Food Chemistry 370 (2022) 130910

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Identification of volatile biomarkers for high-throughput sensing of soft rot

and Pythium leak diseases in stored potatoes
Worasit Sangjan a, Afef Marzougui a, D. Scott Mattinson b, Brenda K. Schroeder c,
Austin A. Bates c, Lav R. Khot a, d, Sindhuja Sankaran a, d, *
a
Department of Biological Systems Engineering, Washington State University, 1935 E. Grimes Way, PO Box 646120, Pullman, WA, USA
b
Department of Horticulture, PO Box 646414, Washington State University, Pullman, WA, USA
c
Department of Entomology, Plant Pathology and Nematology, University of Idaho, 875 Perimeter Drive MS 2329, Moscow, ID, USA
d
Center for Precision and Automated Agricultural Systems, Washington State University, 24106 N. Bunn Road, Prosser, WA, USA

A R T I C L E I N F O A B S T R A C T

Keyword: Soft rot and Pythium leak are postharvest storage diseases of potato tubers that can cause substantial crop losses
Potato rot in the US. This study focused on detecting volatile organic compounds (VOCs) associated with rot inoculated
Disease detection tubers during storage (up to 21 days) using headspace solid-phase microextraction (SPME) coupled to gas
Volatile organic compounds
chromatography (GC) with mass spectrometry (MS) and flame ionization detector (FID) analysis. Russet Burbank
Headspace sampling
Solid-phase microextraction
and Ranger Russet tubers were inoculated with the rot pathogens. Static sampling with 50 min trapping time
Gas chromatography followed by GC–MS and GC–FID analysis identified 23 and 30 common VOCs from the pathogen inoculated
tubers. Overall, n,n–dimethylmethylamine, acetone, 1–undecene, and styrene, occurred frequently and repeat­
ability in inoculated samples based on GC–MS analysis, with the latter two found using GC–FID analysis as well.
Identification of such biomarkers can be useful in developing high-throughput VOC sensing systems for early
disease detection in potato storage facilities.

1. Introduction the process industry and consumer demands. However, it relies on

Potato (Solanum tuberosum L.) is a high-value crop with remarkably
high yield potential and relevant to global food security (Devaux, Kro­
mann, & Ortiz, 2014; Drewnowski, Rehm, & Zhang, 2013). It is
commonly produced in the temperate zone of the northern hemisphere
(Food and Agriculture Organization of the United Nations (FAO), 2019).
About 65% of the produce is used for human consumption, 13% as an­
imal feed and about 10% are reserved as seed. The remaining 12% is
applied in other sectors, such as in ethanol production, paper factories,
and the chemical industry (Devaux et al., 2020; Izmirlioglu & Demirci,
2015). In the US, potato is one of the leading horticultural crops with a
total annual production yield of over 22 million tons (~49.70 t/ha)
(United States Department of Agriculture (USDA) — National Agricul­
tural Statistics Service (NASS), 2019). Potato has a nutrient density,
which can contribute to a balanced diet. Also, it has massive demand in
the processing industry to produce frozen potato products (41%) and
chips (21%) (Bohl & Johnson, 2010; National Potato Council, 2020).
The year-round availability of potato tubers is thus crucial to meet
effective postharvest storage management. The bulk storage losses of
tubers in the U.S. can be up to 7.5% annually, with significant losses due
to storage rots (Lui et al., 2005). These storage rot pathogens also
negatively affect tuber quality. The major storage rot diseases include
pink rot (caused by Phytophthora erythroseptica), dry rot (caused by
Fusarium sambucinum), late blight (caused by Phytophthora infestans),
and Pythium leak (caused by Pythium ultimum) (Olsen, Miller, & Nolte,
2006). In addition, Pectobacterium carotovorum subsp. carotovorum
[formerly Erwinia carotovora subsp. carotovora] that causes soft rot is one
of the top ten most devastating storage diseases (Mansfield et al., 2012).
This bacterium often exhibits opportunistic nature, can spread rapidly,
and serve as a secondary invader after the initial infection by other
pathogens (Olsen & Kleinkopf, 2020).
Effective in-field disease control, storage facility management, and
early rot detection—as potato tubers can be stored up to 12 months
depending on location and the demand–supply cycle—are key factors to
reducing these losses. Potato tubers can be infected in many ways, and
many of the pathogen infections do not typically originate in the storage

* Corresponding author at: Department of Biological Systems Engineering, Washington State University, 1935 E. Grimes Way, PO Box 646120, Pullman, WA, USA.
E-mail address: sindhuja.sankaran@wsu.edu (S. Sankaran).

