Table S1). The three fungal isolates were identified as A. alternata based on their morphological and genetic data. To conduct Koch's postulates, the representative isolate HE4 was used. Smooth bromegrass seed was soaked in water for four days and sown in potting soil contained in plastic pots (10 cm diameter × 15 cm height, five seeds/pot) in a greenhouse under a 16-h photoperiod at temperatures between 20 to 25°C and 60% relative humidity. When the plants reached a height of approximately 20 cm, the plants in three pots (replicates) were sprayed with a spore suspension (106 conidial/ml) at 10 ml/pot, and three pots were sprayed with sterile water for control. Five days after inoculation, the plants exhibited leaf spot symptoms similar to those previously described, while the control plants remained unaffected. The causative fungus was successfully re-isolated from the diseased plants and confirmed morphologically and molecularly on its identity as described above. This experiment was independently conducted three times. This is the first report of A. alternata causing leaf spot on smooth bromegrass in China. Since there is risk that the disease could seriously reduce the yield of the forage crop smooth bromegrass, further research is needed.

Biological control potential of two Beauveria bassiana isolates against the stink bugs Nezara viridula L. and Piezodorus guildinii Westwood (Hemiptera: Pentatomidae) in common bean

BackgroundThe stink bugs, Nezara viridula L. and Piezodorus guildinii Westwood (Hemiptera: Pentatomidae) are the most important and widespread species of polyphagous stink bugs in the tropical and subtropical regions of Latin America, which affect the quality and yield of the common bean (Phaseolus vulgaris L.). The use of synthetic chemical insecticides is the major control strategy to manage stink bugs in common beans and alternatives are needed. In this study, mortality and median Lethal Time (LT50) of two Cuban isolates of the entomopathogenic fungus Beauveria bassiana (18 S-O-R and 96 P-O-E), as well as one commercial Cuban isolate (Bb-18), at a concentration of 1 × 108 conidia/ml were evaluated. These evaluations were conducted against both stink bug species using Petri dish bioassays and a semi-field experiment in common beans.ResultsIn Petri dish bioassays, the isolates 18 S-O-R and 96 P-O-E caused 100% mortality of both N. viridula and P. guildinii. This was significantly higher than for isolate Bb-18, which caused 86.3% N. viridula and 81.3% P. guildinii mortality. In the semi-field experiment, when pooling both stink bug species, total mortality after 14 days was 91.3% for 18 S-O-R, 80.0% for 96 P-O-E and 73.8% for Bb-18 isolates. LT50 value for isolate 18 S-O-R tested under laboratory conditions was 6.04 ± 0.18 days for N. viridula and 5.32 ± 0.14 days for P. guildinii at the same concentration of 1 × 108 conidia/ml. LT50 value for isolate 18 S-O-R in semi field was 6.79 ± 0.37 days for N. viridula and 7.71 ± 0.32 days for P. guildinii at 1 × 108 conidia/ml.ConclusionThe study highlights the potential of B. bassiana 18 S-O-R as a promising candidate for control of stink bugs in common bean under tropical conditions as an alternative to conventional chemical insecticides in integrated pest management (IPM) programs. Moving forward, further research should focus on validating the efficacy under diverse field conditions and integrating application methods into practical IPM approaches. Future use of B. bassiana will enhance sustainability and reduce environmental impacts associated with pesticide use.

Derivation and long-term maintenance of porcine skeletal muscle progenitor cells

Culture of muscle cells from livestock species has typically involved laborious enzyme-based approaches that yield heterogeneous populations with limited proliferative and myogenic differentiation capacity, thus limiting their use in physiologically-meaningful studies. This study reports the use of a simple explant culture technique to derive progenitor cell populations from porcine muscle that could be maintained and differentiated long-term in culture. Fragments of semitendinosus muscle from 4 to 8 week-old piglets (n = 4) were seeded on matrigel coated culture dishes to stimulate migration of muscle-derived progenitor cells (MDPCs). Cell outgrowths appeared within a few days and were serially passaged and characterised using RT-qPCR, immunostaining and flow cytometry. MDPCs had an initial mean doubling time of 1.4 days which increased to 2.5 days by passage 14. MDPC populations displayed steady levels of the lineage-specific markers, PAX7 and MYOD, up until at least passage 2 (positive immunostaining in about 40% cells for each gene), after which the expression of myogenic markers decreased gradually. Remarkably, MDPCs were able to readily generate myotubes in culture up until passage 8. Moreover, a decrease in myogenic capacity during serial passaging was concomitant with a gradual increase in the expression of the pre-adipocyte markers, CD105 and PDGFRA, and an increase in the ability of MDPCs to differentiate into adipocytes. In conclusion, explant culture provided a simple and efficient method to harvest enriched myogenic progenitors from pig skeletal muscle which could be maintained long-term and differentiated in vitro, thus providing a suitable system for studies on porcine muscle biology and applications in the expanding field of cultured meat.

