Introduction

Bacterial diseases of potato, blackleg and soft rot, caused by pectinolytic Pectobacterium and Dickeya spp. are responsible for major losses in seed and ware potato production in Europe and worldwide (Pérombelon and Salmond 1995; Pérombelon 2002; Toth et al. 2011). In Europe, the Dickeya spp. outbreaks have increased substantially since 2001, when the isolates of the new Dickeya species (D. solani) have been found in potato for the first time (Slawiak et al. 2009ab). This pathogen has never before been observed in the European climate zone or elsewhere (van der Wolf et al. 2014; Tsror et al. 2009; Slawiak et al. 2009; Laurila et al. 2008). Since 2005, the presence of D. solani has been reported in potato in many European countries including The Netherlands, Finland, Poland, Germany, Belgium, France, United Kingdom and Sweden, as well as outside Europe, in Israel and Georgia. The species has become the predominant pathogen responsible for the blackleg incidences in Europe (Toth et al. 2011; Tsror et al. 2011; van der Wolf et al. 2014). D. solani seems to be virulent in potato and possesses features allowing its easy spread and survival under the temperate climate in Europe (Czajkowski et al. 2009, 2012a).

The potential to control pectinolytic Dickeya spp. in potato is limited (Czajkowski et al. 2012b). Up to now, only an integrative strategy, combining the use of certified seed tubers descending from pathogen-free minitubers, hygienic practices during harvesting and grading, and avoidance of pathogen introduction during planting and in storage, is partially efficient but has not resulted in a broad eradication of the pathogens from the potato production chain (Perombelon and Kelman 1980; Toth et al. 2011).

Reduction of Pectobacterium and Dickeya spp. populations by treatment of potato tubers is ineffective and consequently rarely used in practical applications in fields (van der Wolf and de Boer 2007). Both, physical treatments including hot air and hot water, UV radiation and solarization, and utilization of chemical agents combining disinfectants and antibiotics, contribute to reduction or eradication of only superficial bacterial populations, whereas, they will not affect bacteria located deeper inside the tubers (Czajkowski et al. 2012b). Pectobacterium and Dickeya spp. are vascular pathogens, able to infect potato tubers internally (Perombelon and Kelman 1980; Czajkowski et al. 2009, 2010) and due to the ability of inhabiting the inner tuber tissues they will not be affected by the superficial treatments, thus the physical measures and chemical control agents will not effectively contribute to the control of Pectobacterium and Dickeya spp. in potato.

The use of elicitors, inducing natural resistance in plants against bacterial infections, can be considered as a promising alternative to chemical and physical treatments. It is well established that plants have evolved a range of different defence mechanisms to combat invasion and infections caused by bacterial pathogens. Despite the presence of passive physical and chemical barriers, different mechanisms are also activated specifically upon pathogen introduction and infection (Kessmann et al. 1994). The induced resistance (IR) may be restricted to the site of the pathogen entrance, or it may spread systemically and develop also in other (healthy) distant parts of the plant (Durrant and Dong 2004).

The two best characterized defence mechanisms in plants are: induced systemic resistance (ISR) and systemic acquired resistance (SAR) (Vallad and Goodman 2004). Activation of the ISR and SAR leads to similar phenotypic responses, however, the molecular and biochemical pathways that are activated in these defence mechanisms are different. SAR is mainly induced by the exposure of roots or foliar tissues (leaves and haulms) to abiotic and biotic elicitors and depends on the salicylic acid signal molecule (Ryals et al. 1994). Activation of SAR leads to production and accumulation of specific pathogenesis-related proteins (PR proteins) (Métraux et al. 2002). In contrast, ISR is prompted by the presence of plant-growth promoting rhizosphere bacteria in plant rhizospheres and is mediated by the plant hormones, ethylene and jasmonic acid (Van Loon et al. 1998). Also, ISR is not associated with production of PR proteins (Heil and Bostock 2002).

