Development of Rapid Detection Methods for Fusarium oysporum f. sp. melonis in Melon Seeds.

Melon (Cucumis melo L.) is a global commercial crop that is sensitive to seed-borne wilt infections caused by Fusarium oxysporum f. sp. melonis (Fom). To address the challenge of detecting Fom contamination, we designed a probe-based real-time PCR method, TDCP2, in combination with rapid or column-based DNA extraction protocols to develop reliable molecular detection methods. Utilizing TDCP2, the detection rate reached 100% for both artificially Fom-inoculated (0.25-25%) and pod-inoculated melon seeds in conjunction with DNA samples from either the rapid or column-based extraction protocol. We performed analyses of precision, recall, and F1 scores, achieving a maximum F1 score of 1 with TDCP2, which highlights the robustness of the method. Additionally, intraday and interday assays were performed, which revealed the high reproducibility and stability of column-based DNA extraction protocols combined with TDCP2. These metrics confirm the reliability of our developed protocols, setting a foundation for future enhancements in seed pathology diagnostics and potentially broadening their applicability across various Fom infection levels. In the future, we hope that these methods will reduce food loss by improving the control and management of melon diseases.

Rapid Detection of Fusarium oxysporum Using Insulated Isothermal PCR and a Rapid, Simple DNA Preparation Protocol.

We developed an insulated isothermal PCR (iiPCR) method for the efficient and rapid detection of Fusarium oxysporum (Fo), which is a fungus that infects various hosts and causes severe crop losses. The Fo iiPCR method was sensitive enough to detect up to 100 copies of standard DNA template and 10 fg of Fo genomic DNA. In addition, it could directly detect 1 pg of mycelium and 10 spores of Fo without DNA extraction. Our study compared the performance of Fo iiPCR to that of three published in planta molecular detection methods-conventional PCR, SYBR green-based real-time PCR, and hydrolysis probe-based real-time PCR-in field detection of Fo. All diseased field samples yielded positive detection results with high reproducibility when subjected to an Fo iiPCR test combined with a rapid DNA extraction protocol compared to Fo iiPCR with an automated magnetic bead-based DNA extraction protocol. Intraday and interday assays were performed to ensure the stability of this new rapid detection method. The results of detection of Fo in diseased banana pseudostem samples demonstrated that this new rapid detection method was suitable for field diagnosis of Fusarium wilt and had high F1 scores for detection (the harmonic mean of precision and recall of detection) for all asymptomatic and symptomatic Fo-infected banana samples. In addition, banana samples at four growth stages (seedling, vegetative, flowering and fruiting, and harvesting) with mild symptoms also showed positive detection results. These results indicate that this new rapid detection method is a potentially efficient procedure for on-site detection of Fo.

Blue light irradiation triggers the antimicrobial potential of ZnO nanoparticles on drug-resistant Acinetobacter baumannii.

Photodynamic inactivation (PDI) is a non-invasive and safe therapeutic method for microbial infections. Bacterial antibiotic resistance is caused by antibiotics abuse. Drug-resistant Acinetobacter spp. is a serious problem in hospitals around the world. These pathogens from nosocomial infections have high mortality rates in frailer people, and Acinetobacter spp. is commonly found in immunocompromised patients. Visible light is safer than ultraviolet light (UV) for PDI of nosocomial pathogens with mammalian cells. Zinc oxide nanoparticles (ZnO-NPs) were used in this study as an antimicrobial agent and a photosensitizer. ZnO is recognized as safe and has extensive usage in food additives, medical and cosmetic products. In this study, we used 0.125 mg/ml ZnO-NPs combined with 10.8 J/cm2 blue light (BL) on Acinetobacter baumannii (A. baumannii) that could significantly reduce microbial survival. However, individual exposure to ZnO-NPs does not affect the viability of A. baumannii. BL irradiation could trigger the antimicrobial ability of ZnO nanoparticles on A. baumannii. The mechanism of photocatalytic ZnO-NPs treatment for sterilization occurs through bacterial membrane disruptions. Otherwise, the photocatalytic ZnO-NPs treatment showed high microbial eradication in nosocomial pathogens, including colistin-resistant and imipenem-resistant A. baumannii and Klebsiella pneumoniae. Based on our results, the photocatalytic ZnO-NPs treatment could support hygiene control and clinical therapies without antibiotics to nosocomial bacterial infections.