Volatile Metabolic Markers for Monitoring Pectobacterium carotovorum subsp. carotovorum Using Headspace Solid-Phase Microextraction Coupled with Gas Chromatography-Mass Spectrometry

Abstract

Identifying the extracellular metabolites of microorganisms in fresh vegetables is industrially useful for assessing the quality of processed foods. Pectobacterium carotovorum subsp. carotovorum (PCC) is a plant pathogenic bacterium that causes soft rot disease in cabbages. This microbial species in plant tissues can emit specific volatile molecules with odors that are characteristic of the host cell tissues and PCC species. In this study, we used headspace solid-phase microextraction followed by gas chromatography coupled with mass spectrometry (HS-SPME-GC-MS) to identify volatile compounds (VCs) in PCC-inoculated cabbage at different storage temperatures. HS-SPME-GC-MS allowed for recognition of extracellular metabolites in PCC-infected cabbages by identifying specific volatile metabolic markers. We identified 4-ethyl-5-methylthiazole and 3-butenyl isothiocyanate as markers of fresh cabbages, whereas 2,3-butanediol and ethyl acetate were identified as markers of soft rot in PCC-infected cabbages. These analytical results demonstrate a suitable approach for establishing non-destructive plant pathogen-diagnosis techniques as alternatives to standard methods, within the framework of developing rapid and efficient analytical techniques for monitoring plant-borne bacterial pathogens. Moreover, our techniques could have promising applications in managing the freshness and quality control of cabbages.

Keywords: Cabbage; Pectobacterium; soft rot; solid-phase microextraction; volatile metabolic marker.

Fig. 1. Comparison of Pectobacterium carotovorum subsp. carotovorum (PCC) bacterial counts and soft rot development between fresh cabbages and PCC-inoculated cabbages under different storage temperatures after 3 days.

Fresh cabbage samples stored at 0°C, 10°C, 20°C, and 30°C are denoted as F0, F10, F20, and F30, respectively. PCC-inoculated cabbages stored at 0°C, 10°C, 20°C, and 30°C are denoted as PCC0, PCC10, PCC20, and PCC30, respectively.

Fig. 2. Principal component analysis (PCA) score plot of fresh and Pectobacterium carotovorum subsp. carotovorum (PCC)-infected cabbages.

(A) Three-class discrimination using the headspace solid-phase microextraction followed by gas chromatography coupled with mass spectrometry (HS-SPME-GC-MS) analysis. (B) Fresh cabbage samples stored at 0°C, 10°C, 20°C, and 30°C are denoted as F0, F10, F20, and F30, respectively. PCC-inoculated cabbages stored at 0°C, 10°C, 20°C, and 30°C are denoted as PCC0, PCC10, PCC20, and PCC30, respectively. Control is denoted as Bpellet.

Fig. 3. Correlation analysis clustering with Pearson’s correlation matrix heatmap (A) and the dendrogram (B) of different cabbage samples.

Fresh cabbage samples stored at 0°C, 10°C, 20°C, and 30°C are denoted as F0, F10, F20, and F30, respectively. PCC-inoculated cabbages stored at 0°C, 10°C, 20°C, and 30°C are denoted as PCC0, PCC10, PCC20, and PCC30, respectively. Bpellet, the bacterial suspension pellet, was stored at 30°C as the control.

Fig. 4. Clustering heatmap of the concentration of volatile compounds in cabbage samples at different storage temperatures.

Fresh cabbage samples stored at 0°C, 10°C, 20°C, and 30°C are denoted as F0, F10, F20, and F30, respectively. PCC-inoculated cabbages stored at 0°C, 10°C, 20°C, and 30°C are denoted as PCC0, PCC10, PCC20, and PCC30, respectively. Bpellet, the bacterial suspension pellet, was stored at 30°C as the control.

Fig. 5. Partial least squares-discriminant analysis (PLS-DA) of headspace solid-phase microextraction followed by gas chromatography coupled with mass spectrometry (HS-SPME-GC-MS) analysis for each cabbage sample at different storage temperatures.

Loading plot of PLS-DA (A) and PLS-DA scores scatter plot (B). Fresh cabbage samples stored at 0°C, 10°C, 20°C, and 30°C are denoted as F0, F10, F20, and F30, respectively. PCC-inoculated cabbages stored at 0°C, 10°C, 20°C, and 30°C are denoted as PCC0, PCC10, PCC20, and PCC30, respectively. Bpellet, the bacterial suspension pellet, was stored at 30°C as the control.

Fig. 6. (A) Variable importance in projection (VIP) scores showing major volatile metabolites that are discriminatory and (B) random forest (RF) analysis showing the top 15 volatile metabolites responsible for classification as mean decreased accuracy values.

Metabolite names provided in red font are shared between both analyses. Fresh cabbage samples stored at 0°C, 10°C, 20°C, and 30°C are denoted as F0, F10, F20, and F30, respectively. PCC-inoculated cabbages stored at 0°C, 10°C, 20°C, and 30°C are denoted as PCC0, PCC10, PCC20, and PCC30, respectively. Bpellet, the bacterial suspension pellet, was stored at 30°C as the control.