Certain chemicals, e.g. salicylic acid (SA), benzothiadiazole (BTH), acybenzolar-S-methyl (BION), 2,6-dichloroisonicotinic acid or β-aminobutyric acid (BABA) can activate the SAR defence mechanism without the plant’s interaction with pathogens (Gozzo 2003). Whereas ISR most often requires the presence of living microorganisms or their fragments (e.g. pathogen proteins, pathogen cell walls components) for induction (Pieterse et al. 2001). ISR can also be enhanced by applying exogenous plant hormones: jasmonic acid and ethylene, or their derivatives (Shoresh et al. 2005; Pieterse et al. 2000). For example, Luzzatto et al. (2007a) demonstrated that calla lily (Zantedeschia spp.) plants exposed to exogenous BION or methyl jasmonate elicitors produced high levels of antimicrobial phenolics. It was also shown that elicitation of these plants with methyl jasmonate but not with BTH induces priming and consequently resistance against P. carotovorum via induction of oxido-reductases (Luzzatto-Knaan et al. 2014).

Although SA and its analogs have been extensively used in research on systemic resistance in different plants, relatively little information can be found on its role in protection against the pectinolytic bacteria. Palva et al. (1994) reported that addition of the SA to the growth medium of axenically-growing tobacco seedlings causes almost complete resistance to infection by P. carotovorum subsp. carotovorum and to our knowledge, there is no information available on the effect of SA on Dickeya spp., particularly the D. solani infections of potato.

The aim of this work was to investigate the effect of application of SA on symptom development caused by D. solani on in vitro grown potato plants. In replicated experiments the potato plants grown on medium supplemented with SA were investigated for the phytotoxic effects. In line with this, potato plants grown on a medium supplemented with SA and stem base-inoculated with GFP-tagged D. solani (Czajkowski et al. 2010) were investigated for the blackleg development, bacterial population dynamics in stems and colonization of plant roots by the GFP-tagged bacteria.

Materials and methods

Bacterial strains and media

GFP-tagged D. solani IPO2222 (IPO2254) (Czajkowski et al. 2010) was grown at 28 °C for 24–48 h on tryptone soya agar (TSA) (Oxoid) or nutrient agar (NA) (Oxoid), supplemented with 150 μg ml−1 ampicillin (Sigma) prior to use. Liquid cultures were prepared in nutrient broth (NB) (Oxoid) and/or in tryptic soya broth (TSB), if required – also supplemented with ampicillin and grown at 28 °C for 24 h with agitation (200 rpm).

Salicylic acid (SA)

Salicylic acid (Sigma) was prepared as hundred or thousand times concentrated stock in 50 % ethanol prior to use. The SA was added to the MS growth medium to the final concentration of 25 or 50 μM.

Assessment of D. solani IPO2254 morphology in transmission electron microscopy (TEM) upon treatment with SA

To assess the morphology of D. solani cells treated with 50 μM SA, bacteria were grown overnight in TSB at 28 °C with shaking (200 rpm) in the presence of 50 μM SA. For the TEM analysis bacteria were adsorbed onto carbon-coated grids (Sigma) stained with 1.5 % uranyl acetate and directly examined with electron microscope (Philips CM100, EM, FEI Company) as described earlier (Szpitter et al. 2014). The experiment was repeated independently one time with the same setup.

Measuring the antibacterial effect of salicylic acid

To check the possible direct effect of SA on D. solani growth in vitro, an overnight IPO2254 bacterial culture with a density of ca. 109 cells ml−1 in NB supplemented with 150 μg ml−1 ampicillin was diluted 50 times in the same medium supplemented with SA to the final concentration of 50 μM. The prepared bacterial cultures were grown at 28 °C with agitation (200 rpm). For negative control, bacteria were prepared under the same conditions, without SA. Growth rate was determined by measuring the optical density (OD600) for a period of 12 h each hour and one time after 24 and after 48 h and by comparing the growth of SA treated bacterial cultures with the growth of control cultures. Bacterial densities were estimated from the OD600 reads assuming that OD600 = 0.1 is equal to 108 cfu ml−1. The experiment was repeated twice with the same set-up with two replicates for each experiment. The results were averaged.

