Article

pubs.acs.org/JAFC

Identification of Volatile Markers in Potato Brown Rot and Ring Rot

by Combined GC-MS and PTR-MS Techniques: Study on in Vitro and
in Vivo Samples
Sonia Blasioli,*,† Enrico Biondi,† Devasena Samudrala,§ Francesco Spinelli,† Antonio Cellini,†
Assunta Bertaccini,† Simona M. Cristescu,§ and Ilaria Braschi†
†
Department of Agricultural Sciences, University of Bologna, Viale G. Fanin 44, 40127 Bologna, Italy
§
Molecular and Laser Physics, IMM, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands
*
S Supporting Information

ABSTRACT: Ralstonia solanacearum (Rs) and Clavibacter michiganensis subsp. sepedonicus (Cms) are the bacterial causal agents
of potato brown and ring rot, respectively, and are included in the A2 list of quarantine pathogens in Europe. Identification by
GC-MS analysis of volatile organic compounds from Rs or Cms cultured on different nutrient media was performed. GC-MS and
PTR-MS analysis were carried out also on unwounded potato tubers infected with the same pathogens. Infected tubers were
produced by experimental inoculations of the plants. In in vitro experiments, Rs or Cms emitted volatile compounds, part of
which were specific disease markers of potato (2-propanol and 3-methylbutanoic acid), mainly originating from bacterial
metabolism (i.e., amino acid degradation, carbohydrate and fatty acid oxidation). In potato tubers, pathogen metabolism
modified the volatile compound pattern emitted from healthy samples. Both bacteria seem to accelerate metabolic processes
ongoing in potatoes and, in the case of Rs, disease markers (1-hepten-3-ol, 3,6-dimethyl-3-octanone, 3-ethyl-3-methylpentane, 1-
chloroctane, and benzothiazole) were identified.
KEYWORDS: Ralstonia solanacearum, Clavibacter michiganensis subsp. sepedonicus, volatile compounds

■ INTRODUCTION
Brown rot and ring rot caused by bacteria Ralstonia solanacearum
race 3 biovar 2 (Rs) and Clavibacter michiganensis subsp.
sepedonicus (Cms), respectively, are among the most severe
potato diseases worldwide. Rs occurs mostly in Solanaceous
plants (e.g., Capsicum spp., eggplant, potato, and tomato) and
many weeds and wild plants,1 and it is spread wordwide.2,3 Cms
has a host range restricted to potato, tomato, eggplant, and some
Solanaceous weeds; it occurs in northern America, northeastern
Europe, and Asia. Both bacteria are included in the A2 list of
quarantine pathogens in Europe and are subject to EU directives
(2006/63/EC and 2006/56/EC for Rs and Cms, respectively).
Tuber internal symptoms of potato brown rot (see symptoms
shown in Table 1) are discoloration and decay localized at
vascular ring and a bacterial slime oozing from the area until the
complete destruction of tubers.4 When tubers, infected with
potato ring rot, are cut and squeezed, a cheesy cream comes out
from the vascular ring; as the infection develops, destruction of
vascular tissue occurs and cracks appear on the tuber surface.5
The absence of internal symptoms does not exclude the absence
of the pathogens (latent infections).
It is well-known that both microorganisms and plants emit
volatile compounds, some of which may have odors that are
characteristic of the species. As reported in Turner and Magan,6
the microbial species, as well as the type of culture media and
growth time, may influence the amount and pattern of volatile
compounds that are produced. In case these compounds are
formed in the presence of pathogens, they can be used as markers
for their presence. No studies about the volatile molecules
emitted from bacterial cultures of Rs and Cms are reported in the
literature.
Potato flavor has been extensively studied due to its
importance in nutrition: volatile compounds predominantly
include aldehydes, alcohols, ketones, acids, esters, hydrocarbons,
amines, furans, and sulfur compounds. The pattern and the
number of volatile components obtained from potatoes can be
quite different, depending on whether potatoes are raw or
cooked and the cooking method used to prepare them.7 All
studies reported in the literature on raw potato flavor are
conducted on sliced or peeled tubers to favor smell diffusion.
Fungi, prokaryotes (bacteria and mollicutes), parasitic plants,
viruses and viroids, nematodes, and protozoa can modify the
volatile pattern emitted from diseased species.8 Potatoes
inoculated with Pectobacterium carotovorum ssp. carotovorum
and P. carotovorum ssp. atrosepticum,9,10 Phytophthora infestans,
Pythium ultimum, and Botrytis cinerea,11,12 and Fusarium
sambucinum13 produce volatile molecules that can be considered
markers of infection. Potatoes used for these studies have been
wounded before the experimental inoculation. Stinson et al.14
found that potatoes infected with Rs and Cms emit molecules
associated with disease: 3-methyl-2-pentanone has been
indentified as a marker of ring rot, and the variation of peak
intensity of short-chain alcohols and ketones is indicative of
brown rot disease. In this study, potatoes were at the final stage of
Received: August 2, 2013
Revised: December 7, 2013
Accepted: December 8, 2013
Published: December 8, 2013

