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Identication 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. Identication by GC-MS analysis of volatile organic compounds from Rs or Cms cultured on dierent 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 specic 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 modied 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, 1chloroctane, and benzothiazole) were identied. 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 inuence 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
2013 American Chemical Society

emitted from bacterial cultures of Rs and Cms are reported in the literature. Potato avor 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 dierent, 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 avor are conducted on sliced or peeled tubers to favor smell diusion. 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 indentied 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 nal stage of
Received: Revised: Accepted: Published:

August 2, 2013 December 7, 2013 December 8, 2013 December 8, 2013 | J. Agric. Food Chem. 2014, 62, 337347

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 aect the visual symptom analysis, making dicult 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 EUs 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 identication of volatile markers or specic ngerprints of the diseases using gas chromatography and proton transfer reaction coupled with mass spectrometry (GC-MS and PTR-MS) analysis. For the rst 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.

Chemicals. Reference compounds were injected, when available, to conrm identication of volatile compounds detected. Methanol, acetaldehyde, 2-propanone, 2-propanol, 2-butanone, propanoic acid, 3-methylbutanal, 2-methylpropanoic acid, toluene, dimethyl disulde, 3methylbutanoic acid, benzaldehyde, methyl 2-methylbutanoate, benzothiazole, 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 M.


Bacterial Strains. The virulent strains IPV-BO 5836 of Rs and IPVBO 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 7296 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 specic 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 specic 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 mL1 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 L 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. | J. Agric. Food Chem. 2014, 62, 337347

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Rs and Cms Experimental Inoculations. For Rs, ca. 100 potato plants were grown in the eld following standard agronomical procedures; within the end of blooming time (after 2 months from the tuber seeding), 30 L of water suspension containing the pathogen (ca. 109 CFU mL1) 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 thirdfourthleaf 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 L of pathogen water suspension (ca. 109 CFU mL1) 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 eld. The remaining plants (about fty) were experimentally inoculated at the stem in the thirdfourth-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 elds 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 Teon 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 Teon cap. CAR/PDMS (Supelco) SPME bers 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 ushed with 2 L h1 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 lled to volume and sealed with Teon 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

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) bers. Supelco 75 m carboxen/polydimethylsiloxane bers were used to monitor gases emitted from Rs or Cms cultures and infected potatoes. SPME bers 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 identication. GC-MS Analyses. Volatile compounds adsorbed by SPME bers 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 Scientic, 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 bondedphase capillary column Supelco SPB-5 poly (5% diphenyl/95% dimethylsiloxane) 30 0.32 mm; 0.25 mm lm thickness (SigmaAldrich Co. LLC, USA). Volatile compounds were separated by applying the following GC oven temperature program: 40 C for 3 min, raised at 3 C min1 to 130 C, raised at 10 C min1 to 260 C, and held at 260 C for 10 min. The identication 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 ushed with 2 L h1 hydrocarbonfree air (Figure 1c). An automatic valve system was used to connect up to ve 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 custombuilt 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 anity (PA) greater than that of water (PA = 691 kJ mol1) undergo ionmolecule reactions by receiving a proton from the hydronium cation, H3O+. The protonated compounds are mass ltered with a quadrupole mass spectrometer and quantied by a secondary electron multiplier as mass to charge ratio, m/z. The calibration was performed with dierent 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), mxylene (m/z 107), and -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 Identication. Sample Processing. After the symptom visual analysis, from each tuber, at the | J. Agric. Food Chem. 2014, 62, 337347

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Table 2. Volatile Compounds Emitted from Rs and Cms Cultured on Dierent Agar Media at Dierent 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 TZA dimethyl disulde LPGA 2-propanone dimethyl disulde 2-butanone 3-methylbutanoic acid 2-furancarboxaldehyde propanoic acid methyl 2methylbutanoate unknown (m/z 84) 3-methylbutanoic acid PDA NPDA Rs Bacterium MS, RS MS, RS MS, RS MS, RS MS (tentatively identied) MS, RS MS, RS potato volatile compd identicationb

methyl 2methylbutanoate dimethyl trisulde

styrene 1-hepten-3-ol 3,6-dimethyl-3-octanone 3-ethyl-3-methylpentane 1-chloroctane benzothiazole 2,2,3,4-tetramethylpentane 2,3,4-trimethylhexane/4methyloctane 4-methyl-2-propyl-1-pentanol not available 3-methylbutanal dimethyl trisulde Cms Bacterium 2-propanol 2-methylpropanoic acid (3-methylbutanoic acid) 2-hydroxy-3pentanone 3-methyl-3-buten-2-one toluene