Assessment of D. solani IPO2254 maceration ability on potato tuber slices upon treatment with SA

A potato slice assay (Czajkowski et al. 2010) was used to check if incubation of D. solani IPO2254 with SA prior to inoculation of potato tuber slices with the bacteria would affect its ability to macerate potato tuber tissue. For this, bacteria were grown for 12 h in TSB in the presence of 50 μM SA with agitation (200 rpm) at 28 °C. After this time, bacteria were centrifuged for 5 min. at 6000 × g and washed two times with 1/4 Ringer’s buffer to remove the elicitor. Bacterial densities were adjusted to ca. 106 cfu ml−1 with sterile demineralized water. For control, bacteria were grown in TSB under the same conditions as described above but without SA supplementation. Ware potato tubers of cv. Bryza (obtained at the local supermarket in Gdansk, Poland) were rinsed with running tap water, surface-sterilized with 70 % ethanol for 10 min, rinsed with running tap water and dried with tissue paper. Potato tubers were cut into ca. 0.7 cm-thick transverse disks using a sterile table knife. Three wells (5 × 5 × 5 mm) per potato slice were made using a sterile cork borer and these were filled with 50 μl of bacterial suspension containing 106 cfu ml−1 in sterile demineralized water. For disease development slices were incubated at 28 °C for 72 h in a humid box. Per treatment, three potato slices derived from three different potato tubers were used. For the negative control 50 μl aliquots of sterile demineralized water were used instead of bacterial suspension. For the positive control, bacteria prepared in the same way but not treated with SA were used. The effect of SA on the ability of D. solani IPO2254 to macerate potato tuber tissue was measured by comparing the ratio of the average diameter of the rotting potato tissue around wells inoculated with SA-treated D. solani IPO2254 with the average diameter of rotting tissue around wells of the positive control (wells inoculated with D. solani IPO2254 not treated with SA prior to inoculation). Two repetitions were made per treatment and the entire experiment was independently repeated on time with the same set-up. Results from both experiments were averaged.

Growth and propagation of in vitro potato plants

In vitro potato plants cv. Kondor were obtained from the Laboratory of Seed Production and Potato Protection, Plant Breeding and Acclimatization Institute - National Research Institute, Bonin, Poland. Plants were grown on Murashige and Skoog (MS) medium (Murashige and Skoog 1962) with 20 g l−1 sucrose and 7 g l−1 agar. Potato plantlets were grown at a temperature of 20–22 °C under white fluorescent light with a 16 h photoperiod (white cool fluorescent light, Philips, TLD 58 W/84o, 30–35 μmol m−2 s−1) for 4 weeks.

The experiments with SA were conducted on micropropagated plants in individual culture tubes (Duchefa Biochemie b.v) using single-node cuttings on MS medium containing 25 or 50 μM SA with 10 g l−1 sucrose and 7 g l−1 agar, pH 5.8. The plantlets were grown at 20–22 °C for 20 days prior to inoculation with bacteria under the same conditions as described above. The control plants were micropropagated as described above, without SA.

Inoculation of in vitro-grown potato plants with D. solani IPO2254

For inoculation of potato plants, 10 μl of bacterial suspension in water containing 105 cfu (colony forming units) ml−1 of D. solani IPO2254 was pipetted into the interspace between the stem base and MS medium of each individual plant. For negative control, instead of bacterial suspension, 10 μl of sterile demineralized water was used. Inoculated plants were grown at a temperature of 20–22 °C under white fluorescent light with a 16 h photoperiod (white cool fluorescent light, Philips, TLD 58 W/84o, 30–35 μmol m−2 s−1). Six treatments were applied on potato plants: (treatment a) plants grown on MS medium and inoculated with water (negative control), (treatment b) plants grown on MS medium and inoculated with D. solani IPO2254 (positive control), (treatment c) plants grown on MS medium supplemented with 25 μM SA and inoculated with water, (treatment d) plants grown on MS medium supplemented with 25 μM SA and inoculated with IPO2254, (treatment e) plants grown on MS medium supplemented with 50 μM SA and inoculated with water and (treatment f) plants grown on MS medium supplemented with 50 μM SA and inoculated with D. solani IPO2254. Per treatment, 20 plants grown in individual culture tubes were used and the entire experiment was repeated independently one time with the same set-up. Plants were visually inspected after 2, 5 and 10 days post inoculation (dpi) for symptom development and were sampled 14 dpi.

