Ng μL Research Articles

Effects of energy-gathered and energy-divergent ultrasound on structure, DNA extraction and adulterated quantification of sweet potato vermicelli and its starch.

The identification of adulterated sweet potato vermicelli faces significant challenges, seriously hindering the development of the vermicelli industry. Herein, we investigate effects of energy-gathered ultrasound (EGU) and energy-divergent ultrasound (EDU) (30, 40 and 50 W L-1) on structure, DNA extraction and adulterated quantification of sweet potato vermicelli and its starch, thereby exploring their potential in adulteration of sweet potato vermicelli. EGU-assisted modified cetyltrimethylammonium bromide (CTAB) protocol with β-mercaptoethanol significantly improved DNA extraction from sweet potato vermicelli (223.7-249.2 ng μL-1) and its starch (133.4-186.4 ng μL-1), followed by EDU-assisted DNA extraction from sweet potato vermicelli (115.1-209.3 ng μL-1) and its starch (33.4-61.0 ng μL-1). Both EGU and EDU treatments resulted in the destruction of microstructure and crystalline structure, as well as changes in pasting and thermal properties of sweet potato vermicelli and its starch. Real-time polymerase chain reaction (PCR) amplification results revealed that EGU and EDU enhanced the efficiency of DNA amplification, and EDU showed smaller cycle threshold (Ct) values than EGU. In addition, EDU-assisted CTAB protocol combined with real-time PCR could detect levels of less than 1% of cassava and maize starches in sweet potato vermicelli. EDU-assisted CTAB protocol combined with real-time PCR shows promise as a sensitive and reliable analytical tool for vermicelli adulteration. © 2024 Society of Chemical Industry.

Probe-Based Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Two Clarireedia spp., the Causal Agent of Dollar Spot of Turfgrass.

Dollar spot is a major fungal disease affecting turfgrass worldwide and can quickly destroy turfgrass swards. An assimilating probe-based loop-mediated isothermal amplification (LAMP) assay was developed to detect Clarireedia monteithiana and C. jacksonii, the causal agents of dollar spot within the continental United States. Five LAMP primers were designed to target the calmodulin gene with the addition of a 6-carboxyl-fluorescein florescent assimilating probe, and the temperature amplification was optimized for C. jacksonii and C. monteithiana identification. The minimum amount of purified DNA needed for detection was 0.05 ng μl-1. Specificity assays against host DNA and other turfgrass pathogens were negative. Successful LAMP amplification was also observed for dollar spot-infected turfgrass field samples. Further, a DNA extraction technique via rapid heat-chill cycles and visualization of LAMP results via a florescent flashlight was developed and adapted for fast, simple, and reliable detection in 1.25 h. This assimilating probe-based LAMP assay has proved successful as a rapid, sensitive, and specific method for the detection of C. monteithiana and C. jacksonii in pure cultures and from symptomatic turfgrass leaves blades. The assay represents a promising technology to be used in the field for on-site, point-of-care pathogen detection.

Establishment of transient and stable gene transformation systems in medicinal woody plant Acanthopanax senticosus

BackgroundTransient and stable gene transformation systems play a crucial role in elucidating gene functions and driving genetic improvement in plants. However, their application in medicinal woody plants has been hampered by inefficient procedures for isolating protoplasts and regenerating plants in vitro.ResultsEmbryogenic callus protoplast isolation and transient transformation system were successfully established. The highest yield of protoplasts was approximately 1.88 × 106 cells per gram with a viability of 90% under the combination of 1.5% cellulase and 0.2% macerozyme, with enzymatic digestion for 6 h in darkness followed by centrifugation at 400×g for 5 min. The transient transfection rate of protoplast reached 45.56% at a PEG 4000 concentration of 40%, a transfection time of 40 min, 16 h of dark incubation, a plasmid concentration of 1.5 ng μL−1, and 25 min heat shock at 45 °C. In addition, 15 Agrobacterium tumefaciens-mediated GUS-positive seedlings were obtained through the somatic embryogenetic pathway under the optimized conditions.ConclusionThis study successfully established both transient and stable genetic transformation systems, paving the way for future molecular biology research on A. senticosus.Graphical

RNA interference-based strategies to control Botrytis cinerea infection in cultivated strawberry