https://doi.org/10.1016/j.foodchem.2021.130910
Received 5 January 2021; Received in revised form 16 August 2021; Accepted 17 August 2021
Available online 4 September 2021
0308-8146/© 2021 Elsevier Ltd. All rights reserved.
W. Sangjan et al.
but are carried into storage as latent infections (Olsen, 2014; Rutolo,
Clarkson, Harper, & Covington, 2018). The amount of inoculum, i.e.,
spores or bacteria present on the tubers, and factors such as improper
ventilation, temperature, humidity, and carbon dioxide level in a stor­
age facility can influence disease development (Alamar, Tosetti, Land­
ahl, Bermejo, & Terry, 2017; Potato Council, 2018). Pathogens gain
entry into the tuber through wounds that occur during harvest (Wang,
Naber, & Crosby, 2020). The storage pathogens cause potato tubers to
rot and decay. This process results in a release of moisture that needs to
be removed from the storage facilities, as high humidity will enhance
soft rot development (Olsen & Kleinkopf, 2020). Identification of the
initiation of rots and spread in a pile is done by visual monitoring by
observing sinkage in the piles and associated rotting smell in the facility
by the managers. Such assessments of large potato piles for early rot
detection are often subjective and possibly lately for remedial measures.
Hence, the industry needs sensing derived from early rot detection
approaches. Using volatile organic compounds (VOCs) released by the
rotting tubers could be a viable sensing approach. The VOCs can be
utilized as a sign/an indicator or biomarker to examine the stored crop’s
morphological and physiological status without destruction (Sankaran,
Mishra, Ehsani, & Davis, 2010; Toivonen, 1997). Researchers have
developed several techniques to trap and adsorb VOCs and apply iden­
tification methods such as gas chromatography–mass spectrometry
(GC–MS), GC–flame ionization detector (GC–FID), gas sensors, and field
asymmetric ion mobility spectrometry (FAIMS) for the early disease
detection in commercial potato storage conditions. Supplementary Data,
Table S1, reports the information on these analytical techniques used for
rot detections in potato tubers.
In this study, two analytical methods, GC–MS and GC–FID, were
evaluated to identify and quantify the VOCs released from Russet Bur­
bank and Ranger Russet potato tubers inoculated with P. carotovorum
subsp. carotovorum or P. ultimum causing soft rot and Pythium leak under
bulk storage conditions. These two techniques were utilized as a paral­
lel/alternative approach to sample the volatile profiles. The hypothesis
was that tubers would release some key VOCs at variable conditions
(multiple potato varieties, pathogens, storage conditions) that can be
used as biomarkers for early rot detection. The GC–MS and GC–FID re­
sults were validated using the standard chemical compounds to obtain
consistent, reliable, and accurate measurement data. This study also
reports headspace sampling protocol optimization to capture VOCs as
well as the sensitivity of solid-phase microextraction (SPME) fibers with
either of carboxen–polydimethylsiloxane (CAR/PDMS) or poly­
dimethylsiloxane–divinylbenzene (PDMS/DVB) coatings. Headspace
sampling using the SPME technique is a simple yet highly sensitive
method for VOC extraction. Nevertheless, it is vital to optimize the
extraction process before sampling, especially on a complex matrix. The
two modes of headspace sampling, static or dynamic, were optimized.
The static mode involves the headspace air not being disturbed between
the SPME’s fiber and the sample during the VOC trapping. Contrarily,
the headspace air is circulated in the dynamic sampling mode (Razote,
Jeon, Maghirang, & Chobpattana, 2002).
In our previous studies (Sinha, Khot, Schroeder, & Si, 2017; Sinha,
Khot, Schroeder, & Sankaran, 2018), a portable FAIMS was used to
detect soft rot disease in Russet Burbank potato tubers stored at room
temperature. The FAIMS was able to reveal the development of storage
rot infections in tubers (P. carotovorum subsp. carotovorum) with the
ability to detect rot symptoms within a day after inoculation. To further
develop the FAIMS technique for early rot detection, we need to
customize the instrument to identify specific biomarkers associated with
storage rot conditions. Therefore, the rationale behind this study was
that understanding the release and identification of specific biomarkers
will allow the development of a high-throughput volatile sensing system
targeting these biomarker compounds for early disease detection in
potato storage facilities.
2
Food Chemistry 370 (2022) 130910
2. Materials and methods
2.1. Sample preparation
Potato tubers of Russet Burbank and Ranger Russet varieties, rep­
resenting major certified seed potato and commercial variety acreage in
the US (National Potato Council, 2019), were obtained from grower/
seed cooperators in Washington and Montana (2018 harvest season).
Both varieties are often bulk stored for up to 12 months after harvest.
Potato tubers appearing healthy and uniform in size (150–200 g) were
selected, washed to remove dirt and excess soil, and surface-sterilized in
10% sodium hypochlorite solution for five minutes. The tubers were
cleaned and rinsed once again with sterile water for five minutes, dried
under a laminar flow, placed in a plastic bin, and kept overnight in the
dark at room temperature before inoculation.
Soft rot pathogen, Pectobacterium carotovorum subsp. carotovorum
(isolate Ec101, Dung, Johnson, & Schroeder, 2014) was grown on
nutrient broth yeast extract in agar plates overnight at 28 ◦ C (Vidaver,
1967). Five mL of sterile water was added per plate, and the plates were
