Identification of Volatile Markers in Potato Brown Rot and Ring Rot by

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, 1chloroctane, and benzothiazole) were identified. KEYWORDS: Ralstonia solanacearum, Clavibacter michiganensis subsp. sepedonicus, volatile compounds

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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 © 2013 American Chemical Society

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: Revised: Accepted: Published: 337

August 2, 2013 December 7, 2013 December 8, 2013 December 8, 2013 dx.doi.org/10.1021/jf403436t | J. Agric. Food Chem. 2014, 62, 337−347

Journal of Agricultural and Food Chemistry

Article

Table 1. Phytopathometric Classes of Symptomatic Diseased Potato Tubers; Severity Classes Are Laddered on the Basis of Vascular Ring Symptoms

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 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.

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.

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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, 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 μM. 338

dx.doi.org/10.1021/jf403436t | J. Agric. Food Chem. 2014, 62, 337−347


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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−fourthleaf 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

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/polydimethylsiloxane 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 bondedphase capillary column Supelco SPB-5 poly (5% diphenyl/95% dimethylsiloxane) 30 × 0.32 mm; 0.25 mm film 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 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 hydrocarbonfree 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 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 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), mxylene (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

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 4 339



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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 TZA

LPGA

PDA

NPDA

potato

volatile compd identificationb

Rs Bacterium dimethyl disulfide

2-propanone dimethyl disulfide 2-butanone 3-methylbutanoic acid 2-furancarboxaldehyde

methyl 2methylbutanoate

3-methylbutanoic acid

propanoic acid methyl 2methylbutanoate unknown (m/z 84)

dimethyl trisulfide 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 trisulfide

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

Cms Bacterium 2-propanol 2-methylpropanoic acid

2-propanol

MS, RS MS, RS MS, RS MS, RS MS (tentatively identified) MS, RS MS, RS

MS (tentatively identified) MS (tentatively identified) MS, RS MS (tentatively identified) MS (tentatively identified) MS (tentatively identified) MS, RS MS (tentatively identified) MS (tentatively identified) MS (tentatively identified) MS, RS MS (tentatively identified) MS, RS MS, RS

MS, RS

(3-methylbutanoic acid) 2-hydroxy-3pentanone

benzaldehyde 3-methyl-3-buten-2-one toluene

MS (tentatively identified) MS, RS MS (tentatively identified) 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. 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 modified, and the use of the endogenous control primers was avoided to increase the sensitivity of the assay.

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 Identification 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 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

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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 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 340



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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

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 2methylbutanoate. 2-Propanone, DMDS, and methyl 2-methylbutanoate 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 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.28−33 The short-chain alcohols, acids, aldehydes, and ketones detected are known as degradation products of carbohydrates.7 As far as methyl 2-

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 no.× 10−5 min−1)

generation no. bacterial pathogen

TZ

LPG

NPD

TZ

LPG

NPD

Rs Cms

14

13 6

0.8 6

49

45 20

3 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 341



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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 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-methylpropanal, 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

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 342



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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

Rs-infected sample

Cms-infected sample

av TIC abundance (g−1 × 104)

appearance frequency



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

0.73 (0.15) 1.33 (0.21)

2 3

1.08 (0.47) 2.29 (1.16)

4 5

0.94 (0.13)

3

1.38 (0.43)

5

1.13 (0.19)

3

1.96 (0.35)

5

1.12 (0.00) 1.66 (1.19)

1 2

2.31 (0.06) 1.56 (0.59)

3 5

toluene

2.60 (0.00)

1

3.01 (0.34)

2

volatile

a



volatile compd identificationa MS, RS MS (tentatively identified) MS (tentatively identified) MS (tentatively identified) MS, RS MS (tentatively identified) MS, RS

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), 2,2,3,4tetramethylpentane (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.)

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.

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



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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 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 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 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 5. 344



Article

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. Identification 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 field strength expressed as V cm−1 and N is the density of the neutral molecules expressed as molecule number cm−3 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 sulfide (DMS, m/z 63), and dimethyl disulfide (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 specific to Cms-diseased tubers and were attributed to 2-propanol and toluene, respectively, as confirmed 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 identified 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 (identified after the measurements). These volatile compounds could be considered specific markers for Rs and Cms infection, respectively. The PTR-MS analysis provided a profile of low molecular mass volatile compounds complementary to the GC-MS analysis. However, 2-butanone, dimethyl disulfide, 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 identification of specific 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 2-

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 Specific Volatile Compounds, and Related Literature diseased tubers (% abundance) possible compd/ major fragment

Rs

Cms

33

methanol

83

65

Waterer and Pritchard10

IA, RS

43

2-propanol

55

55

Waterer and Pritchard40 Stinson et al.14

MS (tentatively identified)

45 47

acetaldehyde ethanol

89

95 35

59

2-propanone

67

65

61

acetic acid

5

26

m/z

intercomparison GC-MS studies

Stinson et al.14

volatile compd identificationa

IA, RS IA (tentatively identified) IA, RS MS (tentatively identified)

ethyl acetate 63

dimethyl sulfide

55

73

2-butanone

55

13

83

cyclohexene

66

65

Stinson et al.

14

IA (tentatively identified) MS (tentatively identified) MS (tentatively identified)

hexanal 85

3-methyl-3buten-2-one

55

61

87

3-methyl-2buten-1-ol 2,3-butanedione 2-pentanone

28

48

93 95

toluene dimethyl disulfide

this study

MS (tentatively identified) MS (tentatively identified)

Stinson et al.14 18 54

this study

MS, RS IA (tentatively identified)

a

IA, identification by isotopic ratio analysis;41 MS, identification by comparison with NIST mass spectrum; RS, identification 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 findings represent the first 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. 345



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ASSOCIATED CONTENT

S Supporting Information *

Additional figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

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

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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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Identification of Volatile Markers in Potato Brown Rot and Ring Rot by

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