Key messageGene silencing of BcDCL genes improves gray mold disease control in the cultivated strawberry.Gene silencing technology offers new opportunities to develop new formulations or new pathogen-resistant plants for reducing impacts of agricultural systems. Recent studies offered the proof of concept that the symptoms of gray mold can be reduced by downregulating Dicer-like 1 (DCL1) and 2 (DCL2) genes of Botrytis cinerea. In this study, we demonstrate that both solutions based on dsRNA topical treatment and in planta expression targeting BcDCL1 and BcDCL2 genes can be used to control the strawberry gray mold, the most harmful disease for different fruit crops. 50, 70 and 100 ng μL−1 of naked BcDCL1/2 dsRNA, sprayed on plants of Fragaria x ananassa cultivar Romina in the greenhouse, displayed significant reduction of susceptibility, compared to the negative controls, but to a lesser extent than the chemical fungicide. Three independent lines of Romina cultivar were confirmed for their stable expression of the hairpin gene construct that targets the Bc-DCL1 and 2 sequences (hp-Bc-DCL1/2), and for the production of hp construct-derived siRNAs, by qRT-PCR and Northern blot analyses. In vitro and in vivo detached leaves, and fruits from the hp-Bc-DCL1/2 lines showed significantly enhanced tolerance to this fungal pathogen compared to the control. This decreased susceptibility was correlated to the reduced fungal biomass and the downregulation of the Bc-DCL1 and 2 genes in B. cinerea. These results confirm the potential of both RNAi-based products and plants for protecting the cultivated strawberry from B. cinerea infection, reducing the impact of chemical pesticides on the environment and the health of consumers.

Revealing the effects of amino acid, organic acid, and phytohormones on the germination of tomato seeds under salinity stress.

Salinity accumulation poses a threat to the production and productivity of economically important crops such as tomatoes (Solanum lycopersicum L.). Currently, salt tolerance breeding programs have been limited by insufficient genetic and physiological knowledge of tolerance-related traits and a lack of an efficient selection domain. For that purpose, we aimed to determine the ability of tomato cultivars to tolerate salt based on seed traits by multiple biochemical pathways. First, we tested three tomato cultivars according to their response to different sodium chloride (NaCl) concentrations (0, 6.3, 9.8, 13.0, and 15.8 dS m-1) and then we analysed their amino acids, organic acids, and phytohormones. Considering the results of germination traits, it is possible to conclude that cultivar H-2274 was more tolerant to salt stress than others. As a result, multivariate discriminant analysis including principal component analysis and two-way hierarchical clustering analyses were constructed and demonstrated that tomato cultivars were separated from each other by the amino acid, organic acid, and phytohormone contents. Considering germination traits of tomato seeds, cv. 'H-2274' was more tolerant to salinity than others depending on high proline (29 pmol µl-1) and citric acid (568 ng µl-1) assays. Biochemical variability offers a valuable tool for investigating salt tolerance mechanisms in tomatoes, and it will be appreciated to find high-tolerant tomato cultivar(s) to saline conditions. Also, the findings of this study have significant potential for practical applications in agriculture, particularly in developing salt-tolerant tomato cultivars to enhance productivity in saline environments and address socio-economic challenges.

Comparative Evaluation of PCR-Based, LAMP and RPA-CRISPR/Cas12a Assays for the Rapid Detection of Diaporthe aspalathi.

Southern stem canker (SSC) of soybean, attributable to the fungal pathogen Diaporthe aspalathi, results in considerable losses of soybean in the field and has damaged production in several of the main soybean-producing countries worldwide. Early and precise identification of the causal pathogen is imperative for effective disease management. In this study, we performed an RPA-CRISPR/Cas12a, as well as LAMP, PCR and real-time PCR assays to verify and compare their sensitivity, specificity and simplicity and the practicality of the reactions. We screened crRNAs targeting a specific single-copy gene, and optimized the reagent concentrations, incubation temperatures and times for the conventional PCR, real-time PCR, LAMP, RPA and Cas12a cleavage stages for the detection of D. aspalathi. In comparison with the PCR-based assays, two thermostatic detection technologies, LAMP and RPA-CRISPR/Cas12a, led to higher specificity and sensitivity. The sensitivity of the LAMP assay could reach 0.01 ng μL-1 genomic DNA, and was 10 times more sensitive than real-time PCR (0.1 ng μL-1) and 100 times more sensitive than conventional PCR assay (1.0 ng μL-1); the reaction was completed within 1 h. The sensitivity of the RPA-CRISPR/Cas12a assay reached 0.1 ng μL-1 genomic DNA, and was 10 times more sensitive than conventional PCR (1.0 ng μL-1), with a 30 min reaction time. Furthermore, the feasibility of the two thermostatic methods was validated using infected soybean leaf and seeding samples. The rapid, visual one-pot detection assay developed could be operated by non-expert personnel without specialized equipment. This study provides a valuable diagnostic platform for the on-site detection of SSC or for use in resource-limited areas.