© 2013 American Chemical Society 337 dx.doi.org/10.1021/jf403436t | J. Agric. Food Chem. 2014, 62, 337−347
Journal of Agricultural and Food Chemistry
Table 1. Phytopathometric Classes of Symptomatic Diseased Potato Tubers; Severity Classes Are Laddered on the Basis of
Vascular Ring Symptoms
infection when the whole tuber rots away; at this stage, other
secondary microorganisms such as Fusarium sp. and Erwinia spp.
or other saprophytic microrganisms can contribute to the
degradation of the tuber tissues15,16 and can affect the visual
symptom analysis, making difficult the discrimination of brown
and ring rot from other tuber rots.
The present work describes part of the results of the Q-Detect
project performed in the EU’s seventh Framework Program
(FP7); its aim was to develop reliable detection methods for
quarantine pests and pathogens to be used by National Plant
Protection Organizations (NPPO) and Inspection Services also
through the detection of volatile organic compounds emitted
from diseased species.
The work was focused on detection of brown rot and ring rot
of potato, by the identification of volatile markers or specific
fingerprints of the diseases using gas chromatography and proton
transfer reaction coupled with mass spectrometry (GC-MS and
PTR-MS) analysis. For the first time, volatile compounds
emitted from (i) bacterial cultures of Rs and Cms (in vitro
assays) and (ii) unwounded potatoes (in vivo assays) without
external disease symptoms and with a low disease severity (to
avoid cross-contaminations due to the presence of other
secondary microorganisms) were investigated.
■
MATERIALS AND METHODS
Chemicals. Reference compounds were injected, when available, to
confirm identification of volatile compounds detected. Methanol,
acetaldehyde, 2-propanone, 2-propanol, 2-butanone, propanoic acid,
3-methylbutanal, 2-methylpropanoic acid, toluene, dimethyl disulfide, 3-
methylbutanoic acid, benzaldehyde, methyl 2-methylbutanoate, benzo-
thiazole, and 1-hepten-3-ol were purchased as pure or analytical
standards from Sigma-Aldrich Co. LLC (USA). Reference volatile
concentration for GC-MS analysis was 100 μM.
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Bacterial Strains. The virulent strains IPV-BO 5836 of Rs and IPV-
BO 7695 of Cms were routinely grown on tetrazolium (TZ)17 and yeast
dextrose calcium carbonate (YDC)18 media at 27.0 ± 0.1 °C for 72−96
h, respectively. The IPV-BO 5836 (Mazzucchi and Traversa,
unpublished data) and IPV-BO 7695 (Mazzucchi and Mucini,
unpublished data) strains were isolated from potato tubers.
In Vitro Assays. Rs and Cms in Solid Media. Preliminary studies
were carried out to identify volatile compounds produced by the
metabolism of Rs and Cms cultured on specific substrates chosen as a
model for their growth. The Rs and Cms strains were streaked on agar
media such as TZ, “levure” peptone glucose (LPG),19 potato dextrose
(PDA; Difco, Becton, Dickinson and Co., USA), and PD prepared using
peeled potato tubers,20 referred to hereafter as natural PD (NPD). The
plates were incubated at 27.0 ± 0.1 °C for appropriate time durations,
based on the growth rate on each specific solid medium: 5 days for Rs on
TZ-agar (TZA; Cms was not cultured on TZA); 2 days on LPG-agar
(LPGA), 11 days on PDA, and 12 days on NPD-agar (NPDA) for both
Rs and Cms. Agar media without pathogens have been used as a negative
control. All experiments were carried out in duplicate.
Rs and Cms in Liquid Media. To follow the development of volatile
compounds as a function of bacterial growth, time course studies were
performed on broth cultures of the pathogens. RS or Cms bacterial
strains were inoculated in TZ (only Rs), LPG, PD, and NPD broths to
obtain an initial suspension (time point = 0 h) of ca. 106 CFU mL−1 for
each pathogen; sterile deionized water (SDW) was added as a negative
control. The inoculated broths were incubated in a rotary incubator at
27.0 ± 0.1 °C at 80 rpm. At days 2 and 7, 1 mL of suspension was
collected, and 10-fold dilutions of the suspension were plated (10 μL
drops) on LPGA and incubated at 27.0 ± 0.1 °C. Each suspension
concentration was determined by counting the number of Rs and Cms
colonies grown after 3 and 5 days of incubation, respectively, when by a
naked eye each single colony was visible. Experiments were carried out
in duplicate.
In Vivo Assays. Infected potatoes were obtained by experimental
inoculations with both bacterial pathogens in potato plants; cultivars
‘Spunta’ and ‘Kennebec’ were chosen as sensitive hosts and inoculated
with Rs and Cms, respectively.
dx.doi.org/10.1021/jf403436t | J. Agric. Food Chem. 2014, 62, 337−347
Journal of Agricultural and Food Chemistry
Rs and Cms Experimental Inoculations. For Rs, ca. 100 potato
plants were grown in the field following standard agronomical
procedures; within the end of blooming time (after 2 months from
the tuber seeding), 30 μL of water suspension containing the pathogen
(ca. 109 CFU mL−1) was experimentally injected into a wound made at
the stem (one-third to two-thirds of the total stems). Cms was
inoculated in two distinct phenological phases (seed and third−fourth-
leaf stage) to increase the disease incidence and the spectrum of
variability of the disease severity in the daughter tubers. Half of the total
plants were experimentally inoculated at the seed stage (around 50
potato tubers) by injection of 180 μL of pathogen water suspension (ca.
109 CFU mL−1) in a well, made at the heal end. The inoculated tubers
were then placed in a dark chamber at 95% humidity for 48 h and then
left for an additional 24 h at 23 ± 1 °C to dry the tubers. The next day the
inoculated tubers were seeded in the field. The remaining plants (about
fifty) were experimentally inoculated at the stem in the third−fourth-leaf
stage (two-thirds of the total stems) following the procedure used for Rs
inoculation. SDW was employed as a negative control.
At approximately 4 months after the inoculation, the tubers from
experimental fields were harvested and stored in a dark climatic chamber
at 4 ± 1 °C. Negative controls and tubers inoculated with the pathogens
were harvested and stored separately to avoid cross-contaminations. At