scraped with a sterile hockey stick. The bacterial slurry liquid was then
pipetted into a sterile 50 mL centrifuge tube, and sterile water was added
to 50 mL in total, resulting in an inoculum concentration of about 6 × 108
colony forming units (CFU) per mL. This concentration of inoculum
standardized in this fashion was applied to ensure consistent rot devel­
opment in the tubers. Ten mL aliquots were then transferred into five
sterile 15 mL centrifuge tubes (Dung, Johnson, & Schroeder, 2014), and a
new tube was used to inoculate each replicate for reducing the chance of
cross-contamination between the replicates. The injection sites on the
tubers’ surface were sterilized using 70% ethanol. A sterile #11 scalpel
(Bard-Parker 371611) was immersed in the 15 mL centrifuge tube con­
taining P. carotovorum subsp. carotovorum and used to puncture the
periderm of the tuber. The scalpel was inserted into the tuber to a depth
of 3 cm, and ten insertions were made about 1 cm apart on a single potato
tuber. Each insertion introduced about 3 × 107 to 3 × 108 CFU per tuber.
The scalpel blade was cleaned with a cotton swab soaked in 70% ethanol
between stab inoculations and changed with every replicate of five tu­
bers. Potato tubers were inoculated with sterile water using the same
protocol, which served as a control treatment (non-inoculated control).
Pythium leak pathogen, Pythium ultimum (isolate Pu17, Kothawade,
Sankaran, Bates, Schroeder, & Khot, 2020) was grown on potato dextrose
agar modified with 4 g/L casamino acids vitamin assay (Difco Sigma
Aldrich, USA) plates at room temperature under black light on a 12 h on
and off-cycle. A sterilized size three-core borer (7 mm) was applied to
make mycelial plugs that was used to inoculate potato tubers. A similar
sterilization method of the tuber’s surface before inoculation was per­
formed. The scalpel was cleaned between inoculations and changed be­
tween each replication as described above. The only difference was the
technique of applying inoculum; a sterile scalpel (#11, refers to the long
triangular blade used for shallow stabbing incisions, Sigma Aldrich, USA)
was used to cut a small circle of tuber tissue 2 cm in diameter and 1 cm
deep into the tissue. The scalpel was then utilized to transfer the mycelial
plug to the tuber wound, and the potato plug was placed on the mycelial
plug and pushed back in place. Tubers were inoculated with agar plugs
with no mycelium to serve as a control treatment.
Following the inoculum preparation and inoculation processes,
inoculated or control tubers were then transferred to replicates of glass
sampling jars. All 3.79 L glass jars/chambers (Specialty Bottle, WA, USA)
were sterilized in an autoclave before experimentation. A customized
Petri dish containing 50 mL of sterile water was placed at the chamber’s
bottom to generate a saturated atmosphere to enhance disease develop­
ment. Sterile stainless-steel supports were used to hold the tubers above
the Petri dish to avoid contact with the water (Supplementary Data,
Fig. S1). Five potato tubers from the inoculated treatment (P. carotovorum
subsp. carotovorum or P. ultimum) were placed in the glass chamber and
considered as one replicate sample of inoculated treatment. Similarly,
another five tubers inoculated with sterile water/agar plugs with no
W. Sangjan et al.
mycelium were placed in the chamber, considered as one replicate
sample of the control treatment. The chambers were then sealed using a
food-grade cling film to create aerobic conditions during the storage.
2.2. Sampling protocol optimization
Soft rot pathogen and Russet Burbank potato variety were utilized for
sampling protocol optimization, as shown in Fig. 1. In the optimization
experiment, the potato tubers were prepared and inoculated with
pathogen to induce soft rot on Russet Burbank stored at 24 ◦ C, as
described in the previous section. There were eight glass chambers,
consisting of four replicates of inoculated and four replicates of sterile
water control treatment samples. The pathogen development was
monitored on 7 and 21 days after inoculation (DAI). The two modes,
static and dynamic headspace sampling, coupled with GC–FID analysis,
were used to find a reasonable method for trapping VOCs.
During headspace sampling, the cover to seal the chamber was
changed from the cling film to a sterilized customized metal cap con­
taining one septum port for injection of the SPME with a coating of 0.65
µm PDMS/DVB (Supelco Co., PA, USA). After one hour of headspace
accumulation at room temperature, the SPME was injected in the
chamber as presented in Supplementary Data, Fig. S1, and the fiber was
exposed to the headspace for 30 or 50 min (referred to as Ht hereafter)
and immediately desorbed in the GC–FID injector. The static (Supple­
mentary Data, Fig. S1a) and dynamic (Supplementary Data, Fig. S1b)
headspace sampling modes were tested in the volatiles’ extraction pro­
cess. The difference between the two modes was a filtered airflow (25
mL/min) circulated inside the chamber during the dynamic headspace
sampling, allowing volatile molecules to be adsorbed to the SPME fiber.
The different headspace trapping times (Ht: 30 and 50 min) were also
evaluated because the time influences the partition of solutes between
the headspace and the fiber coating, directly influencing the SPME
performance (Reto, Figueira, Filipe, & Almeida, 2007).
In another optimization experiment, two specific SPME coatings, i.e.,
CAR/PDMS (24-gauge needle size with 0.566 mm outer diameter and
1.93 µL/25.40 mm dead volume, and 75/85 µm sorbent film thickness)
and PDMS/DVB (24-gauge needle size and 65/70 µm sorbent film thick­
