Development of PMA-qPCR assay to accurately and reproducible quantify viable bacteria of Paenibacillus polymyxa.

Paenibacillus polymyxa is an important biocontrol bacterium. The combination of propidium monoazide (PMA) and quantitative polymerase chain reactionq (qPCR) has proven effective in quantifying live bacteria from various microorganisms. The objective was to create a PMA-qPCR assay to precisely and consistently measure the number of living bacteria of biocontrol P. polymyxa. The primers were designed for the spo0A gene of P. polymyxa HY96-2. The optimal conditions for treating the target strain with PMA were a PMA concentration of 15 µg/mL, an incubation time of 5 min, and an exposure time of 10 min. The PMA-qPCR method had a limit of quantification (LOQ) of 1.0 × 103 CFU/mL for measuring the amount of viable P. polymyxa bacteria. The PMA-qPCR method is more sensitive than the qPCR method in detecting viable bacteria in the mixtures of viable and dead bacteria. The accuracy and reproducibility of quantifying viable P. polymyxa bacteria using the PMA-qPCR method were higher compared to the plate count method.

A new PMA-qPCR method for rapid and accurate detection of viable bacteria and spores of marine-derived Bacillus velezensis B-9987.

Marine-derived Bacillus velezensis B-9987 is an important biocontrol bacterium with a broad-spectrum antibacterial effect. The traditional plate counting method is widely used for quantitative detection of viable bacteria and spores but has some disadvantages such as being laborious and time-consuming (at least 24-48 h). This study aimed to develop a new PMA-qPCR method for rapid and accurate detection of viable bacteria and spores of B-9987. The specific primers were designed for qPCR amplification based on the conserved region of the bmmA gene (encoding a malonyl CoA-ACP transacylase) of B-9987. According to the characteristic that propidium monoazide (PMA) dye can distinguish viable and dead bacteria, the optimal PMA concentration of 10 µg/ml and optimal exposure time of 10 min were achieved under PMA treatment conditions. The B-9987 spores' genomic DNA was successfully extracted after the spore coat was removed and spore germination was induced. The quantification limits of the PMA-qPCR method were determined for viable B-9987 bacteria, spores in pure culture, and spores in marine Bacillus wettable powder (marine Bacillus WP) and were 1.5 × 103 CFU/ml, 6.5 × 102 CFU/ml, and 103 CFU/ml, respectively. Compared with the qPCR method, the PMA-qPCR method could sensitively detect viable bacteria in the viable/dead bacterial mixture. In this study, the developed PMA-qPCR method was found to have excellent sensitivity and specificity in the context of a pure culture of B-9987 strain, which could accurately and rapidly detect viable B-9987 bacteria within 3-4 h and viable B-9987 spores in marine Bacillus WP within 4-6 h.

Enhanced production of questin by marine-derived Aspergillus flavipes HN4-13.

Questin has favorable applications. Fractional factorial design, Box-Behnken design, and response surface methodology were adopted to optimize the fermentation conditions of the marine-derived fungus, Aspergillus flavipes HN4-13, thereby enhancing questin production. Optimal fermentation conditions in a 500-mL conical flask with 200 mL of medium were 4% soluble starch, 0.9% beef extract, 4% NaCl, 0.05% Na2HPO4, pH 6, 2% inoculum size, and shaking at 28 â„ƒ and 160 rpm/min for 7 days. The production of questin can achieve 64.93 ± 4.55 mg/L, with no significant difference from the predicted value (66.27 mg/L). Thus, this optimized process of questin production is feasible. Such production is 17-fold higher than that of the basal Sabouraud's dextrose medium. Results indicate the potential of A. flavipes HN4-13 in the large-scale production of questin through fermentation.

Antibacterial activity and action mechanism of questin from marine Aspergillus flavipes HN4-13 against aquatic pathogen Vibrio harveyi.

This study investigated the antibacterial activity and mechanism of questin from marine Aspergillus flavipes HN4-13 against aquatic pathogenic Vibrio harveyi. The minimal inhibitory concentration and minimal bactericidal concentration of questin against V. harveyi strain SZ-1 and 1.8690 were determined by Oxford cup and tube dilution methods. The mechanism of action of questin against V. harveyi 1.8690 was investigated by bacterial growth curve analysis, ultraviolet absorption, Mo-Sb-Vc colorimetry, alkaline phosphatase and scanning electron microscopy. Results showed that questin exhibited favourable antibacterial and bactericidal activity against V. harveyi by disrupting the cell wall and membrane, which caused the destruction of permeability and integrity of cell wall and membrane, resulting in the leakage of intracellular biological components and change of cell morphology. This paper is the first to report the mechanism of action of questin against the aquatic pathogen V. harveyi.

Optimized extraction process and identification of antibacterial substances from Rhubarb against aquatic pathogenic Vibrio harveyi.

Response surface optimization was applied for the extraction of antibacterial substances from Rhubarb (ASR) against aquatic pathogenic Vibrio harveyi. Based on the experimental results of single factors, the optimal extraction conditions were determined by Box-Behnken design combined with response surface methodology with conditions: 100% ethanol as extraction solvent, liquid/material ratio of 29 mL/g and extraction temperature at 88 °C for 148 min. The factual value of inhibition zones can reach 21.31 ± 0.95 mm and is not different from the predicted value (21.74 mm), which showed that the response surface methodology applied to the extraction optimization of antibacterial substances from Rhubarb against V. harveyi is feasible. Moreover, the yield of ASR was 30.29 ± 2.27%. Five compounds, namely, aloe-emodin, rhein, emodin, chrysophanol and physcion, were identified in ASR by comparing the HPLC chromatogram of the reference mixtures and the sample. Contents of the five compounds were 0.68 ± 0.02, 0.24 ± 0.05, 0.78 ± 0.07, 6.68 ± 0.97 and 0.58 ± 0.15%, respectively. The minimal inhibitory concentration (MIC) values of ASR, aloe-emodin, rhein, emodin, chrysophanol and physcion were 0.625, 0.125, 0.015, > 1, > 1, and > 1 mg/mL, respectively, which indicated that aloe-emodin and rhein are the main antibacterial compounds of Rhubarb.