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Keywords:

  • antibiosis;
  • biological control;
  • Erwinia chrysanthemi;
  • root colonization;
  • soil;
  • survival

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Interactions between Serratia plymuthica A30 and a blackleg-causing biovar 3 Dickeya sp. were examined. In a potato slice assay, S. plymuthica A30 inhibited tissue maceration caused by Dickeya sp. IPO2222 when co-inoculated at a density at least 10 times greater than that of the pathogen. In glasshouse experiments, population dynamics of the antagonist and of the pathogen in planta were studied by dilution plating and confocal laser scanning microscopy (CLSM) using fluorescent protein-tagged strains. Pathogen-free minitubers were vacuum-infiltrated with DsRed-tagged Dickeya sp. IPO2222 and superficially treated during planting with a water suspension containing GFP-tagged S. plymuthica A30. A30 reduced the blackleg incidence from 55% to 0%. Both the pathogen and the antagonist colonized the seed potato tubers internally within 1 day post-inoculation (dpi). Between 1 and 7 dpi, the population of A30 in tubers increased from 101 to c. 103 CFU g−1 and subsequently remained stable until the end of the experiment (28 dpi). Populations of A30 in stems and roots increased from c. 102 to c. 104 CFU g−1 between 7 and 28 dpi. Dilution plating and CLSM studies showed that A30 decreased the density of Dickeya sp. populations in plants. Dilution plating combined with microscopy allowed the enumeration of strain A30 and its visualization in the vascular tissues of stem and roots and in the pith of roots, as well as its adherence to and colonization of the root surface. The implications of these finding for the use of S. plymuthica A30 as a biocontrol agent are discussed.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Blackleg and soft rot bacterial diseases caused by Dickeya and Pectobacterium species can result in significant losses in seed potato production in Europe (Toth et al., 2011). The importance of Dickeya spp. as potato pathogens has increased recently (Sławiak et al., 2009; Toth et al., 2011). This increase has been associated with the presence of a newly identified clade of a biovar 3 Dickeya sp. which appeared recently in Europe and which could not be classified into one of the known six species described so far (Tsror et al., 2008; Sławiak et al., 2009). The new clade may constitute a new Dickeya species, provisionally called ‘D. solani’ (Toth et al., 2011). Its occurrence has been reported in potato in several European countries including the Netherlands, Finland, Poland, Germany, Belgium, France, the UK and Sweden, as well as in Israel (Toth et al., 2011).

Control measures for Dickeya and Pectobacterium species in potato are limited (Czajkowski et al., 2011b). They include the use of certified seed derived initially from pathogen-free minitubers, hygienic measures to avoid introduction and dissemination of the bacterial pathogens, avoidance of wounding of tubers, and field soil drainage to avoid oxygen depletion, which can impair tuber resistance to rotting. However, despite an integrative strategy involving these control measures, an acceptable reduction of blackleg and soft rot problems has not been achieved consistently.

Tuber treatments to reduce bacterial inoculum are rarely used in practice. Physical treatments, such as hot water, hot air and radiation, as well as chemical control agents, may reduce superficial bacterial populations on tubers, but have little effect on internally located bacteria. Dickeya spp. are vascular pathogens capable of colonizing vascular tissues following root or stem infections (Czajkowski et al., 2010a,b). Consequently, relatively high populations of Dickeya spp. are frequently found at the stolon end of progeny tubers (Czajkowski et al., 2009). Not surprisingly, disinfection with chemical and physical treatments is not effective against these internal populations. At present, no systemic bactericides are available which could eliminate these bacteria inside plant vascular tissues.

As an alternative to seed tuber disinfection procedures, use of antagonistic bacteria has been attempted, but generally with little consistent success, mainly because of the inability of the bacteria to invade and survive within the host tissues. However, endophytic bacteria isolated from within surface-sterilized plant tissues were able to colonize plants systemically when applied artificially (Lodewyckx et al., 2002). These bacteria are, by definition, non-pathogenic to and exert no adverse effect on the host plant while interacting with pathogens present (Hallmann et al., 1997). It has already been demonstrated that some endophytes can act as antagonists and that their presence can have a direct positive effect on plant fitness (Chen et al., 1995; Adhikari et al., 2001).

