Simple rapid in vitro screening method for SARS-CoV-2 anti-virals that identifies potential cytomorbidity-associated false positives

The international SARS-CoV-2 pandemic has resulted in an urgent need to identify new anti-viral drugs for treatment of COVID-19 patients. The initial step to identifying potential candidates usually involves in vitro screening. Here we describe a simple rapid bioassay for drug screening using Vero E6 cells and inhibition of cytopathic effects (CPE) measured using crystal violet staining. The assay clearly illustrated the anti-viral activity of remdesivir, a drug known to inhibit SARS-CoV-2 replication. A key refinement involves a simple growth assay to identify drug concentrations that cause cellular stress or “cytomorbidity”, as distinct from cytotoxicity or loss of viability. For instance, hydroxychloroquine shows anti-viral activity at concentrations that slow cell growth, arguing that its purported in vitro anti-viral activity arises from non-specific impairment of cellular activities.

A rapid bioassay for measuring nematode suppressive potential of anaerobic digestate

Summary Anaerobic digestate is a byproduct of anaerobic digestion of organic materials for biogas production. Land application of digestates may provide plant nutrition and suppression of plant-parasitic nematodes. The characteristics of digestates may vary by organic substrates and digestion conditions. To measure nematode-suppressive potential, the rigour and simplicity of a radish bioassay with Heterodera schachtii was expanded. In a three-factor factorial design, three incubation environments, two growth containers and two nematode life stages as inoculum were tested. Containers with 50 g of dry sandy loam soil were inoculated with 500 second-stage juveniles (J2) of H. schachtii or with cysts with equivalent hatchable J2. One seed of radish Raphanus sativus ‘Cherry Belle’ was planted into each container, and 1 ml of drench treatment was applied. After 64 degree days (4-5 days), roots were washed and stained with acid fuchsin for nematode counting. In three experiments, food waste digestate permeate suppressed nematode root penetration in the growth chamber, glasshouse and laboratory, in cups and tubes, and following inoculation with cysts and J2. However, root penetration by J2 was more greatly reduced after cyst inoculation than after J2 inoculation. Investigations in preserving nematode cysts showed that J2 within cysts remained viable after incubation in sandy loam soil at 23°C in sealed tubes in the laboratory for 35 days. Results of the radish assay predicted nematode-suppressive potential against Pratylenchus vulnus on peach rootstock ‘Nemaguard’ in a glasshouse experiment. In summary, a straightforward rapid bioassay for measuring nematode suppressiveness of organic liquids was developed for routine use.

Regional Survey of Diamondback Moth (Lepidoptera: Plutellidae) Response to Maximum Dosages of Insecticides in Georgia and Florida

We conducted maximum dose bioassays of insecticide for the control of diamondback moth (DBM), Plutella xylostella (Linnaeus), in cole crops, from 2016 to 2019 at several commercial locations in Georgia and Florida. The nominal maximum dose was defined as the highest labeled rate of an insecticide at the beginning of the survey in the equivalent of 935 liters/ha dilution. The results indicated low insecticide efficacy for high labeled rates of the following insecticides by common name (Insecticide Resistance Action Committee group number in parentheses). Our 4-yr survey identified very low levels of DBM larval control (<47%) by lambda-cyhalothrin (3), methoxyfenozide (18), pyriproxyfen (7C), novaluron (15), bifenthrin (3), chlorantraniliprole (28), indoxacarb (22A), and methomyl (1A). The best products for DBM control (>74%) listed in decreasing average levels of efficacy were naled (1B), cyclaniliprole (28), tolfenpyrad (21A), emamectin benzoate (6), and cyantraniliprole (28). Intermediate levels of control (61–71%) were obtained with Bacillus thuringiensis subspecies aizawai (11A), Bacillus thuringiensis, subsp. kurstaki, strain ABTS-351 (11A), and spinetoram (5). This rapid bioassay provided the grower with a ranking of insecticide efficacy for the control the DBM population for that farm site. These data allowed growers to make an informed decision on control quickly and plan for resistance management rotations for DBM that season.