Symptom development in in vitro-grown potato plants

In vitro plants were sampled 14 dpi. Potato plants were assessed for wilting, typical blackleg, stem desiccation and plant death. The plants were aseptically removed from the in vitro plastic tubes and per plant, 1-cm long stem fragments taken 1 cm above the stem base were separately suspended in 1 ml of 1/4-strength Ringer’s buffer (Merck) supplemented with 0.02 % diethyldithiocarbamic acid (Arcos Organics) in 2-ml Eppendorf tubes. Three sterile glass beads (Merck Chemicals) were added to each tube and the suspended stem fragments were vigorously vortexed (ca. 300 rpm) for 60 min to grind plant tissues and release bacteria from plant fragments. One hundred μl of the undiluted and serial diluted stem extracts were plated in duplicates on CVP (Hélias et al. 2011) and/or TSA plates supplemented with ampicillin (100 μg ml−1) (Sigma). Plates were incubated for bacterial growth and cavity formation at 28 °C for 24–48 h. The resulting cavity forming, GFP positive, ampicillin resistant colonies were counted and the colony forming units per mg of stem tissue were calculated for each sample.

Assessment of D. solani IPO2254 virulence on in vitro grown potato plants upon treatment with SA

To check the effect of SA on the virulence of D. solani IPO2254 on in vitro grown potato plants, bacteria were grown for 12 h in TSB supplemented with 50 μM SA or in TSB at 28 °C with agitation (200 rpm). After this time, bacteria were centrifuged for 5 min. at 6000 × g and washed two times with 1/4 Ringer’s buffer. Bacterial densities were adjusted to ca. 105 cfu ml−1 with sterile demineralized water. In vitro potato plants of cultivar Kondor were propagated in a similar way as described above. The plantlets were grown at 20–22 °C for 20 days prior to inoculation with bacteria. For inoculation, 10 μl of bacterial suspension in water containing 105 cfu ml−1 of D. solani IPO2254 or 105 cfu ml−1 of D. solani IPO2254 pretreated with SA were pipetted into the interspace between the stem base and MS medium of each individual plant. For negative control, instead of bacterial suspension, 10 μl of sterile demineralized water was used. Inoculated plants were grown at a temperature of 20–22 °C under white fluorescent light with a 16 h photoperiod (white cool fluorescent light, Philips, TLD 58 W/84o, 30–35 μmol m−2 s−1) as described above. In vitro plants were sampled 14 dpi. Potato plants were visually assessed for wilting, typical blackleg, stem desiccation and plant death and the percentage of blackleg-diseased plants was calculated per each treatment. Per treatment 10 individual plants were used and the experiment was repeated one time with the same setup. Per treatment and per experiment, five randomly chosen stem fragments with the length of 1 cm collected 1 cm above the MS medium were assessed for D. solani IPO2254 densities in stems by counting serially diluted in Ringer’s buffer GFP-positive ampicillin resistant colonies on TSA as described above.

Presence of D. solani strain IPO2254 on the surface of potato roots and stem bases assessed with confocal laser scanning macroscope

To check the presence of D. solani IPO2254 on roots and stem bases of inoculated plants, at 14 dpi, four individual plants of each treatment (in total 16 plants per experiment: four plants grown on MS medium and inoculated with sterile water (treatment a), four plants grown in MS medium and inoculated with D. solani IPO2254 (treatment b), four plants grown in MS supplemented with 25 μM SA and inoculated with IPO2254 (treatment d), and four plants grown in MS supplemented with 50 μM SA and inoculated with IPO2254 (treatment f)) were removed from the culture tubes, washed briefly in sterile water and examined for the presence of a GFP signal using a confocal Leica TCS LSI macro confocal microscope (Leica) at magnification 1× and 5×. For this, plants were removed from culture tubes, placed on square transparent glass slides and examined directly with a macroscope without any further processing or incubation. Photographs were taken with a Leica Digital System (Leica) connected to the macroscope. The plants grown on MS medium supplemented with 25 or 50 μM SA but not inoculated with bacteria (treatment c and treatment e) were not examined with the macroscope.