Localized Surface Plasmon Resonance Optical Biosensor for Simple Detection of Deoxyribonucleic Acid Mismatches

Optical biosensors are optical technologies that evaluate changes in the refractive index as they monitor non‐covalent molecular interactions in real time. These make use of unsophisticated, label‐free analytical approaches, which do not require dyes to produce a visible signal. In this study, the efficiency of localized surface plasmon resonance (LSPR) biosensor in detecting a single nucleotide mismatch in deoxyribonucleic acid is examined. The detection is based on the hybridization of a target DNA at 100 ng μL−1 with a complementary biotinylated probe as well as a partially complementary biotinylated with one nucleotide mismatch probe on a gold‐coated surface. Both probes are used at a concentration of 0.1 μm. The LSPR exhibited sensitivity by differentiating sample M+ from sample C+ through varying transmission intensities of 0.28 and 0.26 μA, respectively. Based on these findings, this approach demonstrates a great potential due to its ability to distinguish samples that differ with a single base pair, and its efficiency will be explored in the development of a point‐of‐care device as a simpler and cost‐effective approach for detection of various biologically and medically significant mutations such as antimicrobial resistance mutations. More work is underway to determine the robustness of the LSPR biosensor using the biotin–neutravidin approach.

Isopropanol-promoted DNA extraction by polydopamine functionalized magnetic particles based on metal coordination

High-quality DNA is an important guarantee to start downstream experiments in many biological and medical research areas. Magnetic particle-based DNA extraction methods from blood mainly depend on electrostatic adsorption in a low-pH environment. However, the strong acidic environment can influence the DNA stability. Herein, a polydopamine-functionalized magnetic particle (PDA@Fe3O4)-based protocol was developed for DNA extraction from whole blood samples. In the protocol, Mg2+ and Ca2+ were utilized to bridge the adsorption of DNA by PDA@Fe3O4 via the metal-mediated coordination. Isopropanol was found to efficiently promote DNA adsorption by triggering the change of the conformation of DNA from B-form to more compact A-form. In 50 % isopropanol solution, the DNA adsorption efficiency was nearly 100 % in the presence of 0.5 mM Ca2+ or 1.5 mM Mg2+. The role of metal ions and isopropanol in DNA adsorption was explored. The protocol averts the strong acidic environment and PCR inhibitors, such as high concentrations of salt or polyethylene glycol. It demonstrates superiority in DNA yield (59.13 ± 3.63 ng μL−1) over the commercial kit (27.33 ± 4.98 ng μL−1) and phenol-chloroform methods (37.90 ± 0.47 ng μL−1). In addition, to simplify the operastion, an automated nucleic acid extraction device was designed and fabricated to extract whole genomic DNA from blood. The feasibility of the device was verified by extracting DNA from cattle and pig blood samples. The extracted DNA was successfully applied to discriminate the beef authenticity by a duplex PCR system. The results demonstrate that the DNA extraction protocol and the automated device have great potential in blood samples.

Universal and Naked-Eye Diagnostic Platform for Emetic Bacillus cereus Based on RPA-Assisted CRISPR/Cas12a.

Emetic Bacillus cereus (B. cereus), which can cause emetic food poisoning and in some cases even fulminant liver failure and death, has aroused widespread concern. Herein, a universal and naked-eye diagnostic platform for emetic B. cereus based on recombinase polymerase amplification (RPA)-assisted CRISPR/Cas12a was developed by targeting the cereulide synthetase biosynthetic gene (cesB). The diagnostic platform enabled one-pot detection by adding components at the bottom and cap of the tube separately. The visual limit of detection of RPA-CRISPR/Cas12a for gDNA and cells of emetic B. cereus was 10-2 ng μL-1 and 102 CFU mL-1, respectively. Meanwhile, it maintained the same sensitivity in the rice, milk, and cooked meat samples even if the gDNA was extracted by simple boiling. The whole detection process can be finished within 40 min, and the single cell of emetic B. cereus was able to be recognized through enrichment for 2-5 h. The good specificity, high sensitivity, rapidity, and simplicity of the RPA-assisted CRISPR/Cas12a diagnostic platform made it serve as a potential tool for the on-site detection of emetic B. cereus in food matrices. In addition, the RPA-assisted CRISPR/Cas12a assay is the first application in emetic B. cereus detection.

