A fluorescence-based viability assay for Phytophthora agathidicida oospores

Viability staining is an essential tool in many fields of microbiology. In this study, we aimed to establish a dual fluorescence method for detecting the viability of Phytophthora oospores. Phytophthora is a genus of plant pathogens in the class Oomycete that cause disease in a wide range of agriculturally and ecologically important plants. During the Phytophthora disease cycle, thick-walled oospores can be produced via sexual reproduction. These oospores are essential for long-range dispersal and long-term survival of the pathogen, and therefore methods for the study of oospores are of great interest. In this study, we tested five fluorescent dyes for their ability to stain Phytophthora agathidicida oospores: SYTO 9, FUN-1, fluorescein diacetate, propidium iodide, and TOTO-3 iodide. These dyes represented a selection of total, viable and non-viable cell stains Each dye was assessed individually, and then the best dyes were combined for dual viability staining. In this study, we have identified three dyes — SYTO 9, fluorescein diacetate and TOTO-3 — that can be used to report total, viable and non-viable oospores respectively. We further demonstrate that fluorescein diacetate and TOTO-3 can be used together for dual viability staining. This new method is quantitative and compatible with automated image analysis, allowing oospores to be rapidly and accurately assessed for viability.

Growth Response of Ginger (Zingiber officinale), Its Physiological Properties and Soil Enzyme Activities after Biochar Application under Greenhouse Conditions

This study aimed to investigate the effects of biochar (1%, 2%, and 3%) on seed germination, plant growth, root morphological characteristics, and physiological properties of ginger (Zingiber officinale) and soil enzymatic activities. Pot experiments under greenhouse conditions at 24 °C (day) and 16 °C (night) showed after six weeks that biochar additions of 2% and 3% significantly increased seed germination, plant height, leaf length, leaf number, as well as shoot and root dry weights compared to the control. Total root length significantly increased by 30%, 47%, and 74%, with increasing biochar contents (1%, 2%, and 3%) compared to the control. Root surface area, projected area, root diameter, and root volume reached a maximum at the 3% biochar treatment. The treatment with 2% biochar significantly increased fluorescein diacetate hydrolase and phenoloxidase activities by 33% and 59% compared to the control; so did the addition of 3% biochar, which significantly increased fluorescein diacetate hydrolases, phenoloxidase, and acid and alkaline phosphomonoesterase activity in soil compared to the control. Treatment with 3% biochar increased relative water content by 8%, chlorophyll content by 35%, and carotenoid content by 43% compared to the control. These results suggest that biochar can improve the performance of the rhizome of ginger and increase the activity of soil enzymes, thereby improving soil nutrient supply.

Fluorescein diacetate and rapid molecular testing for the early identification of rifampicin resistance in Mali

BACKGROUND: Non-conversion on auramine smear microscopy indicates a lack of treatment response, possibly associated with initial rifampicin-resistant tuberculosis (RR-TB). However, dead bacteria still stain positive and may be detected. Fluorescein diacetate smear microscopy (FDA) shows live mycobacteria only. Therefore, we studied the potential of 2-month (2M) FDA for the identification of initial RR-TB.METHODS: Between 2015 and 2018, we enrolled new smear-positive pulmonary TB patients from five local centres in Bamako, Mali. After baseline screening, sputum samples were collected at 1M, 2M, 5M and 18M. We used rpoB sequencing to identify initial RR-TB.RESULTS: Of 1359 patients enrolled, 1019 (75%) had rpoB sequencing results. Twenty-six (2.6%, 95%CI: 1.7–3.7) had mutations conferring rifampicin resistance. Most frequent rpoB mutations were located at the codons Asp435Val (42.4%) and Ser450Leu (34.7%). Among patients with initial RR-TB, 72.2% were FDA-negative at 2M (P = 0.2). The positive and negative predictive value of 5M FDA for culture-based failure was respectively 20.0% and 94.7%.CONCLUSION: FDA did not identify the majority of patients with initial RR-TB or culture-based failure. As the full spectrum of mutations identified on sequencing was identified using Xpert, our data support its rapid universal implementation in Mali.