Volatile-Based Diagnosis for Pathogenic Wood-Rot Fungus Fulvifomes siamensis by Electronic Nose (E-Nose) and Solid-Phase Microextraction/Gas Chromatography/Mass Spectrometry

Abstract

Wood rot fungus Fulvifomes siamensis infects multiple urban tree species commonly planted in Singapore. A commercial e-nose (Cyranose 320) was used to differentiate some plant and fungi volatiles. The e-nose distinctly clustered the volatiles at 0.25 ppm, and this sensitivity was further increased to 0.05 ppm with the use of nitrogen gas to purge the system and set up the baseline. Nitrogen gas baseline resulted in a higher magnitude of sensor responses and a higher number of responsive sensors. The specificity of the e-nose for F. siamensis was demonstrated by distinctive clustering of its pure culture, fruiting bodies collected from different tree species, and in diseased tissues infected by F. siamensis with a 15-min incubation time. This good specificity was supported by the unique volatile profiles revealed by SPME GC-MS analysis, which also identified the signature volatile for F. siamensis-1,2,4,5-tetrachloro-3,6-dimethoxybenzene. In field conditions, the e-nose successfully identified F. siamensis fruiting bodies on different tree species. The findings of concentration-based clustering and host-tree-specific volatile profiles for fruiting bodies provide further insights into the complexity of volatile-based diagnosis that should be taken into consideration for future studies.

Keywords: 1,2,4,5-tetrachloro-3,6-dimethoxybenzene; Cyranose 320; Fulvifomes siamensis; SPME GC-MS; concentration specific clustering; electronic nose (e-nose); fungi-host interaction; nitrogen for baseline; signature volatile; volatile-based diagnosis.

Figure 1

Experiment flow for this study.

Figure 2

Nitrogen for the baseline improves e-nose sensitivity and specificity. CDA plots with Interclass M-Distance readings for 0.25 ppm of Farnesene (FAR0.25), Turpentine (TUR0.25), Acetic Acid (AA0.25), and air from biosafety cabinet (AIR) volatile samples using (A) ambient air and (B) nitrogen as the baseline gas (samples have additional suffix –n); and for 0.05 ppm VOC samples (FAR0.05, TUR0.05, AA0.05) using (C) ambient air and (D) nitrogen as the baseline gas. Samples inside a dashed-line ellipse indicate that the Interclass M-Distance is greater than 5.000 and thus form a distinctive cluster.

Figure 3

Better sensor responses using nitrogen as the baseline gas. Mean response ΔR/R_o of the selected active sensors 6, 23, 24, 31, and 32 for volatile chemicals Farnesene (FAR0.05), Turpentine (TUR0.05), Acetic Acid (AA0.05) at 0.05 ppm and air from biosafety cabinet (AIR) using (A) ambient air and (B) nitrogen as the baseline gas (samples have additional suffix -n).

Figure 4

E-nose specifically detects F. siamensis samples. (A): CDA plot of e-nose clustering with nitrogen as the baseline gas for fifteen F. siamensis pure fungal mycelia cut plug samples (F-15) against that of R. microporus (R-15); (B): cultures of F. siamensis (FS) and R. microporus (RM); (C): CDA plot of F. siamensis fruiting bodies (CE149FB, CE151FB) and diseased tissue (CE152DT) against G. australe fruiting body (CE153FB), R. microporus fruiting body (P175FB1) with 15 min of incubation; (D): CDA plot for the same samples after a 5-day incubation period. An orange-dashed line ellipse indicates a distinctive cluster for a single sample, with Interclass M-Distance greater than 5.000 from any other sample. A blue dashed-line ellipse indicates a cluster of two samples with Interclass M-Distance less than 5.000, which is also highlighted in the table.

Figure 5

Real-time field testing with the e-nose. (A) The e-nose setup with the nitrogen pouch; (B) In-field identification of a F. siamensis fruiting body with high confidence after 15-min incubation time.

Figure 6

Volatile quantity-based clustering by the e-nose. CDA plots with Interclass M-distances for different concentrations of AA (A) and TUR (B), and for different numbers (five, ten, and fifteen) of fungal mycelia cut plugs for F. siamensis (C) and R. microporus (D). An orange-dashed line ellipse indicates a distinctive cluster for a single sample, with Interclass M-Distance greater than 5.000 from any other sample. A blue dashed-line ellipse indicates a cluster of two samples with Interclass M-Distance less than 5.000, which is also highlighted in the table.