Statistical analysis

Bacterial count data were analyzed with ordinary linear regression using the statistical software package Statistica ver. 10 (Statsoft, http://www.statsoft.com/). To achieve approximate normality, the data were log transformed after adding a value 1 to avoid taking logs of zero. Results were considered to be significant at P = 0.05 and pair-wise differences were obtained using the t-test. Data were analyzed according to the experimental design e.g. two replicated in vitro experiments with treatments of 20 replications (plants) each. The linear model considered was a complete block design with replicates as complete block.

Results

Antibacterial effect of salicylic acid

No difference in D. solani cell morphology was observed in bacterial cultures grown in TSB with and without SA supplementation in transmission electron microscopy analysis (Fig. 1a). Treatment of D. solani IPO2254 with salicylic acid in concentration of 50 μM for a period of 12 h measured every hour and one time after 24 and 48 h did not affect significantly the bacterial growth in comparison with control as determined by measuring the OD600 (Fig. 1b).

figure 1

Effect of salicylic acid on D. solani IPO2254 virulence

The ability of SA-treated D. solani IPO2254 to macerate potato tuber tissue was investigated using a potato slice assay. After incubation of slices for 3 days at 28 °C, the diameter of the rotting tissues caused by the SA-treated bacteria was not significantly different from that of the non-treated with SA D. solani IPO2254 (Fig. 2a).

Fig. 2
figure 2

The effect of 50 μM SA on the D. solani IPO2254 ability to macerate potato tuber tissue (a) and the ability to cause blackleg symptoms in vitro grown potato plants (b). a To assess the ability of D. solani IPO2254 to macerate potato tuber tissue a potato slice assay was used. The diameter of rotting tissue (in mm) of potato slices was measured after 72 h incubation at 28 °C in a humid box. Wells of potato slices were filled up with 50 μl of sterile water, with 50 μl of bacterial suspension in water containing 106 cfu ml−1 of D. solani IPO2254 or with 50 μl of bacterial suspension in water containing 106 cfu ml−1 of h D. solani IPO2254 pretreated with 50 μM SA for 12 h and washed two times in Ringer’s buffer prior to potato slice assay. Three potato slices containing 3 wells each and derived from three different tubers were used per treatment. The experiment was independently repeated one time and the results were averaged. b To assess the ability of D. solani IPO2254 to cause blackleg symptoms, in vitro grown potato plants of cv. Kondor were inoculated with 10 μl of D. solani IPO2254 suspension in water containing 105 cfu ml−1 or with 10 μl of D. solani IPO2254 suspension pretreated with SA for 12 h and containing the same inoculum. For negative control, instead of bacterial suspension, 10 μl of sterile demineralized water was used. Inoculated plants were grown at a temperature of 20–22 °C under the same conditions as described above. At 14 dpi potato plants were visually assessed for blackleg symptoms and plant death. Ten plants were used per treatment and the entire experiment was independently repeated one time with the same setup

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The ability of SA-treated D. solani IPO2254 to infect potato plantlets was compared with the ability of non-treated with SA D. solani IPO2254 to establish an infection in potato plants and was investigated using the in vitro potato plants of cv. Kondor. Plants infected with SA-treated D. solani IPO2254 displayed similar infection characteristics as plants infected with non-treated D. solani IPO2254, indicating that the SA-treated bacteria were able to colonize and cause infection of in vitro grown potato plants and that their virulence was not affected by incubation with 50 μM SA for 12 h at 28 °C prior to infection studies (Fig. 2b). Bacterial densities in stems infected with SA-treated and SA-untreated D. solani IPO2254 were not statistically different and on average densities of 103–104 cfu g−1 of stems were found (data not shown).

Growth chamber experiments with in vitro grown potato plants

Fitness of potato plants and disease development

Experiments with in vitro plants were conducted in December 2013 – January 2014 (experiment 1) and January – February 2014 (experiment 2). In each experiment the effect of 2 concentrations of SA (25 and 50 μM) on plant fitness and disease progression caused by D. solani IPO2254 in inoculated plants (n = 20) was investigated (Fig. 3).

