VOLATILE ANALYSIS TECHNIQUES WITH HIGH TIME RESOLUTION
A detailed understanding of regulatory mechanisms governing plant VOC biosynthesis and emissions such as circadian or diurnal control regimes require analysis techniques that monitor volatile emission changes with appropriate time resolution. Computer assisted and online dynamic headspace trapping systems are capable of collecting volatiles in hourly or shorter time intervals. In a commercially available online thermal desorption system (Gerstel Online-TDS-G, Gerstel, Germany) volatile samples are drawn automatically and, following a cryofocusing step, compounds are flash heated and directly injected on the column. The time resolution depends on the time necessary to collect sufficient amounts of material from the emitting plant tissue for analysis, the compounds themselves, and the time for chromatographic separation on the GC-MS. The TDS-G system can be connected to collecting containers with plant samples as described in detail by Vercammen et al. (2001). This method allows for a high time resolution, depending on the compounds and a high sensitivity, but requires an extensive laboratory setup. Moreover, analysis by GC presents a time limiting factor. Described below are alternative analytical systems that have been developed to allow faster and sensitive volatile analyses and hence represent promising tools for continuous monitoring of plant VOC emissions.
1.6.1 Transportable GC
To allow for fast real-time analysis of plant VOCs in the laboratory and the field, portable GC instruments have been developed (Yashin and Yashin, 2001). An example of a portable GC is the zNose™ (Electronic Sensor Technology, Newbury Park, CA, USA), which has been applied for the analysis of floral scent and induced VOC emissions from herbivore-damaged plants (Kunert et al., 2002; Oh, 2018). The zNose™ separates compounds by fast gas chromatography and operates with a highly sensitive surface acoustic wave (SAW) quartz microbalance detector, which drastically reduces the volatile sampling and pre-concentration time of the system. As a drawback, the SAW detector does not allow structure elucidation. Therefore, volatiles need to be analyzed by GC-MS prior to calibration of the system with authentic standards. Moreover, the short gas chromatography column reduces the compound resolution. Thus, monitoring changes of volatiles with similar elution profiles is limited.
As another example for a GC system used in the field, Wong et al. (2019) combined SPME with a portable GC containing a low-thermal mass (LTM) column in combination with a miniature ion trap mass spectrometer. LTM columns allow efficient heating and cooling within a short analytical time. This system as well as the zNose™ are applicable for screenings of natural variants or mutant populations and suitable for monitoring kinetics of volatile emissions from floral and vegetative tissues under different endogenous and environmental conditions.
1.6.2 Proton-Transfer Reaction Mass Spectrometry (PTR-MS)
The PTR-MS analysis technology was developed ca. 20 years ago at the University of Innsbruck by Lindinger and coworkers (Lindinger et al., 1998). PTR-MS systems operate independently of gas chromatographic separation and allow online measurements of VOCs with concentrations in the pptv range. Originally developed for monitoring changes of VOCs in the atmosphere, in food control and medical analyses, PTR-MS has been applied for real-time analysis of volatile emissions from plants above- and belowground. PTR-MS instruments are still relatively expensive and their operation requires extended training by experienced researchers.
For detection by PTR-MS, volatiles undergo a chemical ionization by proton-transfer reactions with H,0+ ions. Differences in proton affinities allow a proton transfer from H,0+ ions to a large number of organic volatiles but prevent a reaction of H ,0+ ions with the main constituents of the air. The proton-transfer reaction takes place under defined conditions in a homogeneous electric field applied to a drift tube (Figure 1.4). Ions exiting the tube are finally mass analyzed by a quadrupole or TOF mass spectrometer (Graus et al., 2010). The soft ionization of compounds by protonation causes only low fragmentation, hence mainly one product ion species occurs for each reactant. The extremely fast time response of the instrument results from less than one second that volatiles spend in the drift tube.
