An Improved Method for Quantification of Viable Fusarium Cells in Infected Soil Products by Propidium Monoazide Coupled with Real-Time PCR

Abstract

Fusarium is a soil-borne pathogen that causes root rot disease in cucumber. To date, quantitative real-time PCR (qPCR) is a common tool to detect the content of Fusarium in soil. However, qPCR cannot distinguish between viable and nonviable cells. The aim of this study was to develop a detection technique to pretreat tissue fluid with propidium monoazide (PMA) followed by extract DNA, and then to quantify viable Fusarium cells in contaminated soil. In this work, the specific primer pair F8-1/F8-2 was designed based on the translation elongation factor (EF) gene and a PMA-qPCR assay was established to amplify and quantify soils of viable Fusarium cells. The PMA pretreatment test was optimized, which indicated that the optimal PMA concentration and light exposure time were 50 mmol L−1 and 15 min, respectively. The lowest limit of viable cells in suspension detected and soil by PMA-qPCR were 82 spore mL−1 and 91.24 spore g−1, respectively. For naturally contaminated soil, viable Fusarium cells were detected in eight of the 18 samples, and the Fusarium amount ranged from 104 to 106 spore g−1. In conclusion, the PMA-qPCR method has the characteristics of high sensitivity, efficiency, and time saving, which could support nursery plants to avoid Fusarium infection and agro-industry losses.

The calcium cyanamide and polyethylene blocks the secondary transmission and infection of vegetable leaf diseases

“…A 20 μL reaction volume of qPCR containing template DNA (1 μL), SuperReal PreMix Plus (TIANGEN Biotech) (10 μL), 10 μmol·L −1 primer (each) and 50×ROX Reference Dye (0.4 μL) was used. The amplification procedure was consistent with the method described by Chen et al. (2022) .…”

“…C. cassiicola was inoculated into PDA medium, and the culture was activated at 28.5°C for 5 d. The spores on the plate were washed with sterile water to prepare spore suspensions with concentrations of 10 7 spores·mL -1 . Standard curves were constructed, and the sensitivity was determined according to the method described by Chen et al. (2022) .…”

“…The PMA concentration and exposure time were optimized according to the method described by Chen et al. (2022) .…”

“…The PMA concentration and exposure time were optimized according to the method described by Chen et al (2022). A shaker was used to mix thoroughly to obtain a mixture of PMA, dead and viable cells with final concentrations of 0, 10, 20, 40, 80 and 100 mmol•L −1 .…”

“…It can provide a suitable environment for the growth, reproduction and accumulation of pathogens, leading to plant diseases ( Govaerts et al., 2007 ; Yuan et al., 2021 ). Pathogens can survive as resting hyphae in the soil or diseased bodies for at least five years ( Chen et al., 2022 ). Under favorable conditions, the resting hyphae are activated, after which they colonize tissues and kill the young seedlings before or soon after emergence.…”