storing temperature, Rs just survives,3,4,21 whereas Cms decreases its
activity.5,22 Before volatile compound analysis, to promote the
reactivation and, indeed, the bacterial growth of both pathogens, the
tubers were left at room temperature from 1 to 10 days.
Volatile Compounds Headspace Sampling for GC-MS
Analysis. Volatile compounds analysis on in vitro samples were
performed on slices of medium with the grown Rs and Cms strains,
which were placed in glass tubes sealed with Teflon caps (Figure 1a). A
Figure 1. In vitro and in vivo sample preparation for GC-MS analysis:
(a) slices of Rs or Cms cultured agar medium in glass tubes and (b)
unwounded potato tubers infected with Rs or Cms in jars. Both
container types were sealed with a Teflon cap. CAR/PDMS (Supelco)
SPME fibers were used for headspace sampling. In vivo sample
preparation for PTR-MS analysis is reported in (c), where single potato
tubers were placed in glass cuvettes and flushed with 2 L h−1 air for
headspace analysis. Panel d shows a view of the PTR-MS instrument
measuring low molecular weight volatile compounds from tubers placed
in glass cuvettes.
slice of medium without the pathogen was used as a negative control.
Unwounded tubers (healthy or Rs/Cms experimentally inoculated)
without external visible symptoms of diseases, were placed in 500 mL
jars, and the jars were filled to volume and sealed with Teflon caps
(Figure 1b). Due to the low intensity of measured signals, analysis was
carried out on pooled tubers. Eight pooled Rs-diseased samples with 4
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Article
healthy samples as control, and 10 pooled Cms-diseased samples with 5
control samples were analyzed.
Detection of volatile compounds emitted from in vivo or in vitro
samples was performed by sampling the headspace with solid phase
microextraction (SPME) fibers. Supelco 75 μm carboxen/polydime-
thylsiloxane fibers were used to monitor gases emitted from Rs or Cms
cultures and infected potatoes. SPME fibers were exposed for 24 h to
headspace of in vitro solid media and in vivo samples and for 30 min to
headspace of broth samples.
After GC-MS analysis, tubers have been examined by visual analysis of
symptoms on the vascular ring and by bacterial strain reisolation and
identification.
GC-MS Analyses. Volatile compounds adsorbed by SPME fibers
from in vitro and in vivo samples were thermally desorbed in the GC
injector (kept at 250 °C) of a Finnigan Mat ion trap gas chromatograph
GC-MS (Thermo Fisher Scientific, USA) and analyzed for the masses.
Transfer line and source temperatures were kept at 250 and 200 °C,
respectively. Mass spectra were recorded with a 1 s scan time in the m/z
range from 20 to 350 using electronic ionization (ionization energy, 70
eV) as a source. The carrier gas was helium (pressure, 35 kPa).
Chromatographic separation was performed on a fused-silica bonded-
phase capillary column Supelco SPB-5 poly (5% diphenyl/95%
dimethylsiloxane) 30 × 0.32 mm; 0.25 mm film thickness (Sigma-
Aldrich Co. LLC, USA). Volatile compounds were separated by
applying the following GC oven temperature program: 40 °C for 3 min,
raised at 3 °C min−1 to 130 °C, raised at 10 °C min−1 to 260 °C, and held
at 260 °C for 10 min. The identification of volatile compounds was
achieved by comparing their mass spectra with the mass database stored
in the National Institute of Standards and Technology U.S. Government
library (NIST, 1998).
Quantitative analysis of detected volatiles emitted by in vitro samples
was done by integration of peak area, whereas, for in vivo samples, by
area normalization to sample weight. Data were statistically processed
using the semimaximal dispersion (maximal error) or the standard
deviation.
Volatile Compound Headspace Sampling for PTR-MS
Analysis. Potato tubers experimentally inoculated with Rs (18 samples)
and Cms (23 samples) and controls (mocked or healthy, 18 tubers as Rs
and 23 tubers as Cms control) were shipped from Bologna (Italy) and
stored in the refrigerator at Radboud University, Nijmegen (The
Netherlands). A few hours before the PTR-MS measurements, they
were kept in the laboratory at 20 °C. A single unwounded potato was
placed in a leaktight glass cuvette and flushed with 2 L h−1 hydrocarbon-
free air (Figure 1c). An automatic valve system was used to connect up
to five cuvettes to the PTR-MS (Figure 1d) in alternate sequences of 30
min.23 After the volatile compound analyses, the samples were returned
to the Bologna laboratory for establishment of the symptomatic and
asymptomatic features by expert visual inspection.
PTR-MS Analysis. PTR-MS analysis was carried out with a custom-
built mass spectrometer at the Trace Gas Research Group at the
Radboud University, Nijmegen (The Netherlands). A detailed
description of the instrument has been given elsewhere.24,25 Trace
quantities of volatile compounds are sampled directly, and those having
proton affinity (PA) greater than that of water (PA = 691 kJ mol−1)
undergo ion−molecule reactions by receiving a proton from the
hydronium cation, H3O+. The protonated compounds are mass filtered
with a quadrupole mass spectrometer and quantified by a secondary
electron multiplier as mass to charge ratio, m/z. The calibration was
performed with different concentrations ranging from 0.035 to 1 ppmv
(parts per million volume) obtained from a reference mixture of 1 ± 0.05
ppmv (of methanol (m/z 33), acetaldehyde (m/z 45), 2-propanone (m/
z 59), isoprene (m/z 69), benzene (m/z 79), toluene (m/z 93), m-
xylene (m/z 107), and α-pinene (m/z 137) in nitrogen dilution gas
(Linde, Dieren, The Netherlands).
Symptom Analysis. After GC-MS and PTR-MS analyses, each
tuber was cut in half, and the disease symptoms on the vascular ring were
visually analyzed and photographed: to better evaluate the disease
severity, a phytopathometric class ladder was built (Table 1).
Bacterial Pathogen Reisolation and Identification. Sample
Processing. After the symptom visual analysis, from each tuber, at the
dx.doi.org/10.1021/jf403436t | J. Agric. Food Chem. 2014, 62, 337−347
Journal of Agricultural and Food Chemistry Article