ness) were assessed for their sensitivity analysis using GC–MS technique,
as described in Fig. 1. The Russet Burbank tubers were inoculated with
P. carotovorum subsp. carotovorum following a similar protocol described
above using static sampling mode with Ht of 50 min. The tubers were
stored at 24 ◦ C for up to 7 DAI, and two replicates of inoculated and two
replicates of control treatment samples were used to extract VOCs with
two SPME coatings. The samples were analyzed with the GC–MS system to
evaluate these two SPME coatings’ performance. GC–MS was applied
because it can identify the VOCs used to analyze the SPME coatings’
performance and cross-check with the VOCs from previous studies (Table
S1). The VOC trapping protocol was developed from the above experi­
mental methods and used in data collection as described below.
Fig 1. Summary of data acquisition and analysis protocol utilized in this study.
3
Food Chemistry 370 (2022) 130910
2.3. Experimental protocol for VOC data collection
The summary of the experiments and data collection process was as
reported in Table 1. Each experiment included both treatments with
inoculated and control samples. The potato tubers were inoculated with
P. carotovorum subsp. carotovorum or P. ultimum as presented in the
previous section. The samples were stored at 24 ◦ C condition. Pertinent
to each sample, VOC profiles were monitored non-invasively at different
sampling time points on 0, 7, 14, and 21 DAI. The collection of VOCs
released by sample replicates was conducted following static headspace
sampling mode at Ht of 50 min. The samples were characterized by both
GC–MS and GC–FID analysis. In addition, the experiment from Russet
Burbank and Ranger Russet potato tubers inoculated with
P. carotovorum subsp. carotovorum as inoculated treatment and sterile
water as control treatment stored at 10 ◦ C was also conducted with the
same protocol as described above. The related sampling time points
were 0, 14, and 28 DAI for this reduced temperature condition.
2.4. GC–MS and GC–FID analysis
At the beginning of every sampling time point, the SPME fiber was
pre-conditioned for approximately five minutes in the GC injector at
100 ◦ C prior to starting the headspace sampling. Additionally, static
headspace sampling using SPME with an empty sealed chamber (storage
setup unit without potatoes) was completed and analyzed under the
same conditions for both GC analyses to ascertain the possibility of other
volatile compounds being emitted from the experiment chamber (blank
sample).
GC–MS was utilized to identify VOCs using an Agilent 6890 GC
equipped with an Agilent 5973 quadrupole mass spectrometer (Agilent
Technologies, Santa Clara, CA, USA) and a ZB–1 MS capillary column
(60 m × 0.32 mm; film thickness 1.00 µm) (Phenomenex, Torrance, CA,
USA) by sampling two replicates for each treatment and at each sam­
pling time point. The splitless injector’s temperature and the transfer
line were kept constant at 250 ◦ C and 150 ◦ C, respectively. The column
temperature was held at 33 ◦ C, and the fiber was retrieved after 4 min,
followed by an increase to 50 ◦ C at a rate of 2 ◦ C/min, followed by 5 ◦ C/
min to a final temperature of 225 ◦ C/min and held for 5 min. Electron
impact mode was set at 70 eV, and the molecular weight values were set
in the range of 30 to 250 atomic mass units. Compounds were identified
by matching their mass spectra with the Wiley/NIST library of standard
compounds (John Wiley & Sons Inc., Hoboken, NJ) and qualified using
Agilent ChemStation software (Agilent Technologies, Santa Clara, CA,
USA). Over 85% match threshold was utilized during compound
identification.
During GC–FID analysis, the SPME was injected directly into a
Hewlett Packard 5890 Series II GC (Agilent Technologies, Santa Clara,
CA, USA) coupled with a flame ionization detector. Both GC–MS and
GC–FID systems were set in the splitless injection mode. The separation
W. Sangjan et al.
was achieved on a DB–1 MS capillary column (60 m × 0.32 mm; film
thickness 0.50 µm) (Agilent Technologies, Santa Clara, CA, USA). The
same temperature program as the GC–MS analysis was utilized during
the GC–FID analysis. The temperature increased at the rate of 2 ◦ C/min
to 50 ◦ C/min, then at a rate of 5 ◦ C/min until 225 ◦ C was reached and
maintained for 5 min. The total time per SPME run was 50 min with both
the inlet and detector temperatures held constant at 200 ◦ C.
Moreover, the identification using GC–MS and GC–FID analysis were
validated by comparing the GC retention times (RTs) with the standard
chemical compounds, which also were identified by mass spectra, as
shown in Fig. 1. The chemicals were purchased from Sigma-Aldrich
(Sigma-Aldrich, St. Louis, MO, USA). Each compound was further
identified by GC–MS, therefore matching the authentic compound to the
Wiley/NIST library and comparing it to the given compounds per their
elution order. The compounds were individually diluted in distilled
water, mixed, and then further delivered into a 4 mL SPME vial with
30% NaCl. The compounds were analyzed at levels from 4.20 ug/ml for
acetone; 0.86 ug/ml for dimethyl disulfide; 0.49 ug/ml in 50% methanol
for 3–hydroxyl–2–butanone; 0.45 ug/ml for indole; 17 ug/ml for n,
n–dimethylmethylamine and 2–propanol; 0.45 ug/ml for styrene; and at
0.85 ug/ml for 1–undecene and 2–undecanone. In addition, each com­
pound was analyzed a second time by SPME desorption into the GC–FID
system to absolutely relate the retention times of the unknowns in
relation to the GC–MS. Finally, the Kovats retention index (RI) was
calculated for each compound listed here as a standard and reported.