Fig. 3
figure 3

Effect of SA and Dickeya solani IPO2254 on the fresh weight (a) and maximum shoot length (b) of axenically-grown potato plants. Plants were grown for 14 days on MS medium with or without 25 or 50 μM SA prior to inoculation with the pathogen. Plants were analyzed after next 14 days (after 28 days in total) post or without inoculation with D. solani IPO2254. a Average fresh weight of plants determined 14 days post plant inoculation with bacteria. Control plants were inoculated with sterile water. Data from two independent experiments were analyzed separately. Values followed by identical characters are not significantly different (P = 0.05). b Average height of the highest shoot measured 14 days post plant inoculation with bacteria. Control plants were inoculated with sterile water. Data from two independent experiments were analyzed separately. Values followed by identical characters are not significantly different (P = 0.05)

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Treatment (a)

In plants grown on MS medium and inoculated with sterile water, no disease symptoms were observed during the entire course of both experiments. In experiment 1, the average height of plants at 14 dpi was 4.8 cm and the average fresh weight of haulms was 61.7 mg. In experiment 2 the average height of plants was 5.5 cm and the average fresh weight was 62.4 mg.

Treatment (b)

In plants grown on MS medium and inoculated with D. solani IPO2254, the first symptoms appeared 3 days after inoculation in both experiments. These were: colonization of roots observed as an increase of turbidity of MS medium around the roots and induction of stem base rotting and blackening progressing up the stems. In both experiments, after 14 days all plants showed typical blackleg symptoms. The infections resulted in a severe reduction of the plant height of 40 % percent in experiment 1 and 49 % in experiment 2, respectively, and of the fresh weight with 54 % reduction in experiment 1 and 52 % in experiment 2, respectively.

Treatment (c)

In plants grown on MS medium supplemented with 25 μM SA and inoculated with water, no disease symptoms were observed during the entire course of both experiments. The treatment with SA did not significantly affect plant height and fresh weight.

Treatment (d)

In plants grown on MS medium supplemented with 25 μM SA and inoculated with IPO2254 bacteria, the first symptoms appeared 3 days after inoculation, similarly as in Treatment (b). After 3 days, bacteria were present in the rhizosphere which was visualized as an increase of turbidity of the MS medium around plant roots. In the next 2–3 days, decay of the stem base was initiated which gradually extended along the stem. Due to severe infections after 14 days, blackleg symptoms were observed in 85 % of plants in experiment 1 and in 80 % plants in experiment 2. At 14 dpi the reduction of plant weight was 58 % in experiment 1 and 53 % in experiment 2, in comparison with the control plants untreated with IPO2254. Similarly, the shoot length was 76 % and 73 % of the shoots of control plants in experiment 1 and 2, respectively.

Treatment (e)

In plants grown on MS medium supplemented with 50 μM SA and inoculated with water, no disease or other symptoms were observed in both experiments. No statistically significant reduction of shoot length and fresh plant weight was observed in both experiments.

Treatment (f)

In plants grown on MS medium supplemented with 50 μM SA and inoculated with D. solani IPO2254, no blackleg symptoms were observed in both experiments. Three days post inoculation in 10 % plants and 15 % plants (two plants in experiment 1 and three plants in experiment 2) colonization of roots by D. solani was observed. This, however, did not result in development of symptoms during the following 11 days (14 dpi). At the same time, only very low bacterial densities were found in the colonized plants (ca. 1–10 cfu g−1) in both experiments.

Bacterial populations in stems

Fourteen days after inoculation, stem fragments of in vitro potato plants were extracted and plated on TSA and/or CVP supplemented with ampicillin to determine the percentage of infected stems and to quantify internal populations of D. solani IPO2254. In both experiments no bacteria were found in the plants grown on MS medium supplemented with SA and inoculated with water (treatment a, treatment c and treatment e) (Fig. 4a). Simultaneously, the GFP tagged strain was found in all stem fragments collected from plants grown on MS medium and inoculated with D. solani IPO2254 (treatment b), and in all stem fragments collected from plants grown on MS medium supplemented with 25 μM SA and inoculated with D. solani (treatment d). In experiment 1 and 2, 10 % and 15 % stem fragments, respectively, collected from plants grown on MS medium supplemented with 50 μM SA and inoculated with strain IPO2254 (treatment f) harboured GFP-tagged bacteria. Bacterial densities in stems varied largely per plant in treatment b, treatment d and treatment f, and on average the densities of 102–104 cfu g−1 of D. solani were recorded (Fig. 4b).