Molecular characterization of Indian races of Fusarium oxysporum f. sp. lentis (Fol) based on secreted in Xylem (SIX) effector genes and development of a SIX11 gene-based molecular marker for specific detection of Fol.

Fusarium wilt of lentil caused by Fusarium oxysporum f. sp. lentis (Fol) is a destructive pathogen limiting lentil production in India. In the present study, Secreted in Xylem (SIX) effectors genes were explored in Indian races of Fol and also a diagnostic tool for reliable detection of the disease was developed. Four SIX effectors genes, SIX11, SIX13, SIX6 and SIX2 were identified in 12 isolates of Fol belonging to seven races. SIX11 was present in all the races while SIX 13 was absent in race 6 and SIX6 was present only in race 4. The phylogenetic analysis revealed the conserved nature of the SIX genes within the forma specialis and showed sequence homology with F. oxysporum f. sp. pisi. The presence of three effectors, SIX11, SIX13 and SIX6 in race 4 correlates with high disease incidence in lentil germplasms. The in-silico characterization revealed the presence of signal peptide and localization of the effectors. Further SIX11 effector gene present in all the isolates was used to develop Fol-specific molecular marker for accurate detection. The marker developed could differentiate F. oxysporum f. sp. lycopersici, F. solani, F. oxysporum, Rhizoctonia solani and Sclerotium rolfsii and had a detection limit of 0.01ng μL- 1. The effector-based marker detection helps in the unambiguous detection of the pathogen under field conditions.

Development and validation of a versatile non-invasive urinary steroidomics method for wildlife biomonitoring

Wildlife conservation is often challenged by a lack of knowledge about the reproduction biology and adaptability of endangered species. Although monitoring steroids and related molecules can increase this knowledge, the applicability of current techniques (e.g. immunoassays) is hampered by species-specific steroid metabolism and the requisite to avoid invasive sampling. This study presents a validated steroidomics method for the (un)targeted screening of a wide range of sex and stress steroids and related molecules in urine using ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). In total, 50 steroids (conjugated and non-conjugated androgens, estrogens, progestogens and glucocorticoids) and 6 prostaglandins could be uniquely detected. A total of 45 out of 56 compounds demonstrated a detection limit below 0.01 ng μL−1. Excellent linearity (R2 > 0.99), precision (CV < 20 %), and recovery (80–120 %) were observed for 46, 41, and 39 compounds, respectively. Untargeted screening of pooled giant panda and human samples yielded 9691 and 8366 features with CV < 30 %, from which 84.1 % and 83.0 %, respectively, also demonstrated excellent linearity (R2 > 0.90). The biological validity of the method was investigated on male and female giant panda urine (n = 20), as well as pooled human samples (n = 10). A total of 24 different steroids were detected with clear qualitative and quantitative differences between human and giant panda samples. Furthermore, expected differences were revealed between female giant panda samples from different reproductive phases. In contrast to traditional biomonitoring techniques, the developed steroidomics method was able to screen a wide range of compounds and provide information on the putative identities of metabolites potentially important for reproductive monitoring in giant pandas. These results illustrate the advancements steroidomics brings to the field of wildlife biomonitoring in the pursuit to better understand the biology of endangered species.

Application of recombinase polymerase amplification with CRISPR/Cas12a and multienzyme isothermal rapid amplification with lateral flow dipstick assay for Bactrocera correcta.