The RI was performed by running a series of straight-chain alkanes
(C4–C14) on each GC system.
2.5. Data analysis
In the optimization experiment, two headspace sampling modes and
two trapping times were evaluated. Thus, evaluated were 64 GC–FID
profiles (2 sampling modes: static/dynamic × 4 replicate samples × 2
treatments: inoculated/control × 2 sampling time points: 7/21 DAI × 2
Ht: 30/50 min) from this experiment to identify a suitable headspace
sampling mode based on the number of peaks and associated peak in­
tensities. Similarly, during the optimization of fiber coatings, 32 GC–MS
profiles (2 sampling modes: static/dynamic × 2 replicate samples × 2
treatments: inoculated/control × 1 sampling time points: 7 DAI × 2 Ht:
30/50 min × 2 SPME coatings: CAR/PDMS and PDMS/DVB) were
analyzed.
The total number of VOC sample profiles from GC–MS analysis
during tuber experiments (Table 1) was approximately 64 (4 experi­
ments with two pathogens and two tuber varieties × 2 treatments × 2
replicate samples × 4 sampling time points), and the percentage
occurrence of biomarkers was ranked after the dataset was accumulated
and pre-processed. The percent occurrence was computed by dividing
the number of known events (samples with peak presence) with the total
number of events (sample size) and presenting the data as a percentage.
Regarding GC–FID analysis, the total number of VOC sample profiles
was 96 (4 experiments with two pathogens and two tuber varieties × 2
treatments × 4 replicate samples × 3 sampling time points). Each VOC
profile from GC–FID analysis was represented by the RT and the relative
area under each peak curve. However, the VOC profiles varied slightly
Table 1
Summary of experimental details during GC–MS and GC–FID analysis performed to identify potato rot related biomarkers over 21 days storage period.
Experiment Diseasea Potato Varietyb Storage Temperature,◦ C
1 Pcc RB 24
2 Pcc RR 24
3 PU RB 24
4 PU RR 24
a
Pcc: P. carotovorum subsp. carotovorum, PU: P. ultimum; b RB: Russet Burbank, RR: Ranger Russet; c About two inoculated and two control replicate samples were
analyzed per experiment; and d M: Analysis with MS, M/F: Analysis with both MS and FID.
Food Chemistry 370 (2022) 130910
between samples/experiments. Therefore, the dataset was pre-processed
using a custom-developed MATLAB algorithm (version 2017a, The
MathWorks, Natick, MA, USA). Initially, the blank VOC profiles (SPME
fiber and empty chamber sample) were subtracted from all the sample
VOC profiles. A data matrix was then generated by aligning the peaks
between samples and merging for convenient analysis. Finally, the data
were analyzed using a custom-developed algorithm in R Studio (version
3.2.5, R core Team, Vienna, Austria) to identify peaks and their simi­
larities as well as differences, as described in Fig. 1.
In regard to the identification of common peaks from GC–FID anal­
ysis between inoculated and control treatment samples based on the four
experiments (Table 1), the peaks appearing in two or more replicate
samples in both treatments (inoculated and control) at each sampling
time point (e.g., 7, 14, and 21 DAI) were identified and considered for
statistical analysis. Moreover, these peaks were also present in two or
more experiments (of 4 experiments, as described in Table 1). Similarly,
the unique peaks (inoculated samples only) present in at least two
replicate samples and two experiments were also identified and
considered for statistical analysis.
2.6. Statistical analysis
Statistical analysis was performed to compare the area of common
peaks between inoculated and control samples or unique peaks between
two pathogens and varieties. Four replicate samples were prepared for
each condition (varieties, pathogens, and treatment). Initially, the
Shapiro-Wilk’s significance test, the density plot analysis, and the
quantile–quantile plot analysis were performed to test for normality.
Finally, Wilcoxon Rank Test was performed to compare the datasets.
Results were inferred at 5% level of significance. Furthermore, the
principal components analysis (PCA) was applied to detect the coherent
pattern in the GC–FID based response as different clusters between
inoculated and control treatment samples.
3. Results and discussion
3.1. Optimization of sampling protocol
The number of peaks or VOCs detected using GC–FID analysis for
Russet Burbank tubers inoculated with P. carotovorum subsp. carotovorum
varied based on the headspace sampling mode and trapping time, as
presented in Fig. 2a. The GC–FID analyzer run with static sampling mode
for Ht of 50 min detected more than 550 peaks per treatment. On the
other hand, roughly 330 peaks were detected per treatment with dy­
namic sampling mode at Ht of 50 min. Moreover, either of the two
headspace sampling modes with Ht of 50 min generated more peaks than
Ht of 30 min (Fig. 2a). In addition, the peak intensity was affected by the
trapping time (Fig. 2b). The Ht of 50 min resulted in detecting a higher
VOC concentration with over 104 GC area units. The static sampling
mode detected up to 40 peaks in the range of 104 to 105 GC area units,
while the dynamic mode could detect only one peak in a similar range in
the control treatment samples (Fig. 2b). Static headspace sampling mode
could also trap the compounds having the intensity in the range of 1 to 5
× 105, and some were over 5 × 105 GC area units.
No. of Replicates per Treatment No. of Replicates Analyzed using GC Sampling Day, DAI
Inoculated Control MSc FID 0 7 14 21
4 4 4 8 Md M/F M/F M/F
4 4 4 8 M M/F M/F M/F
4 4 4 8 M M/F M/F M/F
4 4 4 8 M M/F M/F M/F
4
W. Sangjan et al. Food Chemistry 370 (2022) 130910