The analysis of volatile mixtures by PTR-MS is limited by the ability to determine only the molecular mass of products. Compounds of the same nominal mass cannot be identified separately using a quadrupole MS. Therefore, systems w'ith additional analysis by GC either in parallel or by coupling to the PTR-MS have been developed or instruments using triple-quad MS analysis have been applied to distinguish between isomeric species (Graus et ah, 2010; Materic et ah, 2015). Moreover, the development of PTR-TOF-MS has allowed the separation of isobaric VOCs in complex mixtures (Graus et ah, 2010).
The PTR-MS technique has been applied in several studies to measure fluctuations of volatile emissions from various plants. Usually whole plants or plant parts are enclosed in glass containers, inert bags or dynamic cuvette systems (Tholl et ah, 2006) with a continuous air stream and controlled temperature, humidity and light conditions, and aliquots of the exiting air are analyzed by PTR-MS. Emissions of volatiles including isoprene and monoterpenes from trees and other plants have been monitored under laboratory and field conditions at different developmental
FIGURE 1.4 Schematic representation of a PTR-TOF-MS instrument according to Graus et ah, (2010). Chemical ionization of VOCs by proton-transfer reaction occurs in the drift tube (DT). In the ToF mass spectrometer ions are pulsed orthogonally toward the reflectron (Reft) and are refocused into the detector (Det) plane. An amplifier/discriminator unit (XCD) preprocesses the signal to generate individual ion counting events, which are further processed by a time to digital-converter (TDC). HC, hollow cathode; SD, source drift region; TMP, turbo molecular pump. (Modified from Graus, M. et ah, J. Am. Soc. Mass Spectrom., 21, 1037-1044, 2010. With permission.) stages and in response to changes of abiotic factors like light and temperature or biotic factors such as herbivory (e.g., Loreto et al., 2006; Brilli et al., 2011; Giacomuzzi et al., 2016; Mozaffar et al., 2018).
Besides measurements of VOCs from aboveground plant tissues, PTR-MS has been used successfully for noninvasive analysis of VOCs from roots. Danner et al. (2012, 2015) measured sulfur containing compounds emitted by Brassica roots after herbivory by positioning a two-part cuvette on top of the soil at the stem-root interface. A constant flow of purified air was applied to flush the root-cuvettes. The instrument was used in SIM mode optimized for measuring sulfur compounds. In a different system developed by Acton et al. (2018) a root glass chamber was designed, in which VOC-free air enters the vessel at its base through a ring of tubing and exits the chamber at a port located above the soil level. Here, a PTR-TOF-MS with selective reagent ionization (SRI-TOF-MS) was used to detect VOCs with high time and mass resolution and identify distinct functional groups of compounds.
1.6.3 Automated Systems for Volatilome Analysis
The emission of VOCs can be considered a phenotypic trait that provides information about the developmental and physiological state of an organism and its exposure to biotic and abiotic stress. Therefore, new systems have been developed to monitor so-called plant “volatilomes” representing the entirety of VOCs emitted by a plant including CO, and water vapor (Jud et al., 2018). The VOC-SCREEN platform established by Schnitzler and coworkers allows the simultaneous, noninvasive online analysis of VOCs emitted from the foliage of 24 plants (Figure 1.5). In a test example, whole potted barley plants were enclosed in flow-through cuvettes. VOCs were sampled
FIGURE 1.5 Schematic representation of an automated multiple cuvette system for volatilome analysis according to Jud et al. (2018). Air from the phytotron chamber is pumped into cuvettes containing individual plants (blue lines). Air exiting the cuvettes (red lines) undergoes analysis by PTR-ToF-MS and other gas analyzers. Sampling occurs from one cuvette at a time. A portion of the air can be sampled for VOCs for GC-MS analysis. IRGA, infrared gas analyzer. (Reprinted from Jud, W. et al., Plant Methods, 14, 109, 2018. With permission.) every 2 h for 5 min from each cuvette and measured quantitatively and in a nontargeted way by PTR-TOF-MS with a high mass resolution and detection limits in the low ppt range.