MS (tentatively identied) MS (tentatively identied) MS, RS MS (tentatively identied) MS (tentatively identied) MS (tentatively identied) MS, RS MS (tentatively identied) MS (tentatively identied) MS (tentatively identied) MS, RS MS (tentatively identied) MS, RS MS, RS

2-propanol 2-methylpropanoic acid unknown (m/z 86) 3-methylbutanoic acid


MS, RS MS (tentatively identied) MS, RS MS (tentatively identied) MS, RS


Specic markers of Rs and Cms are highlighted in bold. Methods used to conrm volatile compound detected are also reported. bMS, identication by comparison with NIST mass spectrum; RS, identication by injection of reference standards. heal end, a core was crushed in 2 mL of SDW and left to settle. After 15 min, 1.5 mL of extract was collected to carry out microbiological and molecular assays. Reisolation and Identication of the Pathogen. Each core extract was centrifuged for 20 min at 10000g at 4 1 C; the pellet was resuspended in 1 mL of SDW, and 50 L of resuspension was inoculated on SMSA agar(for Rs, as specied 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 puried on appropriate media and identied with pathogenicity test (EU Directives). Molecular Assays. The remaining 950 L 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

protocols of Seal et al.26 and Pastrik et al.27 were followed for PCR assays to identify Rs and Cms, respectively (EU Directives). The PCR method of Pastrik et al.27 was slightly modied, and the use of the endogenous control primers was avoided to increase the sensitivity of the assay.

GC-MS Analysis. In Vitro Assays. Volatile compounds emitted from bacterial cultures of Rs or Cms grown on dierent media (TZA, LPGA, PDA, and NPDA) were detected by SPMEGC-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 | J. Agric. Food Chem. 2014, 62, 337347


Journal of Agricultural and Food Chemistry simulate a simplied 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 signicantly dierent 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 dierences were observed. Dimethyl disulde (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 polysuldes (DMDS and dimethyl trisulde) was produced by Rs on LPGA along with 2-propanone and methyl 2methylbutanoate. 2-Propanone, DMDS, and methyl 2-methylbutanoate were markers for Rs on LPGA, whereas dimethyl trisulde 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 identied as markers of pathogen presence: 3-methylbutanal was a compound characteristic of the Cms metabolism on LPGA, and an increase of dimethyl trisulde 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 2methylbutanoate, 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, 2methylpropanoic acid, and 2-hydroxy-3-pentanone concentrations (Figure S2 in the Supporting Information). 3-Methylbutanoic 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.2833 The short-chain alcohols, acids, aldehydes, and ketones detected are known as degradation products of carbohydrates.7 As far as methyl 2341


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 bacteriumbroth 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 conrmed by SPME-GC-MS analysis, which did not reveal dierences 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 Dierent Liquid Media
substrate generation no. bacterial pathogen Rs Cms TZ 14 LPG 13 6 NPD 0.8 6 growth rate (generation no. 105 min1) TZ 49 LPG 45 20 NPD 3 20

in dierent 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 signicantly lower than that of Cms (3 and 20 generation number 105 min1 for Rs and Cms, respectively). As was already observed for Rs cultured in TZA, dimethyl disulde 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 dierent for Rs and Cms because the growth rates of the two bacteria in LPG broth were dierent (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 conrmed 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 dierences were observed between inoculated and control samples: the concentration of dimethyl disulde and an | J. Agric. Food Chem. 2014, 62, 337347

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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 disulde.

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 CmsLPG 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 dierent 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 identied 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 dierences (i.e., an increase of 2-methylpropanal, 3-methylbutanal, and 2-methylbutanal concentrations) were observed after 2 days in RsNPD 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

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 identied volatile. The concentration of 2-propanol in the presence of Cms in NPD broth was about 3 times higher than that measured in RsNPD sample due to the higher growth rate of Cms (20 and 3 105 generation number min1 for Cms and Rs, respectively). The dierent pattern of volatile compounds detected depends on pathogen growth rate as well as chemical composition of the substrate. The large amount of dimethyl disulde released by RsTZ 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 | J. Agric. Food Chem. 2014, 62, 337347

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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 volatile 3-methylbutanoic acid 2,2,3,4tetramethylpentane 2,3,4-trimethylhexane/4methyloctane 4-methyl-2-propyl-1pentanol 2-propanol 3-methyl-3-buten-2-one toluene