Fig 2. Optimization results: (a) number of peaks from tubers inoculated with P. carotovorum subsp. carotovorum using different sampling modes and headspace
trapping time; and (b) the number of peaks with observed peak area range. The optimization experiment had control (C) and inoculated (I) replicate samples where
headspace was sampled using static (S) and dynamic (D) modes with 30 min (30) and 50 min (50) trapping times.

Table 2
Percent occurrence of VOCs detected during the GC–MS analysis of Russet Burbank and Ranger Russet potato tubers inoculated with P. carotovorum subsp. carotovorum
or P. ultimum and stored at 24 ◦ C.
VOC No Total Occurrence, % Volatile Organic Compound RT, min Occurrence at Sampling Period, %

0 DAI 7 DAI 14 DAI 21 DAI

(na = 6) (n = 10) (n = 9) (n = 9)
b c
Common VOC 1 85 Unknown 31.80 100 100 78 67
2 44 Decanal 38.90 100 50 22 22
3 12 3,7–dimethyl–3–octanol 34.20 67 0 0 0
4 9 Dodecanol 45.40 50 0 0 0
5 6 Hexanal 21.00 33 0 0 0

VOC from inoculated treatment only 1 71 N,N–Dimethylmethylamine 3.80 50 50 89 89

2 65 1–Undecene 34.40 50 60 78 67
3 53 Styrene 25.50 0 10 89 100
4 53 Acetone 4.40 0 30 78 89
5 47 3–Hydroxy–2–butanone 13.50 17 60 44 56
6 38 3–Methyl–1–butanol 16.30 33 30 44 44
7 29 2–Undecanone 40.70 0 20 44 44
8 29 Ethanol 4.00 0 30 22 56
9 24 2–Propanol 4.70 0 0 33 56
10 24 1–Octanol 26.00 0 0 33 56
11 24 Indole 40.50 0 0 11 78
12 21 Dimethyl disulfide 16.70 33 20 33 0
13 18 1,2–Dimethoxybenzene 35.30 0 0 11 56
14 18 2–Methyl–1–butanol 16.60 0 20 11 33
15 12 Methoxymethyl–benzene 29.80 0 0 0 44
16 9 3–Octanone 29.40 0 0 22 11
17 9 Methoxybenzene 26.40 0 0 11 22
18 9 4–Ethyl–1,2–dimethoxybenzene 41.40 0 0 11 22
19 9 2–Nonanone 33.80 0 10 0 22
20 6 1–Butanol 11.90 0 10 0 11
21 6 2–Ethyl–1–hexanol 32.50 0 10 0 11
22 3 2–Butanone 8.20 0 0 0 11
23 3 2–Pentanone 12.40 0 0 0 11
a
n: Sample size of inoculated samples; bThe total occurrences (n = 10) in control samples for unknown compound at 31.80 min RT, decanal, 3,7–dimethyl–3–octanol,
dodecanol, and hexanal were 80–100% (0–21 DAI), 60–90% (0–21 DAI), 10–30% (0–7 DAI), 40% (0 DAI), and 30% (0 DAI), respectively; and cCompound not found
from the database in the MS library.