A portion of the VOCs is trapped on adsorbent tubes to allow for GC-MS analysis of different isomers. The cuvette based system has the advantage that CO, assimilation and transpiration rates can be measured simultaneously with the analysis of VOCs and thereby allow correlations of VOC release with the physiological condition of the plant. The screening platform can be installed in controlled environmental chambers and is suitable for monitoring VOC profiles under transient conditions or long term scenarios.
1.6.4 Sensors for VOC Profiling in the Field
Developments in smart farming and precision agriculture are based on obtaining accurate information on crop plants, soil, weather and environmental conditions. Various sensor technologies have been developed to monitor these conditions. Among these, there is growing interest in obtaining information about the physiological state of plants by sensing VOC emissions. VOC sensing is considered a diagnostic tool in determining plant health by recognizing infested or infected plant material at an early stage of outbreak. Targeted VOCs include compounds involved in plant defense as well as VOCs released from pathogens or pests (Cellini et al., 2017).
Electronic noses have been developed for monitoring mixtures of VOCs that are released from different materials. An e-nose operates with an array of sensors, whose surfaces interact with the gas phase and have different sensibilities to volatile molecules. Based on electric signals from the sensors, patterns or profiles corresponding to specific VOC compositions are generated (Wilson, 2013). While e-noses do not identify individual VOC compounds they can be applied for the nondestructive quick monitoring or recognition of VOC blends. Several applications of commercially available e-nose models in plant disease and pest diagnosis have been summarized by Cellini et al. (2017). Parallel testing of an e-nose with GC-MS analysis provides more precise information on the sensor response to specific compounds and their discrimination between different odor sources (Long et ah, 2019).
A challenge of using e-noses is their limited sensitivity, but cartridges with VOC sorbent materials such as Tenax have been coupled with electronic noses (Cellini et ah, 2017). In these systems VOCs are adsorbed and thermally desorbed in a small air volume prior to their detection by the sensor. The adsorbent can provide some selectivity toward VOCs that are of specific interest concerning distinct disease profiles. Another challenge in the application of e-noses is that VOC recognition can be compromised by background noise, natural variation, or variability of VOC blends due to environmental conditions other than those targeted in the analysis. Therefore, it has been suggested that e-nose technology might best be combined with other phenotyping and molecular methods to optimize plant disease diagnostics.
In addition to e-noses, differential mobility spectrometry (DMS) coupled with gas chromatography (GC-DMS) is emerging as a promising tool for VOC based disease diagnostics. DMS is a form of field asymmetric waveform ion mobility spectrometry (FAIMS), that identifies molecules according to their difference in ion behavior under alternating low and high electric fields (Anderson et al., 2008). Combining DMS with GC allows for the profiling of complex VOC blends with high sensitivity and an analysis time faster than that of a standard GC. The system is portable and, in contrast to the e-nose, can build libraries based on chemical standards (Anderson et al., 2008).
Davis and coworkers applied GC-DMS to determine VOC fingerprints characteristic of the early onset of citrus greening disease (Aksenov et al., 2014). The method was found to be about 90% accurate at early stages of infection before trees do show visual symptoms and PCR based detection methods are applied. The measurements were paired with SPME-GC-MS analysis (using Twisters®) to determine the identities of the most discriminating compounds between healthy and infected trees (Figure 1.6).
FIGURE 1.6 VOC monitoring by GC-DMS from the canopy of citrus trees to diagnose citrus greening disease, (a) Field sampling with a GC-DMS device, (b) GC/DMS plots representing VOC signatures for healthy (noninoculated) (top) and inoculated (middle) trees, which were asymptomatic for the disease. VOC signatures show differential abundances of compounds in inoculated and noninoculated trees. The differences are plotted on the Student’s t-test plot (/;<0.05) (bottom). (Reprinted from Aksenov, A.A. et al., Anal. Chem., 86, 2481-2488, 2014. With permission.)