Fig. 4
figure 4

Effect of supplementation of the growth medium with SA on the average percentage of symptomatic plants (showing blackleg or browning and rotting of stem tissue) 14 days post inoculation with bacteria (n = 20 plants per treatment) (a) and on GFP-tagged D. solani IPO2254 bacterial densities in stems (n = 20 plants per treatment) (b). Values followed by identical characters are not significantly different (P = 0.05)

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Confocal laser scanning macroscopy analysis

Plant parts were analyzed with a Leica TCS LSI confocal macroscope (Leica) at a low magnification of 1 and 5 times. At 14 dpi, bright GFP signal was found on all roots and stem bases of sampled plants derived from treatment b (plants grown on MS medium and inoculated with D. solani IPO2254) and in plants of treatment d (plants grown on MS medium with 25 μM SA and inoculated with IPO2254). No GFP signal was observed in the stem bases and roots of treatment a (plants grown on MS medium and inoculated with sterile water) and in stem bases and roots of plants in treatment f (grown on MS supplemented with 50 μM SA and inoculated with bacteria) (Fig. 5).

Fig. 5
figure 5

Colonization of roots of in vitro grown potato plants with GFP-tagged D. solani IPO2254 assessed with confocal laser scanning macroscopy (CLSM). Potato roots were collected 14 days post inoculation and were briefly washed from MS medium with tap water and used immediately for macroscopic analysis. Samples were taken from plants inoculated with D. solani IPO2254, plants grown on MS medium supplemented with 25 μM SA and inoculated with D. solani IPO2254, and plants grown on MS medium supplemented with 50 μM SA and inoculated with D. solani IPO2254. For control, potato plants grown on MS medium were inoculated with sterile water. Green fluorescence was observed on roots and stem bases taken from plants inoculated with IPO2254 and in plants grown on medium supplemented with 25 μM SA and inoculated with IPO2254. No fluorescence was observed in plants grown on medium supplemented with 50 μM SA and inoculated with bacteria

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Discussion

This study was conducted to assess the potential of SA as an elicitor to induce resistance against D. solani in potato. It is the first case dealing with SA treatment used in in vitro potato plants against infection caused by D. solani and also, the first description of a simple screening system combining in vitro (potato) plants, the fluorescently-labelled pathogenic bacteria and (fluorescent) macroscopy, that can be used on large number of (potato) plants and with different chemicals tested as prospective elicitors in vitro.

Elicitation of potato plants against pectinolytic bacteria has been analyzed previously in several studies (Palva et al. 1994; Vidal et al. 1997; Luzzatto et al. 2007b). For example, Palva et al. (1994) showed that the SA treatment together with application of P. carotovorum subsp. carotovorum on tobacco resulted in hypersensitivity reaction and induction of yet-unknown factors able to neutralize the plant cell wall degrading enzymes produced by the pathogen. Vidal et al. (1997) demonstrated that compounds from P. carotovorum subsp. carotovorum may act antagonistically with SA elicitation. SA was observed to inhibit the induction of genes coding for pathogenies-related (PR) proteins elicited by the bacteria, which suggested a cross-talk of SA and bacterial elicitor network. To our best knowledge, there is no information available on elicitation of plants against Dickeya spp. and specifically against D. solani.

Addition of 50 μM SA to the MS medium resulted in strong protection of plants against colonization of and infection with D. solani via developing roots. Plants treated with 50 μM SA were symptomless whereas 100 % of untreated plants showed symptoms. This suggests that the application of SA prevents bacteria from colonizing the root surface and systemic infections of potato plants. Similar observations on SA concentration in plants were reported for rice (Oryza sativa) and pea (Pisum sativum) where it was shown that application of exogenous SA may protect roots from being invaded by pathogens and/or from developing symptoms even up to 100 % (Silverman et al. 1995; Blilou et al. 1999).