Rs-infected sample appearance frequency 2 3 3 3 1 2 1 av TIC abundance (g1 104) 1.08 (0.47) 2.29 (1.16) 1.38 (0.43) 1.96 (0.35) appearance frequency 4 5 5 5

Cms-infected sample av TIC abundance (g1 104) appearance frequency volatile compd identicationa MS, RS MS (tentatively identied) MS (tentatively identied) MS (tentatively identied) MS, RS MS (tentatively identied) MS, RS

av TIC abundance (g1 104) 0.73 (0.15) 1.33 (0.21) 0.94 (0.13) 1.13 (0.19) 1.12 (0.00) 1.66 (1.19) 2.60 (0.00)

2.31 (0.06) 1.56 (0.59) 3.01 (0.34)

3 5 2

MS, identication by comparison with NIST mass spectrum; RS, identication 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), 2,2,3,4tetramethylpentane (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 insignicant in NPD, extracted from peeled potatoes. As has been already reported for fungi,35 these toxic compounds could have an inhibitory eect on bacteria, which explains the slow Rs and Cms growth in PD broth. The yeast extract contained in

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 rst two days) and of carbohydrates (detected at day 7): note that the nal degradation products as CO2 and H2O were not detected because they are not retained and separated from the GC column used. | J. Agric. Food Chem. 2014, 62, 337347

Journal of Agricultural and Food Chemistry In Vivo Assays. Infected tubers produced under eld 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 identied 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 ve 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 dierent times. Some signicant dierences, which increase with the reactivation time, could be observed in the GC-MS chromatograms of Rs-infected potato samples with respect to controls (see the Supporting Information, Figure S5 (left), and Table 2): 1hepten-3-ol (m/z 114), 3,6-dimethyl-3-octanone (m/z 156), 3ethyl-3-methylpentane (m/z 142), 1-chloroctane (m/z 148), and benzothiazole (m/z 135) were identied as markers of brown rot disease (their presence in diseased samples was conrmed 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 3methylbutanoic acid (m/z 102), 2,2,3,4-tetramethylpentane (m/ z 128), 2,3,4-trimethylhexane/4-methyloctane (m/z 128), or 4methyl-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 3methylbutanoic acid, 2,2,3,4-tetramethylpentane, 2,3,4-trimethylhexane/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-tetramethyl344


pentane and the corresponding disappearance of 3-methylbutanoic acid were observed. Disease severity analysis revealed that the samples contained tubers with medium to high symptom level (class 3). No specic markers of ring rot have been identied after GCMS analysis of Cms-diseased samples kept for 1 day at room temperature, but an increase of the relative intensity of two peaks, which were identied as 2-propanol (m/z 60) and 3-methyl-3buten-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 signicant intensity variations of the peak assigned to toluene (m/z 92). Dierent 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 conrmation of the dierent 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 dierences between the average abundances of control and diseased samples were signicant. The trend was conrmed 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 identied 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 diusion 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 eect, 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 t with our ndings. 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 identied as branched-chain alkanes and alcohols with a C atom number >5. | J. Agric. Food Chem. 2014, 62, 337347