Table 2. Volatile Compounds Emitted from Rs and Cms Cultured on Different Agar Media at Different Times from Inoculation (5
Days for Rs Grown on TZA; 2 Days for Rs and Cms on LPGA; 11 Days on PDA; and 12 Days on NPDA) and from Potato Tubers
from Plants That Were Experimentally Inoculated with the Pathogensa
medium
volatile compd
TZA LPGA PDA NPDA potato identificationb
Rs Bacterium
dimethyl disulfide 2-propanone MS, RS
dimethyl disulfide MS, RS
2-butanone MS, RS
3-methylbutanoic acid 3-methylbutanoic acid MS, RS
2-furancarboxaldehyde MS (tentatively
identified)
propanoic acid MS, RS
methyl 2- methyl 2- MS, RS
methylbutanoate methylbutanoate
unknown (m/z 84)
dimethyl trisulfide MS (tentatively
identified)
styrene MS (tentatively
identified)
1-hepten-3-ol MS, RS
3,6-dimethyl-3-octanone MS (tentatively
identified)
3-ethyl-3-methylpentane MS (tentatively
identified)
1-chloroctane MS (tentatively
identified)
benzothiazole MS, RS
2,2,3,4-tetramethylpentane MS (tentatively
identified)
2,3,4-trimethylhexane/4- MS (tentatively
methyloctane identified)
4-methyl-2-propyl-1-pentanol MS (tentatively
identified)
not available 3-methylbutanal MS, RS
dimethyl trisulfide MS (tentatively
identified)
Cms Bacterium
2-propanol 2-propanol 2-propanol MS, RS
2-methylpropanoic acid 2-methylpropanoic MS, RS
acid
unknown (m/z 86)
3-methylbutanoic acid (3-methylbutanoic MS, RS
acid)
2-hydroxy-3- MS (tentatively
pentanone identified)
benzaldehyde MS, RS
3-methyl-3-buten-2-one MS (tentatively
identified)
toluene MS, RS
a
Specific markers of Rs and Cms are highlighted in bold. Methods used to confirm volatile compound detected are also reported. bMS, identification
by comparison with NIST mass spectrum; RS, identification by injection of reference standards.