Bactrocera correcta is a quarantine pest that negatively impacts the fruit and vegetable industry. Differentiating B. correcta from similar species, especially in non-adult stages, remains challenging. Rapid molecular identification techniques, such as recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a and multienzyme isothermal rapid amplification with lateral flow dipstick (MIRA-LFD), play a crucial role in early monitoring and safeguarding agricultural production. Our study introduces two methods for the rapid visual identification of B. correcta. Bactrocera correcta specific RPA primers, CRISPR RNA (crRNA), and the LFD probe were designed based on the cox1 genes. The RPA reaction conditions were optimized (at 37 °C for 8 min) for effective template DNA amplification. Two nucleic acid detection methods were established to visualize RPA. In the RPA-CRISPR/Cas12a system, the optimal LbCas12a/crRNA concentration ratio was 200:400 nmol L-1. Successful amplification was determined by the presence or absence of green fluorescence following 15 min incubation at 37 °C. The MIRA-LFD system achieved precise identification of the target species within 4 min at 37 °C. Both methods exhibited high specificity and sensitivity, allowing for detection from 1.0 × 10-1 ng μL-1 of DNA. Combined with rapid DNA extraction, rapid identification of individual B. correcta at different developmental stages was achieved, enhancing the practicality and convenience of the established methods. Our research findings demonstrate that both the RPA-CRISPR/Cas12a and MIRA-LFD methods for B. correcta detection was accurate and rapid (within 30 min and 10 min, respectively), at 37 °C. Our methods do not rely on expensive equipment, thus possess high practical value, providing improved identification solutions for port quarantine pests and field applications. © 2024 Society of Chemical Industry.

Resistance to both aphids and nematodes in tobacco plants expressing a Bacillus thuringiensis crystal protein.

Bacillus thuringiensis (Bt) and its crystal toxin or δ-endotoxins (Cry) offer great potential for the efficient control of crop pests. A vast number of pests can potentially infect the same host plant, either simultaneously or sequentially. However, no effective Bt-Cry protein has been reported to control both aphids and plant parasitic nematodes due to its highly specific activity. Our study indicated that the Cry5Ba2 protein was toxic to the green peach aphid Myzus persicae, which had a median lethal concentration (LC50) of 9.7 ng μL-1 and fiducial limits of 3.1-34.6 ng μL-1. Immunohistochemical localization of Cry5Ba2 revealed that it could bind to the apical tip of microvilli in midgut regions. Moreover, transgenic tobacco plants expressing Cry5Ba2 exhibited significant resistance to Myzus persicae, as evidenced by reduced insect survival and impaired fecundity, and also intoxicated the Meloidogyne incognita as indicated by a decrease in galls and progeny reproduction. In sum, we identified a new aphicidal Bt toxin resource that could simultaneously control both aboveground and belowground pests, thus extending the application range of Bt-based strategy for crop protection. © 2024 Society of Chemical Industry.

Development of a high-throughput DNA isolation method for livestock and poultry meat based on mesoporous metal-organic framework-coated solid-phase microextraction device

A rapid, portable, simple, low-cost, and high-throughput DNA isolation method was developed for the quantitative identification of meat adulteration based on mesoporous UiO-66 coated solid phase microextraction (SPME) devices. The high-throughput DNA extraction system consists of 96 UiO-66 SPME devices and a polyformaldehyde plate, which can extract DNA from 96 livestock and poultry meat samples simultaneously. At the same time, several parameters of the high throughput DNA isolation method were optimized, including lysis buffer volume, elution buffer volume, lysis time and elution time, etc. Compared with the single DNA isolation device, the 96-SPME high-throughput extraction system maintains the original good universality, reproducibility, chemical stability, and reusability. On this basis, it obtained higher DNA concentration (80 ± 34 ng μL−1), satisfactory DNA purity (A260/A280=2.4) and shorter extraction time (less than 7.2 s per sample), which are much more convenient and efficient than the conventional kit method and the single SPME DNA isolation method. In addition, the conventional multiplex polymerase chain reaction and real-time polymerase chain reaction techniques were used for the qualitative and quantitative detection of meat adulteration. The DNA obtained from this extraction method is highly reproducible and sensitive for the identification of duck ingredients in beef, with a detection range of 1–100 % and a minimum detection limit of 1 % (w/w).

Cross-pathogenicity of Phytophthora palmivora associated with bud rot disease of oil palm and development of biomarkers for detection.