5
W. Sangjan et al.
When GC–MS analysis was performed to assess the fiber perfor­
mance, results revealed that SPME coated with CAR/PDMS trapped
VOCs better, hence the high numbers of peaks compared to PDMS/DVB
(Supplementary Data, Fig. S2). Nevertheless, blank normalized data
showed similar results for both fibers (data not shown). These results
included three VOCs commonly found in rotting potato tubers: dimethyl
disulfide (RT = 16.7 min), styrene (RT = 25.5 min), and 3–octanone (RT
= 29.4 min). Overall, the static headspace sampling mode using the
PDMS/DVB SPME coating with a Ht of 50 min was used in this study.
Fig 3. Statistical analysis of common peaks detected during the GC–FID analysis from control and inoculated samples.
6
Food Chemistry 370 (2022) 130910
3.2. GC–MS analysis
Static headspace sampling followed by SPME coupled with GC–MS
analysis on the samples, detected six typical peaks in both control and
inoculated treatments (major ones reported in Table 2) and one peak
(nonanol) appearing only in the control treatment samples. The five
peaks could be detected early at the 0 DAI, with an unknown compound
at RT of 31.80 min and the decanal that appeared at every sampling time
point on both control and inoculated samples. The analysis of VOCs
W. Sangjan et al.
appearing only from the inoculated treatment samples allowed the
identification of 23 compounds and their repeatability in terms of
occurrence frequency at each sampling time is reported in Table 2. The
two frequently occurring compounds as a result of rot were n,
n–dimethylmethylamine and 1–undecene. Additional consistently
appearing compounds, at each sampling time point, included
3–hydroxy–2–butanone, 3–methyl–1–butanol, and dimethyl disulfide.
In another scenario, GC–MS analysis from two tuber varieties (Russet
Burbank and Ranger Russet) inoculated with P. carotovorum subsp.
carotovorum or sterile water stored at 10 and 24 ◦ C were analyzed. About
21 unique compounds originating from the inoculated treatment were
detected (major peaks summarized in Supplementary Data, Table S2).
Results also show that n,n–dimethylmethylamine and 1–undecene were
the two major VOCs that appeared in all sampling time points, similar to
the results described in Table 2. Furthermore, as observed from Table 2
and Supplementary Data, Table S2, n,n–dimethylmethylamine,
1–undecene, 3–hydroxy–2–butanone, and 3–methyl–1–butanol were
consistently detected at every sampling time point. Nonetheless, there
were some differences in peaks based on the GC–MS analysis of tubers
inoculated with P. carotovorum subsp. carotovorum between samples
stored at 10 and 24 ◦ C. Overall, the number of peaks and peak intensity
were lower at 10 ◦ C, as presented in Supplementary Data, Fig. S3.
In addition to the compounds that were commonly found between
soft rot and Pythium leak inoculated samples, efforts were made to
identify peaks present in inoculated samples of one pathogen, not the
other. GC–MS analysis confirmed 4–ethyl–1,2–dimethoxybenzene and
methoxybenzene as the compounds related to Pythium leak samples
stored at 24 ◦ C, absent in soft rot inoculated samples. Overall, the
dominant VOC based on GC–MS analysis, n,n–dimethylmethylamine
detected only from inoculated treatment, has not been reported previ­
ously. In contrast, other VOCs such as 1–undecene, acetone, ethanol,
dimethyl disulfide, 1,2–dimethoxybenzene, 1–butanol, 2–butanone, and
2–pentanone were previously reported in association with tubers
infected with soft rot pathogen (de Lacy Costello et al., 1999; Lui et al.,
2005; Ouellette, Raghavan, Reeleder, & Greenhalgh, 1990; Varns &
Glynn, 1979; Waterer & Pritchard, 1985).
3.3. GC–FID analysis
3.3.1. Common peaks between inoculated and control treatment samples
Nine common peaks were found from the experiments at 24 ◦ C
storage temperature, as presented in Fig. 3. In addition, the peak at RT of
36.28 min was detected in both tuber varieties inoculated with
P. carotovorum subsp. carotovorum stored at 10 and 24 ◦ C. Supplemen­
tary Data, Table S3, summarizes the results demonstrating that many
common peaks were neither normally distributed nor significantly
different. Three peaks (RT = 25.86, 26.14, and 26.59 min) showed peak
area differences between samples inoculated with P. carotovorum subsp.
carotovorum or P. ultimum and control. Supplementary Data, Fig. S4,
presented the result of different clusters between inoculated and control
treatment samples at 14 and 21 DAI from the experiments at 24 ◦ C
storage temperature condition.
3.3.2. Unique peaks in inoculated treatment samples
There were 30 unique peaks detected from tubers inoculated with
P. carotovorum subsp. carotovorum or P. ultimum that are common be­
tween both pathogens (Supplementary Data, Fig. S5). Two peaks at RT
of 16.99 and 26.75 min occurred in more than 50% samples, and their
average peak area was around 28 × 103 and 41 × 103 GC units,
respectively. The maximum average peak area was 345 × 103 GC units
detected at RT of 3.17 min. In general, the average peak area was about
57 × 103 GC units for these unique peaks (Supplementary Data, Fig. S5).
These 30 unique peaks were filtered to evaluate the differences be­
tween tubers inoculated with different pathogens at each sampling time
point (Supplementary Data, Tables S4 and S5). Overall, there were no
statistical differences between the peaks from tubers inoculated with
7
Food Chemistry 370 (2022) 130910
P. carotovorum subsp. carotovorum and P. ultimum. Comparing different
sampling periods (7, 14, and 21 DAI), two unique inoculated peaks at RT
of 26.75 and 26.93 min were consistently found in all three periods,
which could be used as biomarkers for early detection of both patho­
gens. In addition, peaks with RT of 2.93, 3.00, 3.11, 3.17, 16.99, 27.10,
and 31.67 min were present in at least two sampling time points.
Finally, unique pathogen-specific (P. carotovorum subsp. carotovorum
or Pythium ultimum) peaks released from Ranger Russet and Russet
Burbank tubers were evaluated from the same dataset. Three peaks from
two sampling time points (14 and 21 DAI) were detected as VOCs
released from tubers inoculated with P. carotovorum subsp. carotovorum
(Supplementary Data, Table S6). These peaks were not significantly
different for Russet Burbank and Ranger Russet samples. No such peaks
were detected from tubers of both varieties inoculated with P. ultimum.
3.4. Chemical biomarkers
The GC–MS analysis detected 23 VOCs from inoculated tubers that
were absent in control tubers, while the GC–FID analysis detected 30
VOC peaks. In general, the GC–FID detected more VOCs than GC–MS
analysis. The sensitivity and selectivity comparison of GC–MS and
GC–FID techniques have been reported in some studies. FID has been
reported to be able to detect down to a level of 5 pg with a 107 dynamic
range, whereas MS can detect to a level of 10 pg at a 105 range in the
total ion mode (Buffington & Wilson, 1991). Aparicio-Ruiz, García-
González, Morales, Lobo-Prieto, and Romero (2018) utilized both
GC–MS and GC–FID techniques for analyzing volatile compounds in
virgin olive oil and stated that FID showed good selectivity, linearity,
and applicability at a upper working range; while MS was suitable at a
lower working ranges with better sensitivity and limits of detection and
quantification. FID was reported to be applicable for categorizing virgin
olive oil categories. Van Asten (2002) reported that even if MS detectors
are extremely sensitive, they are not as robust as the FID; thus, sensi­
tivity drift is noticed. Nevertheless, the GC–MS offers the ability to
identify the compounds using the MS database (Kushalappa, Lui, Chen,
& Lee, 2002; Romero, García-González, Aparicio-Ruiz, & Morales, 2015;
Silva et al., 2018).
In this study, the standard chemical compound peaks relating to
major VOCs, as detected by the GC–MS analysis (Table 2), were used for
comparison in both GC–MS and GC–FID analysis. The peak RT of these
chemicals was matched with the corresponding GC–MS and GC–FID
profiles. Table 3 summarizes the results confirming the VOCs associated
with sample potato tubers inoculated with P. carotovorum subsp. car­
otovorum or P. ultimum. These chemicals matched the peaks identified
using GC–MS analysis (Table 2) and GC–FID analysis (Supplementary
Data, Fig. S5).
Overall, the major biomarkers from Russet Burbank and Ranger
Russet tubers inoculated with P. carotovorum subsp. carotovorum or
P. ultimum stored at 24 ◦ C would be n,n–dimethylmethylamine,
1–undecene, acetone, and styrene based on GC–MS analysis. Amongst
these, 1–undecene and styrene had a high occurrence percentage and
repeatability at different sampling time points as detected using both
GC–MS and GC–FID analysis. The compound 1–undecene appeared at
every sampling time point. These results further support our initial hy­
pothesis and pertinent FAIMS work by Sinha, Khot, Schroeder, and Si
(2017), Sinha, Khot, Schroeder, and Sankaran (2018) that VOC bio­
markers can be used as a reliable and potentially rapid approach for
potato tuber rot detection in stored potatoes. Nevertheless, the bio­
markers need to be further validated with more variable conditions
(other types of rot, potato varieties) and biological samples.
It should be noted that the VOCs and their peak intensities are
influenced by the incubation period, where the rot progression and
disease severity will tend to increase with the incubation period that can
be associated with an increase in pathogen activity. The pathogen ac­
tivity will also increase with raised temperature. Nevertheless, as the
goal was towards rot detection at different rot-development stages
W. Sangjan et al. Food Chemistry 370 (2022) 130910