Establishment of a novel microfluidic co-culture system for simultaneous analysis of multiple indicators of gefitinib sensitivity in colorectal cancer cells.

The therapeutic effect of gefitinib on colorectal cancer (CRC) is unclear, but it has been reported that stromal cells in the tumor microenvironment may have an impact on drug sensitivity. Herein, we established a microfluidic co-culture system and explored the sensitivity of CRC cells co-cultured with cancer-associated fibroblasts (CAFs) to gefitinib. The system consisted of a multichannel chip and a Petri dish. The chambers in the chip and dish were designed to continuously supply nutrients for long-term cell survival and create chemokine gradients for driving cell invasion without any external equipment. Using this system, the proliferation and invasiveness of cells were simultaneously evaluated by quantifying the area of cells and the migration distance of cells. In addition, the system combined with live cell workstation could evaluate the dynamic drug response of co-cultured cells and track individual cell trajectories in real-time. When CRC cells were co-cultured with CAFs, CAFs promoted CRC cell proliferation and invasion and reduced the sensitivity of cells to gefitinib through the exosomes secreted by CAFs. Furthermore, the cells that migrated out of the chip were collected, and EMT-related markers were determined by immunofluorescent and western blot assays. The results demonstrated that CAFs affected the response of CRC cells to gefitinib by inducing EMT, providing new ideas for further research on the resistance mechanism of gefitinib. This suggests that targeting CAFs or exosomes might be a new approach to enhance CRC sensitivity to gefitinib, and our system could be a novel platform for investigating the crosstalk between tumor cells and CAFs and understanding multiple biological changes of the tumor cells in the tumor microenvironment.

Effect of Mammea americana Aqueous Extract on the Control of the Coffee Red Spider Mite

The coffee red spider mite, Oligonychus ilicis, stands out as one of the main pests of Conilon coffee. The use of synthetic chemical control has led to an increase in the population of this mite and a reduction in its natural enemies. Aiming to find viable alternatives, this study aimed to evaluate the acaricidal potential of “abricó” peel extract, Mammea americana, in the management of O. ilicis. Each treatment consisted of 7 repetitions, with 10 individuals per repetition. They were placed in Petri dishes (10.0 x 1.2 cm), containing coffee leaf disks (4 cm diameter) inside, and moistened cotton at the bottom of the dish and around the disks. The peels of M. americana were dried, crushed, and diluted in aqueous solution to obtain extracts at concentrations of 2; 2.57; 3.30; 4.24; 5.45; and 7 %. The application was done by spraying, using an Alfa 2 model airbrush connected to a compressor calibrated with a constant pressure of 1.3 psi. 1 mL of solution of each concentration was applied for each repetition. Distilled water and the surfactant Tween® 80 (0.05 % v/v) were used as control treatments. The acaricidal effect was evaluated 24, 48, and 72 hours after spraying, and the lethal concentrations LC50 and LC90 were calculated. The results of the toxicity tests indicated an increase in the mortality of O. ilicis with the increase in the concentrations of aqueous extracts of M. americana, resulting in 100 % mortality. The LC90 was estimated at 3.83 %. In the chemical analysis of the M. americana extract, the presence of phenolic acids, tannins, and flavonoids was observed at concentrations of 39.150, 40.783, and 0.0349 mg-1, respectively. We conclude that the aqueous extract of M. americana peels demonstrated acaricidal potential in a controlled environment for the coffee red spider mite.