Phytophthora palmivora has caused disease in many crops including oil palm in the South America region. The pathogen has had a significant economic impact on oil palm cultivation in Colombia, and therefore poses a threat to oil palm cultivation in other regions of the World, especially in Southeast Asia, the largest producer of the crop. This study aimed to look at the ability of isolates from Malaysia, Colombia, and other regions to cross-infect Malaysian oil palm, durian, and cocoa and to develop specific biomarkers and assays for identification, detection, and diagnosis of P. palmivora as a key component for the oil palm biosecurity continuum in order to contain the disease especially at the ports of entry. We have developed specific molecular biomarkers to identify and detect Phytophthora palmivora using polymerase chain reaction (PCR) and real-time loop mediated isothermal amplification (rt-LAMP) in various sample types such as soil and plants. The limit of detection (DNA template, pure culture assay) for the PCR assay is 5.94 × 10-2 ng µl-1 and for rt-LAMP is 9.28 × 10-4 ng µl-1. Diagnosis using rt-LAMP can be achieved within 30min of incubation. In addition, PCR primer pair AV3F/AV3R developed successfully distinguished the Colombian and Malaysian P. palmivora isolates.

An experimental study on the visual identification of Fritillaria ussuriensis based on LAMP and nucleic acid colloidal gold technique

Fritillaria ussuriensis Maxim is one of the traditional Chinese valuable herbs, which is the dried bulb of Fritillaria, a plant of the lily family. The identification of authenticity about F. ussuriensis is still technically challenging. In this study, visual identification was performed by ring-mediated isothermal amplification and nucleic acid colloidal gold techniques. Firstly, multiple sequence comparative analysis was performed by DNAMAN to find the differential sites of F. ussuriensis and its mixed pseudo-products, and the specific identification primers of F. ussuriensis were designed. Genomic DNA was extracted by the modified CTAB method, and the reaction system and reaction conditions were optimized to construct LAMP for the visual detection of F. ussuriensis, meanwhile, the genuine product was cloned and the extracted plasmid was sequenced. The specificity and sensitivity were detected, and also verified by nucleic acid colloidal gold method, and 20 commercially available samples were tested. The extracted DNA met the requirements of the experiment, and the genuine F. ussuriensis PCR product titrated on a test strip showed two bands on the T and C lines, while the counterfeit and negative control showed only one band on the C line, which matched the LAMP results. The specificity was 100 %, and the sensitivity of LAMP assay was up to 0.01 ng μL−1, while that of colloidal gold assay was 0.1 ng μL−1, thus the LAMP assay had high sensitivity. 14 out of 20 commercially available samples of F. ussuriensis were qualified, and 6 were unqualified, and the results of the two methods of identification were consistent. In this study, the combined detection method of LAMP and colloidal gold for nucleic acid was established to be specific, rapid, precise and visualized, which can provide a new technical idea for the detection of F. ussuriensis.

Assessment of four different DNA extraction methodologies for the molecular detection of phage λ and Bacillus sp. in raw bovine milk samples

Microbiological analysis of milk is essential to guarantee the quality and safety of dairy products. Molecular methods based on identifying nucleic acids, such as PCR, detect a diversity of microorganisms; however, inhibitors such as fats make milk a complex matrix for extracting DNA. In this study, different DNA extraction methodologies were compared for the molecular detection of microorganisms in raw bovine milk samples, evaluating performance through the concentration (ng μL−1) and purity (index 260/280) of the DNA extracted from each milk sample by spectrophotometry, gel electrophoresis, and PCR amplification. The results demonstrated that the commercial QIAamp DNA mini kit method is significantly better when compared with MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit Thermo Scientific ™, salting out, and Chelex™ 100 in terms of concentration and quality.