Journal of Agricultural and Food Chemistry No data are available in the literature to explain the formation of these products. Likely, they might be carbohydrate degradation intermediates, more complex than volatiles with three or four carbon atoms detected in bacterial cultures. As far as the detection of other volatile compounds is concerned, benzothiazole is a degradation product of S-containing amino acid and toluene, as previously reported, along with detected ketones are products of fatty acid degradation. 2-Propanol, toluene, and benzothiazole were also detected in the headspace of tubers infected with Phytophthora infestans and Fusarium solani var. coeruleum.37 In our case infected tubers were chosen to avoid contamination due to other saprophytic organisms such as Fusarium spp. and Pectobacterium spp: moreover, the presence of Phytophthora infestans in tubers used for this study was excluded. PTR-MS in Vivo Analysis. PTR-MS is a useful tool for realtime monitoring of low molecular mass volatile compound emissions, such as ethanol, methanol, propanol, and 2propanone. Most of these volatile compounds are not in the range of the compounds measured with GC-MS. High levels of several m/z were measured in diseased samples of Rs and Cms as compared to the controls; their abundance is given in Table 5. Identication of the measured m/z is not straightforward, as PTR-MS cannot distinguish between product ions with the same mass. However, previous experimental determinations in similar conditions as used here (e.g., E/N = 120 V cm2, where E is the electric eld strength expressed as V cm1 and N is the density of the neutral molecules expressed as molecule number cm3 in the reaction chamber); reaction chamber pressure (2 mbar) together with the isotopic ratio analysis38 allows assignment of several compounds such as methanol (m/z 33), acetaldehyde (m/z 45), ethanol (m/z 47), 2-propanone (m/z 59), dimethyl sulde (DMS, m/z 63), and dimethyl disulde (DMDS, m/z 95). Possible candidates or their major fragment for the ions measured are indicated in Table 5. Two of them, with m/z 43 and 93, were specic to Cms-diseased tubers and were attributed to 2-propanol and toluene, respectively, as conrmed by GC-MS analysis in this study. For some of the remaining compounds the most probable candidates were indicated on the basis of the literature. Most of the volatile compounds were common for tubers infected with Rs as well as for those infected with Cms. 2Butanone and sulfur-containing compounds (DMS and DMDS) were identied only in Rs-diseased tubers (DMDS was also the marker of Rs presence in TZ and LPG solid and liquid media). Ethanol and toluene were mainly produced by the Cms-diseased samples with symptomatic characteristics of class 3 or higher (identied after the measurements). These volatile compounds could be considered specic markers for Rs and Cms infection, respectively. The PTR-MS analysis provided a prole of low molecular mass volatile compounds complementary to the GC-MS analysis. However, 2-butanone, dimethyl disulde, ethanol, ethyl acetate, acetaldehyde, and 2-propanone were also found in the headspace of potatoes inoculated with Pectobacterium carotovorum ssp. carotovorum and atrosepticum.9,39 Hexanal and traces of toluene and acetic acid were reported from tubers inoculated with Phytophthora infestans and Fusarium solani var. coeruleum.37 In conclusion, GC-MS and PTR-MS techniques allowed recognition of potato tubers infected by Rs or Cms through the identication of specic disease markers: 1-hepten-3-ol, 3,6dimethyl-3-octanone, 3-ethyl-3-methylpentane, 1-chloroctane, and benzothiazole were markers of potato brown rot, whereas 2345


Table 5. Possible Volatile Compounds Detected in the Headspace of Rs- and Cms-Diseased Potatoes by PTR-MS Analysis, Percentage of Diseased Tubers That Emitted the Specic Volatile Compounds, and Related Literature
diseased tubers (% abundance) m/z 33 possible compd/ major fragment methanol Rs 83 Cms 65 intercomparison GC-MS studies Waterer and Pritchard10 Waterer and Pritchard40 Stinson et al.14 volatile compd identicationa IA, RS





MS (tentatively identied)

45 47 59 61

acetaldehyde ethanol 2-propanone acetic acid ethyl acetate


95 35 65 26 Stinson et al.14

67 5

IA, RS IA (tentatively identied) IA, RS MS (tentatively identied)

63 73

dimethyl sulde 2-butanone

55 55 13 Stinson et al.

IA (tentatively identied) MS (tentatively identied) MS (tentatively identied)


cyclohexene hexanal




3-methyl-3buten-2-one 3-methyl-2buten-1-ol 2,3-butanedione 2-pentanone toluene dimethyl disulde



this study

MS (tentatively identied) MS (tentatively identied)




Stinson et al.14 18 54 this study MS, RS IA (tentatively identied)

93 95

IA, identication by isotopic ratio analysis;41 MS, identication by comparison with NIST mass spectrum; RS, identication by injection of reference standards.

propanol and toluene were markers of potato ring rot. The techniques provided also a promising alternative to the molecular assays. Within the framework of the development of innovative and rapid techniques for the detection of quarantine pathogens to be used by National Plant Protection Organizations and Inspection Services in the European Union, these ndings represent the rst step toward the realization of plant pathogen noninvasive diagnostic methods alternative to the standard methods reported in EU Directives, which require costly and time-consuming microbiological, serological, and molecular assays. Further studies will be addressed to the volatile compound detection by electronic nose due to the importance of accelerating potato disease analysis. | J. Agric. Food Chem. 2014, 62, 337347

Journal of Agricultural and Food Chemistry



S Supporting Information *

Additional gures. This material is available free of charge via the Internet at


Corresponding Author

*(S.B.) Phone: +39 051 2096207. Fax: +39 051 2096203. E-mail:

This work was nanced as Q-Detect project, which was a part of the EUs 7th Framework Program (FP7-KBBE-2009-3).

The authors declare no competing nancial 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 elds 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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