heal end, a core was crushed in 2 mL of SDW and left to settle. After 15 protocols of Seal et al.26 and Pastrik et al.27 were followed for PCR assays
min, 1.5 mL of extract was collected to carry out microbiological and to identify Rs and Cms, respectively (EU Directives). The PCR method
molecular assays. of Pastrik et al.27 was slightly modified, and the use of the endogenous
Reisolation and Identification of the Pathogen. Each core extract control primers was avoided to increase the sensitivity of the assay.

was centrifuged for 20 min at 10000g at 4 ± 1 °C; the pellet was
resuspended in 1 mL of SDW, and 50 μL of resuspension was inoculated
on SMSA agar(for Rs, as specified in the EU Directives) and YDC or
NCP-88 (for Cms, EU Directives) agar and incubated at 27 ± 1 °C from
3 to 7 days to isolate Rs and Cms pathogens. The Rs-like and Cms-like
colonies were purified on appropriate media and identified with
pathogenicity test (EU Directives).
Molecular Assays. The remaining 950 μL resuspended pellet was
used for DNA extraction using a DNeasy Plant Mini Kit (Qiagen,
Germany). The DNA was then stored at −20 °C for PCR assays. The
340
■
RESULTS AND DISCUSSION
GC-MS Analysis. In Vitro Assays. Volatile compounds
emitted from bacterial cultures of Rs or Cms grown on different
media (TZA, LPGA, PDA, and NPDA) were detected by SPME-
GC-MS analysis (Table 2). These media were chosen to study
the metabolism of Rs or Cms in matrices less complex than that
represented by potato tuber. PDA and NPDA were chosen to
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Journal of Agricultural and Food Chemistry
simulate a simplified potato substrate. Depending on media
characteristics, volatile compound analysis was done when the
single colonies have shown their typical morphology on agar
media: after 5 days for Rs grown on TZA, and after 2, 11, and 12
days for Rs or Cms grown on LPGA, PDA, and NPDA,
respectively (EU Directives). Because no data about the
incubation time on PDA and NPDA (these media are basically
used as standard in mycology but not in bacteriology) are
reported in EU Directives, the required time to evaluate the
morphology of a typical colony was deduced by visual analysis of
Rs or Cms cultured plates.
The microorganism activity in cultured media was related to
the increase of volatile compound concentration in comparison
with that of uncultured media. Indeed, although samples were
prepared in sterile conditions and hermetically sealed, the
occurrence of volatile compounds released by the substrate
incubated at 27 °C could not be avoided.
In Table 2, only volatile compounds emitted in amounts
significantly different from the control or detected in cultured
agar media are reported. Volatile compounds emitted from the
metabolic activity of the two pathogens were broadly similar, and
only minor differences were observed.
Dimethyl disulfide (DMDS) was the main volatile compound
metabolized by Rs on TZA. Cms was not cultured on this
medium that is typical for Rs, and it is also not recommended
because of its slow growth rate.
A mixture of polysulfides (DMDS and dimethyl trisulfide) was
produced by Rs on LPGA along with 2-propanone and methyl 2-
methylbutanoate. 2-Propanone, DMDS, and methyl 2-methyl-
butanoate were markers for Rs on LPGA, whereas dimethyl
trisulfide was a degradation product detected in uncultured
LPGA, the concentration of which increased in the presence of
pathogen. Considering the slow growth of Cms with respect to
Rs, only a few molecules were identified as markers of pathogen
presence: 3-methylbutanal was a compound characteristic of the
Cms metabolism on LPGA, and an increase of dimethyl trisulfide
concentration was revealed in comparison with the control.
Volatile compound pattern of Rs metabolism on PDA was
more complex than that observed on the other agar media as
highlighted from the number of volatile compounds detected
(Table 2 and Figure S1 in the Supporting Information). Among
these, 3-methylbutanoic acid, propanoic acid, methyl 2-
methylbutanoate, and an unknown compound with m/z 84
were detected only in Rs cultured on PDA. Moreover, the
metabolic activity of Rs pathogen on PDA was related to the
increase of 2-butanone, 2-furancarboxaldehyde, and styrene
concentrations. 2-Propanol and 3-methylbutanoic acid were
markers of Cms metabolism cultured on PDA. The pathogen
favored also the increase of 2-methylpropanoic acid, an unknown
compound with m/z 86, and benzaldehyde concentrations.
No disease markers and no variations of volatile compound
concentration were detected in Rs cultured on NPDA. On the
contrary, similar to what was observed on PDA, the metabolism
of Cms on NPDA was related to the variation of 2-propanol, 2-
methylpropanoic acid, and 2-hydroxy-3-pentanone concentra-
tions (Figure S2 in the Supporting Information). 3-Methyl-
butanoic acid was also detected but with a concentration too low
to be taken into consideration as a disease marker on NPDA.
All volatile compounds detected seemingly come from
degradation reactions of amino acids.28−33 The short-chain
alcohols, acids, aldehydes, and ketones detected are known as
degradation products of carbohydrates.7 As far as methyl 2-
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methylbutanoate is concerned, it can be produced by oxidation of
very long chain unsaturated fatty acids.34
Time Course Study on in Vitro Samples. The distribution of
volatile compounds emitted from bacteria kept for 2 and 7 days at
27 ± 1 °C in liquid suspensions was analyzed in relation with
time. Time course studies were carried out in a time range
arbitrarily chosen but adequate to allow the growth of the two
bacterial pathogens. In general, the presence of water coupled
with shaking increases the bacterial growth speed with respect to
that in agar media. In our experimental conditions, the pathogen
growth was maximal at day 2 in all bacterium−broth systems with
the exception of Rs suspended in NPD broth, in which even after
7 days, the bacterium kept growing (data not shown). For Cms in
both LPG and NPD broths, the death phase was observed at day
7. No bacterial growth was observed in PD broth for both Rs and
Cms within 7 days as confirmed by SPME-GC-MS analysis,
which did not reveal differences between inoculated samples and
controls (data not shown). As shown in Table 3, Rs growth rate
Table 3. Number of Cells Generated and Growth Rate
Calculated at Day 2 for Rs or Cms in Different Liquid Media
substrate
growth rate (generation
generation no. no.× 10−5 min−1)
bacterial pathogen TZ LPG NPD TZ LPG NPD
Rs 14 13 0.8 49 45 3
Cms 6 6 20 20
in different broths calculated at 2 days increased in the order TZ
≥ LPG ≫ NPD, whereas for Cms inoculated in LPG and NPD
broths the growth rates were comparable. Rs growth rate in LPG
broth doubled that of Cms in the same liquid medium; in NPD
broth, on the contrary, the Rs growth rate was significantly lower
than that of Cms (3 and 20 generation number × 10−5 min−1 for
Rs and Cms, respectively).
As was already observed for Rs cultured in TZA, dimethyl
disulfide was the marker of Rs metabolism in TZ broth: after 7
days, its concentration increased 5 times compared with that at
day 2 (data reported in the Supporting Information, Figure S3).
In Figure 2, volatile compounds emitted during the Rs and Cms
bacterial growth in LPG broth are reported. The pattern of
detected volatile compounds was different for Rs and Cms
because the growth rates of the two bacteria in LPG broth were
different (the growth rate of Rs doubled that of Cms, Table 3).
The increase of toluene concentration after 2 days from
inoculation of Rs in LPG broth along with the emission of
DMDS confirmed the bacterial growth. Similar to what it was
observed for methyl 2-methylbutanoate, also toluene can be
considered a product of long-chain unsaturated fatty acid
oxidation.34
As expected, for Cms at day 0, control and inoculated broths
produced similar volatile compound patterns. After 2 days and
more markedly at day 7, concentrations of 2-methylpropanal and
3-methylbutanal (both derived from amino acid Strecker
degradation) considerably decreased in the inoculated samples
in contrast with what has been observed in LPGA medium. After
7 days, the Cms growth curve achieved the death phase and the
decrease of volatile compounds concentrations could be due
reasonably to the reduction of living cell number that
metabolized the substrate. At this stage, volatile compound
distribution differences were observed between inoculated and
control samples: the concentration of dimethyl disulfide and an
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Journal of Agricultural and Food Chemistry Article

Figure 2. Volatile compounds emission from Rs or Cms cultured in LPG broth after 0, 2, and 7 days of growth. Volatile compounds emitted from
uncultured LPG broth samples are reported as a control (mean of two replicates; semimaximal dispersions in vertical bars). TIC, total ion current;
DMDS, dimethyl disulfide.

Figure 3. Volatile compound emission from Rs or Cms cultured on NPD broth after 0, 2, and 7 days of growth. Volatile compounds emitted from
uncultured NPD broth samples are reported as a control (mean of two replicates; semimaximal dispersions in vertical bars). TIC, total ion current.

unknown molecule (m/z 83) in the Cms−LPG broth system
increased compared to the control.
The volatile compound pattern detected in the headspace of
Rs or Cms inoculated in NPD broths was very similar (Figure 3)
but different from the patterns observed on NPDA media. A
mixture of methyl-aldehydes containing three or four carbon
atoms were detected in both inoculated and control samples,
whereas a mixture of methyl-carboxylic acids containing three or
four carbon atoms were identified in Cms cultured on NPDA (no
disease markers and no variation of volatile compound
concentration were detected in Rs cultured on the same
medium). Slight differences (i.e., an increase of 2-methylpropa-
nal, 3-methylbutanal, and 2-methylbutanal concentrations) were
observed after 2 days in Rs−NPD broth system compared with
control sample. After 7 days, the metabolism of Rs in NPD broth
changed: the mixture of aldehydes disappeared, and 2-propanol
342
was the only volatile compound to be detected. For the Cms−
NPD system, after 2 days, negligible volatile compound amounts
were detected and, after 7 days, 2-propanol was the only
identified volatile. The concentration of 2-propanol in the
presence of Cms in NPD broth was about 3 times higher than
that measured in Rs−NPD sample due to the higher growth rate
of Cms (20 and 3 × 10−5 generation number min−1 for Cms and
Rs, respectively).
The different pattern of volatile compounds detected depends
on pathogen growth rate as well as chemical composition of the
substrate. The large amount of dimethyl disulfide released by
Rs−TZ solid and liquid media samples might derive from
casamino acids present in TZ composition (BD Biosciences,
USA), which contains cystine (derived from cysteine oxidation).
LPG contains Bacto Peptone (BD Biosciences, USA), an
enzymatic digest of animal proteins that can contain traces of
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Journal of Agricultural and Food Chemistry Article