The efficacy of SA-mediated resistance of in vitro grown potato plants against D. solani appears to be dose dependent. At a concentration of 25 μM SA the level of protection was only minor. In contrast, in a twice as high concentration (50 μM) the protection effect was almost complete, and no blackleg symptoms in tested plants with IPO2254 were observed 14 dpi. Supplementation of 25 or 50 μM SA to MS medium had no adverse effect on plant growth. At higher concentrations, i.e. 150 and 300 μM, salicylic acid caused deterioration of plant growth and abnormalities during leaf (e.g. yellowing and wilting) and root development (e.g. poor rooting) in vitro (data not shown). The dose-dependent phytotoxicity of exogenous SA was reported previously for different plants and growth systems (Raskin 1992; Hayat et al. 2010), however to our knowledge no conclusive summary is present concering the phytotoxic effect of SA in potato plants and tubers (Hayat et al. 2010).

This study shows that axenically-grown potato plants can be used successfully to study the effect of defense elicitation against pectinolytic bacteria. It was reported previously that elicitors are rapidly taken up by the root system of in vitro plants (Palva et al. 1994). In vitro plants are grown under highly controlled conditions and therefore the results were highly repeatable. Thus it may serve as a method-of-choice for the first screening of other compounds for protection of (potato) plants against soft rot bacterial pathogens. The screening method can be scaled up easily if necessary.

The use of in vitro plants of cv. Kondor, highly susceptible to infections with Dickeya spp., resulted in a rapid colonization of plants. The use of the GFP-tagged D. solani strain IPO2254 allowed microscopical observations of bacteria on root and stem base surface as it was described earlier by Golan et al. (2010), who used a GFP-tagged P. carotovorum subsp. carotovorum to follow the fate and population dynamics of bacterial cells upon infection of in vitro grown Ornithogalum dubium plants. This study shows that D. solani is able to enter roots from the medium whereupon the potato plants are systemically colonized as it was reported previously for plants grown under greenhouse conditions in soil (Czajkowski et al. 2010).

It has been reported previously that SA can cause morphological and transcriptional changes in bacterial cells yet this effect was evaluated mainly for human pathogens such as Serratia marcescens, Staphylococcus aureus and Klebsiella pneumoniae (Price et al. 2000; Delaney et al. 1994). Consequently, only very little information is present on its effect on virulence of plant pathogenic bacteria (Raskin 1992). We have found only one study describing the SA effect on P. carotovorum – species close related to Dickeya spp. (Lagonenko et al. 2013). Lagonenko et al. (2013) showed that 25 and 50 mM SA (1000 × times higher concentration than used in our study) negatively influenced important virulence determinants i. e. biofilm formation, motility and quorum sensing mechanism in P. carotovorum. However, it is likely that in our studies the effect of SA was exclusively plant-mediated as no direct antibacterial effect of 50 μM on bacterial growth was observed. The IPO2254 growth in liquid cultures containing the elicitor in the given concentration was similar to the growth of this strain in cultures without SA as evidenced by measuring the optical density of bacterial cultures up to 12 h and after 24 and 48 h. Also, in the transmission electron microscopy analysis no effect of SA on the morphology of D. solani was observed. In line, we have not observed any negative effects of SA on bacterial virulence and ability to macerate potato tuber tissue in comparison with the untreated with SA D. solani IPO2254 using two different approaches – potato slice assays and in vitro grown potato plants. It cannot be excluded however, that higher concentrations of SA may cause morphological and physiological alternations in Dickeya spp. and particularly in D. solani, however at the concentration used in our study (50 μM SA) such effects were not observed. The protection of potato plants against D. solani by SA is therefore probably based on induced resistance of the treated plants, as it was reported earlier for other pathogens (Durrant and Dong 2004).

For a feasible application of SA in the control of potato blackleg caused by D. solani a considerable amount of work still needs to be done. There is a demand for information concerning various factors that may affect the influence of SA, including the time of application, the effect of the plant cultivar, the response against other pathogens causing blackleg and the most importantly, the impact of environmental conditions that would influence the expression of induced resistance in the field (Angelova et al. 2006). There is also a need to understand the mechanism of the protection afforded by SA to potatoes against D. solani. Work involving mass spectrometry analysis of potato-derived compounds elicited upon SA treatment and IPO2254 infection and experiments with hydroponically grown potato plants are now being conducted to understand the molecular basis of the SA-mediated elicitation of potato plants against D. solani and to find the time in which the elicitation effect last for, respectively.