Table 3
Standard chemical compounds related to major VOCs detected using the GC–MS analysis from inoculated tubers. These compounds were validated and compared to
the retention times from both GC–MS and GC–FID analysis, and percent total occurrence of inoculated VOCs/peaks are reported.
Standard Chemical Compound GC–MS Results GC–FID Results

(Rank) RTb, min RIc (Rank) RTe, min RI Occurrence of Unique

Total Occurrencea, % (n = 34) Total Occurrenced, % (n = 48) Inoculated Peakf

7 DAI 14 DAI 21 DAI

N,N–Dimethylmethylamine (1) 71 3.80 517.6 (7) 31 2.93 541.0 Xg X

1–Undecene (2) 65 34.40 1169.2 (1) 56 26.75 1208.0 X X X
Styrene (3) 53 25.50 963.4 (2) 52 16.99 991.6 X X
Acetone (4) 53 4.40 550.5 (6) 33 3.00 544.9 X X
3–Hydroxy–2–butanone (5) 47 13.50 766.1 (21) 13 5.96 786.3
2–Undecanone (7) 29 40.70 1352.6 (12) 23 32.34 1377.7 X
2–Propanol (9) 24 4.70 570.5 (9) 27 3.17 554.4 X X
Indole (11) 24 40.50 1343.6 (8) 29 31.67 1357.5 X X
Dimethyl disulfide (12) 21 16.70 814.0 (29) 8 7.40 819.6
a
Results from Table 2; b Retention time of the standard chemical compound detected from the GC–MS analysis; c RI: Kovats retention index; d Results from Sup­
plementary Materials, Fig. S5; e Retention time of the standard chemical compound detected from the GC–FID analysis; f Results from Supplementary Materials, Tables
S4 and S5, and n = 16 for each DAI; g X: Present in samples.

across environments, biomarkers were identified by integrating the data
across time points, although the number of peaks and peak intensities
tend to increase with the incubation period.
4. Conclusion
The qualitative analysis of VOC peaks detected by the GC–MS and
GC–FID analysis identified 23 and 30 unique peaks released from Russet
Burbank and Ranger Russet inoculated with either P. carotovorum subsp.
carotovorum or P. ultimum stored at 24 ◦ C, respectively. These com­
pounds are indicative of infection occurring in the potato tubers.
Quantitative GC–FID analysis results revealed no significant differences
in the unique inoculated peaks for Russet Burbank and Ranger Russet
samples, The two key findings from this research are: (1) Validation
using standard chemical compounds indicated that n,
n–dimethylmethylamine, 1–undecene, acetone, and styrene would serve
as biomarkers for Russet Burbank and Ranger Russet tubers infected
with P. carotovorum subsp. carotovorum or P. ultimum using GC–MS
analysis, and (2) Static headspace sampling for 50 min using the PDMS/
DVB coated SPME was optimal for extracting VOCs with low to medium
molecular weights and low to medium boiling points. The proposed
optimization protocols (static/dynamic, SPME coatings) can be relevant
and applicable to other researchers utilizing similar techniques (GC–MS,
GC–FID) for volatile sampling to study food chemistry of crop or pro­
cessed food products.
The release of the VOCs can be associated with the potato tuber
biochemistry and the pathogenicity process of the pathogens. Studying
the changes in VOC profiles considering both variabilities in potato
varieties and pathogens, thus, is important to develop VOC-based sensor
technologies for early disease detection in storage facilities with broader
implementations. This would be one of the key contributions of this
study. The biomarkers identified herein can be utilized for the detection
of potato tuber rot caused by P. carotovorum subsp. carotovorum or
P. ultimum. Potato storages could be monitored using the portable FAIMS
system or similar types of sensors for these biomarkers’ presence and
serve as an early indicator of tuber rot development. This equipment and
methods would provide critical information for storage managers to
measure the stored potato tubers’ overall health and enable informed
decisions to ensure the potato stakeholders’ profitability in monitoring
rot conditions.
CRediT authorship contribution statement
Worasit Sangjan: Methodology, Investigation, Validation, Formal
analysis, Data curation, Visualization, Writing - original draft, Writing -
review & editing. Afef Marzougui: Methodology, Formal analysis,
Writing - review & editing. D. Scott Mattinson: Methodology, Formal
analysis, Investigation, Writing - review & editing, Supervision. Brenda
K. Schroeder: Methodology, Formal analysis, Investigation, Writing -
review & editing, Supervision. Austin A. Bates: Methodology, Investi­
gation. Lav R. Khot: Conceptualization, Methodology, Formal analysis,
Investigation, Resources, Writing - review & editing, Project adminis­
tration, Funding acquisition. Sindhuja Sankaran: Conceptualization,
Methodology, Formal analysis, Investigation, Resources, Writing - re­
view & editing, Project administration, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
This study was funded, in part, by the Washington State Department
of Agriculture, Specialty Crop Block Grant # K2296, and supported by
the USDA National Institute of Food and Agriculture Hatch project #
1014919. We would like to thank Blaine Meek from AgriNorthwest for
providing potato tuber samples and additional support during this
project and thank Dr. Mark Pavek for assisting us with the acquisition of
potato tuber samples.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodchem.2021.130910.
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