Table 4. Detected Pathogen Markers, Their Average Total Ion Current (TIC) Abundance Normalized to the Sample Weight (SD
in Parentheses), and Their Frequency of Appearance Related to 4 Control and 8 Diseased Rs Samples and to 5 Control and 10
Diseased Cms Samples, Respectively
control Rs-infected sample Cms-infected sample
av TIC abundance appearance av TIC abundance appearance av TIC abundance appearance volatile compd
volatile (g−1 × 104) frequency (g−1 × 104) frequency (g−1 × 104) frequency identificationa
3-methylbutanoic acid 0.73 (0.15) 2 1.08 (0.47) 4 MS, RS
2,2,3,4- 1.33 (0.21) 3 2.29 (1.16) 5 MS (tentatively
tetramethylpentane identified)
2,3,4-trimethylhexane/4- 0.94 (0.13) 3 1.38 (0.43) 5 MS (tentatively
methyloctane identified)
4-methyl-2-propyl-1- 1.13 (0.19) 3 1.96 (0.35) 5 MS (tentatively
pentanol identified)
2-propanol 1.12 (0.00) 1 2.31 (0.06) 3 MS, RS
3-methyl-3-buten-2-one 1.66 (1.19) 2 1.56 (0.59) 5 MS (tentatively
identified)
toluene 2.60 (0.00) 1 3.01 (0.34) 2 MS, RS
a
MS, identification by comparison with NIST mass spectrum; RS, identification by injection of reference standards.

Figure 4. Total ion current (TIC) relative abundance (peak area normalized to sample weight) of (a) 3-methylbutanoic acid (m/z 102), 2,2,3,4-
tetramethylpentane (m/z 128), 2,3,4-trimethylhexane/4-methyloctane (m/z 128), and 4-methyl-2-propyl-1-pentanol (m/z 144) detected in Rs
infected potato samples and (b) 2-propanol (m/z 60), 3-methyl-3-buten-2-one (m/z 84), and toluene (m/z 92) in Cms samples. (Abundances lower
than 103 are not reported.)