A Rapid Bioassay to Evaluate Efficacy of Hypovirulent Binucleate Rhizoctonia in Reducing Fusarium Crown and Root Rot of Tomato

Background: Fusarium Oxysporum f.sp. Radicis-Lycopersici (FORL) caused Fusarium Crown and Root Rot of tomato (FCRR), it’s a serious constraint on tomato production and contributing to yield losses. Aims/Method: Using a rapid bioassay, Hypovirulent Binucleate Rhizoctonia (HBNR) was tested for their ability to reduce Fusarium Crown and Root Rot (FCRR) of tomato, caused by Fusarium oxysporum f.sp. radicis lycopersici (FORL). Roots of tomato seedlings growing on 2% water agar in plastic boxes were inoculated with living or dead mycelial disks of HBNR. After 24 h, the pathogen was applied at 0, 3, 6, and 9 cm away from the position of the HBNR. Results: When living HBNR was used, the treatments provided significant protection to tomato seedlings from FCRR infection at all distances tested. Tomato plants pre-inoculated with living HBNR at different times (12 h and 24 h before inoculation with the pathogen) and challenged with FORL showed significant reduction of FCRR lesion development. A significant reduction was still observed even when HBNR was inoculated simultaneously with or 12 h after inoculation of a pathogen. Seedlings treated with dead HBNR and culture filtrates also showed significantly reduced FCRR lesion development. When living HBNR were enveloped by a polycarbonate membrane filter, a significant reduction of FCRR lesion development was still observed. Conclusion: In all experiments, reduction of FCRR lesion development in seedlings treated with HBNR tended to decrease with longer distance from the inoculation point of FORL and HBNR. We developed a simple, rapid, and miniaturized bioassay for evaluating the efficacy of HBNR against FORL. The bioassays require only 12 - 18 days, which is at least 12 days less than the soil system employed by previous researchers.

Antibacterial Activities of Metabolites from Vitis rotundifolia (Muscadine) Roots against Fish Pathogenic Bacteria

Enteric septicemia of catfish, columnaris disease and streptococcosis, caused by Edwardsiella ictaluri, Flavobacterium columnare and Streptococcus iniae, respectively, are the most common bacterial diseases of economic significance to the pond-raised channel catfish Ictalurus punctatus industry. Certain management practices are used by catfish farmers to prevent large financial losses from these diseases such as the use of commercial antibiotics. In order to discover environmentally benign alternatives, using a rapid bioassay, we evaluated a crude extract from the roots of muscadine Vitis rotundifolia against these fish pathogenic bacteria and determined that the extract was most active against F. columnare. Subsequently, several isolated compounds from the root extract were isolated. Among these isolated compounds, (+)-hopeaphenol (2) and (+)-vitisin A (3) were found to be the most active (bacteriostatic activity only) against F. columnare, with 24-h 50% inhibition concentrations of 4.0 ± 0.7 and 7.7 ± 0.6 mg/L, respectively, and minimum inhibitory concentrations of 9.1 ± 0 mg/L for each compound which were approximately 25X less active than the drug control florfenicol. Efficacy testing of 2 and 3 is necessary to further evaluate the potential for these compounds to be used as antibacterial agents for managing columnaris disease.

Toxicity Assessment of Produced Water Using Microtox Rapid Bioassay

Aim: The study aimed at employing the Microtox test procedure in the current biological monitoring protocol as a reliable, rapid and ecologically relevant bioassay tool for toxicity assessment in environmental compliance monitoring of produced water discharges. Study Design: Inhibition of bioluminescence by V. fischeri [median effective concentration (EC50)] was employed as the toxicity index. Place and Duration of Study: Microbiology Department of Halden Laboratories, Port Harcourt, Nigeria / one month. Methodology: Percent reduction in bioluminescence by V. fischeri after 15-min exposure to the PW samples was recorded as median effective concentration (EC50) values. Results: The 15 min EC50 values of the untreated and treated produced water samples for V. fischeri was 1.0% and 23.27% respectively. Microtox test indicated the treated and untreated produced water samples were “very toxic” and “extremely toxic” respectively, after 15 min exposure time. Conclusion: These findings emphasize the need for adequate treatment of produced water to meet standard discharge limits of regulatory agencies in Nigeria, as both physicochemical analysis and bioassay (Microtox) suggested that the treated PW was toxic to V. fischeri. This study thus supports the use of Microtox (bacterial toxicity) system as a sensitive and rapid bioassay tool for biological monitoring protocol in Nigeria's petroleum industry.