Antimalarial and Antileishmanial Flavonoids from Calendula officinalis Flowers

Calendula officinalis L. (Asteraceae), commonly known as English or pot marigold, is an herbaceous plant with edible flowers. In this study, UPLC-ESI-MS/MS analysis was used for tentative identification of compounds in marigold flower methanol extract (MFE). In addition, RP-HPLC-DAD analysis was used to quantify the flavonoids hesperidin and rutin in MFE. The antileishmanial potentials of the crude extract and compounds were evaluated against Leishmania major promastigotes and amastigotes. Further, in vivo 4-day antimalarial testing of the extract and compounds was carried out at doses of 25 mg kg−1 per day using mice infected with ANKA strain of Plasmodium berghei, following standard procedure. Molecular docking studies were carried out to assess the binding mode of flavonoids against the vital targets of L. major, including pteridine reductase 1 and farnesyl diphosphate synthase enzymes. The in silico antimalarial potentials of flavonoids were evaluated against wild-type Plasmodium falciparum dihydrofolate reductase-thymidylate synthase and phosphoethanolamine methyltransferase enzymes. Twenty compounds were tentatively identified by UPLC-ESI-MS/MS analysis of MFE, of which, seven flavonoids, six saponins, three phenolic acids, three fatty acids, and a triterpene glycoside were identified. MFE phytochemical analysis revealed that hesperidin content was 36.17 mg g−1 extract, that is, 9.9-fold their content of rutin (3.65 mg g−1 extract). The method was validated to ensure reproducibility of the results. The tested samples exhibited antileishmanial potentials against L. major promastigotes, with IC50 values of 98.62, 118.86, and 104.74 ng µL−1 for hesperidin, rutin, and MFE, respectively. Likewise, hesperidin showed inhibitory potentials against L. major amastigote with an IC50 value of 108.44 ± 11.2 µM, as compared to miltefosine. The mean survival time, parasitemia, and suppression percentages showed similar results for the three samples against ANKA strain of P. berghei. The docking studies showed good binding affinities of rutin and hesperidin with numerous H-bonding and van der Waals interactions. Marigold flowers are nutraceuticals, presenting important sources of bioactive flavonoids with potential against neglected tropical diseases.

Sublethal doses of insecticide reduce thermal tolerance of a stingless bee and are not avoided in a resource choice test.

Insecticides and climate change are among the multiple stressors that bees face, but little is known about their synergistic effects, especially for non-Apis bee species. In laboratory experiments, we tested whether the stingless bee Tetragonula hockingsi avoids insecticide in sucrose solutions and how T. hockingsi responds to insecticide and heat stress combined. We found that T. hockingsi neither preferred nor avoided sucrose solutions with either low (2.5 × 10-4 ng µl-1 imidacloprid or 1.0 × 10-4 ng µl-1 fipronil) or high (2.5 × 10-3 ng µl-1 imidacloprid or 1.0 × 10-3 ng µl-1 fipronil) insecticide concentrations when offered alongside sucrose without insecticide. In our combined stress experiment, the smallest dose of imidacloprid (7.5 × 10-4 ng) did not significantly affect thermal tolerance (CTmax). However, CTmax significantly reduced by 0.8°C (±0.16 SE) and by 0.5°C (±0.16 SE) when bees were fed as little as 7.5 × 10-3 ng of imidacloprid or 3.0 × 10-4 ng of fipronil, respectively, and as much as 1.5°C (±0.16 SE) and 1.2°C (±0.16 SE) when bees were fed 7.5 × 10-2 ng of imidacloprid or 3.0 × 10-2 ng of fipronil, respectively. Predictions of temperature increase, and increased insecticide use in the tropics suggest that T. hockingsi will be at increased risk of the effects of both stressors in the future.

Application of dsRNA of FgPMA1 for disease control on Fusarium graminearum1

Fusarium graminearum is a fungal plant pathogen which causes Fusarium head blight (FHB), a devastating disease on cereal crops. Here we report that FgPMA1 could be a new target to control FHB by the application of double-stranded RNA (dsRNA) of FgPMA1. FgPMA1 was divided into 6 segments to generated RNA interference (RNAi) constructs (FgPMA1RNAi-1, -2, -3, -4, -5, and -6), and these constructs were transformed in F. graminearum strain PH-1. The expression of FgPMA1 reduced by 18.48%, 33.48% and 56.93% in FgPMA1RNAi-1, FgPMA1RNAi-2 and FgPMA1RNAi-5, respectively. FgPMA1RNAi-1, -2, and -5 mutants inhibited fungal development, including mycelium growth, mycelial morphology, asexual and sexual development, and toxin production. The length of lesions on wheat leaves, wheat coleoptiles and wheat ears were shorter after infection with FgPMA1RNAi-1, -2, and -5 mutants than wild-type PH-1. These results showed that three segments (FgPMA1RNAi-1, -2, and -5) exhibited effective silencing effects. After treatment with 25 ng µL-1 dsRNA of these segments in vitro, the growth rate of mycelium growth was significant decreased, mycelium became deformed with bulbous structure at the tip, and the mycelium lost the ability to produce conidia in F. graminearum strain PH-1, Fusarium asiacitum strain 2021 and phenamacril-resistant strain YP-1. After application of FgPMA1RNAi-1-dsRNA and FgPMA1RNAi-2-dsRNA to wheat ears, pathogenicity reduced 34.21-35.40%.