fatty acids, the oxidation of which produces toluene. Both PD
and NPD contain potato derivatives such as starch, sugar,
protein, minerals, and glycoalkaloids. Solanine and chaconine
glycoalkaloids are usually found concentrated in potato peel, and
thus they can be present in commercial PD, but they can be
insignificant in NPD, extracted from peeled potatoes. As has
been already reported for fungi,35 these toxic compounds could
have an inhibitory effect on bacteria, which explains the slow Rs
and Cms growth in PD broth. The yeast extract contained in
343
LPG and the carbohydrates naturally present in potato (i.e., PD
and NPD media) might explain the formation of carbohydrate
degradation products. On the basis of these considerations, it was
possible to explain the occurrence of volatile compounds
produced by the degradation of amino acids and fatty acids
(detected in the first two days) and of carbohydrates (detected at
day 7): note that the final degradation products as CO2 and H2O
were not detected because they are not retained and separated
from the GC column used.
dx.doi.org/10.1021/jf403436t | J. Agric. Food Chem. 2014, 62, 337−347
Journal of Agricultural and Food Chemistry
In Vivo Assays. Infected tubers produced under field
conditions by Rs or Cms experimental inoculations were
analyzed by SPME-GC-MS to identify markers of brown and
ring rot diseases and compared with those identified in in vitro
assays.
Harvested tubers showed a high percentage of disease
incidence but not all were symptomatic: 78 and 91% of tubers
were found infected by Rs and Cms, respectively, 49 and 46% of
which showed the typical tuber symptoms of brown and ring rot.
Among the symptomatic tubers, the severity of diseases was
variable, and the evaluation of the disease severity of the tuber
vascular ring (Table 1) assigned the lowest value to healthy
potatoes and to the class of infected but asymptomatic tubers
(latently infected) and was rated 0 (zero). The remaining classes
were rated between 1 and 5 according to five levels of disease
severity (from “very low” to “very high” level) as shown by the
pictures reported in Table 1. At the highest level of the
phytopathometric scale (corresponding to class 5), symptoms of
the tubers were ascribed to Rs or Cms as well as to other
secondary microorganisms as Fusarium sp. and Erwinia spp. that
contributed to the degradation of potato tissues.15,16,36 All
samples analyzed were composed for the most part by tubers
belonging to class 1 or 2. Moreover, all Rs-infected and 50% of
Cms-infected samples contained at least one tuber with medium
or high symptom level (class 3 or 4).
Chromatograms and corresponding mass spectra were
collected (Figure S4) for infected and control samples kept at
room temperature for different times.
Some significant differences, which increase with the
reactivation time, could be observed in the GC-MS chromato-
grams of Rs-infected potato samples with respect to controls (see
the Supporting Information, Figure S5 (left), and Table 2): 1-
hepten-3-ol (m/z 114), 3,6-dimethyl-3-octanone (m/z 156), 3-
ethyl-3-methylpentane (m/z 142), 1-chloroctane (m/z 148), and
benzothiazole (m/z 135) were identified as markers of brown rot
disease (their presence in diseased samples was confirmed for
about 50% analyzed samples). In addition, the presence of
symptoms of brown rot disease seemed to be related to the
increase of relative intensities of the peaks assigned to 3-
methylbutanoic acid (m/z 102), 2,2,3,4-tetramethylpentane (m/
z 128), 2,3,4-trimethylhexane/4-methyloctane (m/z 128), or 4-
methyl-2-propyl-1-pentanol (m/z 144) (Table 2). The collected
chromatograms showed variations of the relative intensity of
peaks in the entire acquisition range (peaks with m/z 158 and
186 attributable to 2-propyl-1-heptanol and 2-ethyl-1-decanol,
respectively, showed the most evident variations but the
frequency of appearance was <25%).
The average abundance and appearance frequency of 3-
methylbutanoic acid, 2,2,3,4-tetramethylpentane, 2,3,4-trime-
thylhexane/4-methyloctane, and 4-methyl-2-propyl-1-pentanol
are reported in Table 4: these volatile compounds were detected
both in control and Rs-diseased samples with a frequency
percentage of about 75 and 63%, respectively, even if their
average abundance was slightly lower in the control samples than
in diseased ones. Their relative abundances (peak area
normalized to sample weight) are reported in Figure 4a. Samples
named Control3-10d, Rs4-10d, and Rs5-10d were kept at 25 °C
for 10 days before GC-MS analyses. Generally, volatile
compound emission normalized to sample weight was higher
from the diseased samples than controls and seemed to depend
on the reactivation time duration, as clearly shown in volatile
compounds composition emitted from Rs4-10d and Rs5-10d
samples. For these samples, an increase of 2,2,3,4-tetramethyl-
344
Article
pentane and the corresponding disappearance of 3-methylbuta-
noic acid were observed. Disease severity analysis revealed that
the samples contained tubers with medium to high symptom
level (class ≥3).
No specific markers of ring rot have been identified after GC-
MS analysis of Cms-diseased samples kept for 1 day at room
temperature, but an increase of the relative intensity of two peaks,
which were identified as 2-propanol (m/z 60) and 3-methyl-3-
buten-2-one (m/z 84), in diseased samples was observed (see
Supporting Information, Figure S5 (right), and Table 2). Further
analyses performed on Cms suspected diseased samples showed
significant intensity variations of the peak assigned to toluene
(m/z 92). Different from what has been observed in Rs samples,
volatile compound production of Cms-diseased samples did not
seem related to the time of bacterial reactivation (data not
shown) as a confirmation of the different biology of the two
pathogens.3,5,21,22
The average abundance and appearance frequency of volatile
compounds detected are reported in Table 4. The appearance
frequency was low (30, 50, and 20% of total analyzed samples for
2-propanol, 3-methyl-3-buten-2-one, and toluene, respectively),
but the differences between the average abundances of control
and diseased samples were significant. The trend was confirmed
by the relative abundances of the volatile compounds normalized
to each sample weight (Figure 4b): 2-propanol and toluene
allowed discrimination between healthy and Cms-diseased
samples.
The 2-propanol and 3-methylbutanoic acid were already
identified as markers of Cms metabolism in cultured agar media
PDA and NPDA; 3-methylbutanoic acid was also a marker of Rs
metabolism in PDA. The similarity between potato tubers and
PDA in terms of chemical composition can explain the detection
of the same volatile compounds. However, potato is a matrix
more complex than PDA, especially for the presence of
unwounded peel, which creates a barrier between the tuber
internal part and the environment. Peel reduces the potato smell
diffusion and, at the same time, adsorbs on its surface odors from
the environment such as soil or fungi smell, making very complex
the detection of possible diseases markers due to the presence of
these interfering volatile compounds. To minimize this effect,
potato tubers used in our study have been gently brushed to
remove residues of soil and possible spores of saprophytic fungi.
Moreover, the absence of external disease symptoms and low
disease severity in assayed tubers were responsible for the limited
production of volatile compounds characteristic of brown and
ring rot diseases. An attempt to identify brown and ring rot
markers by GC-MS analysis has been made by Stinson et al.14
Unfortunately, these results are not comparable with those
reported in the present paper because they used potatoes with
clear disease symptoms (comparable with our class 5 symptom
level, Table 1), whereas in our study potato tubers belonging to
the lower classes were used. Moreover, a comparison between
the two analytical approaches is not allowed as Stinson et al.14 did
not report details of GC-MS analysis. Similarly, in a recent
review7 on volatile compounds emitted from raw potatoes,
tubers have been analyzed after peeling and for this reason the
results cannot fit with our findings.
Volatile compounds detected in our bacterial cultures are
organic compounds usually produced in the main metabolic
processes. On the contrary, in potato samples, with the exception
of 2-propanol and 3-methylbutanoic acid, volatile compounds
from the metabolism of Rs or Cms were mostly identified as
branched-chain alkanes and alcohols with a C atom number >5.
dx.doi.org/10.1021/jf403436t | J. Agric. Food Chem. 2014, 62, 337−347
Journal of Agricultural and Food Chemistry Article

No data are available in the literature to explain the formation of Table 5. Possible Volatile Compounds Detected in the
these products. Likely, they might be carbohydrate degradation Headspace of Rs- and Cms-Diseased Potatoes by PTR-MS
intermediates, more complex than volatiles with three or four Analysis, Percentage of Diseased Tubers That Emitted the
carbon atoms detected in bacterial cultures. As far as the Specific Volatile Compounds, and Related Literature
detection of other volatile compounds is concerned, benzothia-
diseased
zole is a degradation product of S-containing amino acid and tubers (%
toluene, as previously reported, along with detected ketones are abun-
products of fatty acid degradation. 2-Propanol, toluene, and dance)
benzothiazole were also detected in the headspace of tubers possible compd/ intercomparison volatile compd
infected with Phytophthora infestans and Fusarium solani var. m/z major fragment Rs Cms GC-MS studies identificationa
coeruleum.37 In our case infected tubers were chosen to avoid 33 methanol 83 65 Waterer and IA, RS
Pritchard10
contamination due to other saprophytic organisms such as
Fusarium spp. and Pectobacterium spp: moreover, the presence of
43 2-propanol 55 55 Waterer and MS (tentatively
Phytophthora infestans in tubers used for this study was excluded. Pritchard40 identified)
PTR-MS in Vivo Analysis. PTR-MS is a useful tool for real- Stinson et al.14
time monitoring of low molecular mass volatile compound
emissions, such as ethanol, methanol, propanol, and 2- 45 acetaldehyde 89 95 IA, RS
propanone. Most of these volatile compounds are not in the 47 ethanol 35 IA (tentatively
range of the compounds measured with GC-MS. identified)
High levels of several m/z were measured in diseased samples 59 2-propanone 67 65 Stinson et al.14 IA, RS
of Rs and Cms as compared to the controls; their abundance is
given in Table 5. 61 acetic acid 5 26 MS (tentatively
Identification of the measured m/z is not straightforward, as identified)
PTR-MS cannot distinguish between product ions with the same ethyl acetate
mass. However, previous experimental determinations in similar
conditions as used here (e.g., E/N = 120 V cm2, where E is the 63 dimethyl sulfide 55 IA (tentatively
identified)
electric field strength expressed as V cm−1 and N is the density of 73 2-butanone 55 13 Stinson et al. 14
MS (tentatively
the neutral molecules expressed as molecule number cm−3 in the identified)
reaction chamber); reaction chamber pressure (2 mbar) together
with the isotopic ratio analysis38 allows assignment of several 83 cyclohexene 66 65 MS (tentatively
compounds such as methanol (m/z 33), acetaldehyde (m/z 45), identified)
ethanol (m/z 47), 2-propanone (m/z 59), dimethyl sulfide hexanal
(DMS, m/z 63), and dimethyl disulfide (DMDS, m/z 95).
Possible candidates or their major fragment for the ions 85 3-methyl-3- 55 61 this study MS (tentatively
buten-2-one identified)
measured are indicated in Table 5. Two of them, with m/z 43
and 93, were specific to Cms-diseased tubers and were attributed
87 3-methyl-2- 28 48 MS (tentatively
to 2-propanol and toluene, respectively, as confirmed by GC-MS buten-1-ol identified)
analysis in this study. For some of the remaining compounds the 2,3-butanedione
most probable candidates were indicated on the basis of the 2-pentanone Stinson et al.14
literature.
Most of the volatile compounds were common for tubers 93 toluene 18 this study MS, RS
infected with Rs as well as for those infected with Cms. 2- 95 dimethyl disulfide 54 IA (tentatively
Butanone and sulfur-containing compounds (DMS and DMDS) identified)
a
were identified only in Rs-diseased tubers (DMDS was also the IA, identification by isotopic ratio analysis;41 MS, identification by
marker of Rs presence in TZ and LPG solid and liquid media). comparison with NIST mass spectrum; RS, identification by injection
Ethanol and toluene were mainly produced by the Cms-diseased of reference standards.
samples with symptomatic characteristics of class 3 or higher
(identified after the measurements). These volatile compounds
could be considered specific markers for Rs and Cms infection,
respectively. propanol and toluene were markers of potato ring rot. The
The PTR-MS analysis provided a profile of low molecular mass techniques provided also a promising alternative to the molecular
volatile compounds complementary to the GC-MS analysis. assays.
However, 2-butanone, dimethyl disulfide, ethanol, ethyl acetate, Within the framework of the development of innovative and
acetaldehyde, and 2-propanone were also found in the headspace rapid techniques for the detection of quarantine pathogens to be
of potatoes inoculated with Pectobacterium carotovorum ssp. used by National Plant Protection Organizations and Inspection
carotovorum and atrosepticum.9,39 Hexanal and traces of toluene Services in the European Union, these findings represent the first
and acetic acid were reported from tubers inoculated with step toward the realization of plant pathogen noninvasive
Phytophthora infestans and Fusarium solani var. coeruleum.37 diagnostic methods alternative to the standard methods reported
In conclusion, GC-MS and PTR-MS techniques allowed in EU Directives, which require costly and time-consuming
recognition of potato tubers infected by Rs or Cms through the microbiological, serological, and molecular assays.
identification of specific disease markers: 1-hepten-3-ol, 3,6- Further studies will be addressed to the volatile compound
dimethyl-3-octanone, 3-ethyl-3-methylpentane, 1-chloroctane, detection by electronic nose due to the importance of
and benzothiazole were markers of potato brown rot, whereas 2- accelerating potato disease analysis.
345 dx.doi.org/10.1021/jf403436t | J. Agric. Food Chem. 2014, 62, 337−347
Journal of Agricultural and Food Chemistry
■
ASSOCIATED CONTENT
*
S Supporting Information
Additional figures. This material is available free of charge via the
Internet at http://pubs.acs.org.
■ AUTHOR INFORMATION
Corresponding Author
*(S.B.) Phone: +39 051 2096207. Fax: +39 051 2096203. E-mail:
sonia.blasioli@unibo.it.
Funding
This work was financed as Q-Detect project, which was a part of
the EU’s 7th Framework Program (FP7-KBBE-2009-3).
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We gratefully thank C. E. Gessa and U. Mazzucchi for their
precious work and suggestions, which helped us in setting up the
experiments. We acknowledge A. Galeone for her contribution to
molecular assays. We also thank A. Nastri and S. Vecchi for the
excellent management of the fields used for the tuber production,
S. Grandi for the GC-MS maintenance, and S. Brigati and P.
Bertolini for the use of the refrigerated cells. We are grateful to
the Phytosanitary Service of Emilia Romagna Region for
providing special permissions to export infected tubers to project
partners.
■
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