An endophytic antagonistic Serratia plymuthica strain, A30, which was active against biovar 3 Dickeya spp. in vitro and on potato plants under glasshouse conditions, was recently isolated and described (Czajkowski et al., 2011a). The antagonist was isolated from rotting tissue of superficially disinfected tubers wrapped in plastic foil to induce tuber decay. The strain was selected on the basis of in vitro production of antibiotics against Dickeya spp. and has been extensively characterized in in vitro tests for other features that are potentially involved in antagonism: production of biosurfactants, motility and growth under aerobic and anaerobic conditions at relatively low (10°C) temperatures. In replicated glasshouse experiments, S. plymuthica A30 reduced blackleg symptom expression caused by biovar 3 Dickeya spp. by 100% and colonization of stems by the pathogen by 97% after co-inoculation of tubers by vacuum infiltration (Czajkowski et al., 2011a).

This study aimed to acquire knowledge on the interactions between S. plymuthica A30 and biovar type 3 Dickeya sp. (strain IPO2222) in potato plants as a preliminary step in the possible commercial exploitation of the biocontrol agent (Cook, 1993). This will involve understanding the antagonistic mechanism, ecology of the biocontrol agent, survival in the environment and its ability to colonize potato plants internally and superficially.

The control of Dickeya spp. by S. plymuthica A30 was investigated in a potato slice assay at different inoculum densities. The potato slice assay was also used to study the population dynamics of the pathogen and the antagonist on/in tuber tissue. In repeated glasshouse experiments, tubers were treated with a suspension of the antagonist just before covering tubers with soil, to simulate a seed tuber application procedure in the field. The possibility of the strain colonizing potato plants after tuber treatments was studied to determine the potential of the antagonist for control of biovar 3 Dickeya spp. in internal plant tissues. To enable visualization of bacteria in planta in vascular tissues with microscopical techniques, the biovar 3 Dickeya sp. strain was tagged with plasmid-based red fluorescent protein (DsRed) and S. plymuthica A30 with green fluorescent protein (GFP).

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacterial strains and growth media used

Serratia plymuthica A30 (Czajkowski et al., 2011a) and a Dickeya sp. biovar 3 strain (IPO2222) (Sławiak et al., 2009) were grown at 28°C for 24–48 h on tryptic soya agar (TSA) (Oxoid) or nutrient agar (NA) (Oxoid) prior to use. Liquid cultures were prepared in nutrient broth (NB) (Oxoid) and/or tryptic soya broth (TSB) (Oxoid), grown at 28°C for 24 h with agitation (200 rpm). Strains of GFP-tagged S. plymuthica A30 and DsRed-tagged Dickeya sp. IPO3012 (derived from wildtype strain A30 and IPO2222, respectively) were grown using the same media but supplemented with 40 μg mL−1 tetracycline (Sigma) (these media were designated NAt, TSAt, NBt and TSBt, respectively). When plant extracts were analysed, growth media were supplemented additionally with cycloheximide (Sigma) to a final concentration of 200 μg mL−1 to prevent fungal growth.

Generation of GFP-tagged S. plymuthica A30 and DsRed-tagged Dickeya sp. IPO2222 strains

Plasmids pRZ-T3-gfp and pRZ-T3-dsred (Bloemberg et al., 2000) were used for generation of GFP-tagged S. plymuthica A30 and DsRed-tagged biovar 3 Dickeya sp. IPO3012, respectively. The plasmids carrying genes coding for fluorescent proteins under constitutive promoters were introduced into bacterial cells by electroporation as described in Czajkowski et al. (2010a). Briefly, 50-μL suspensions of competent bacterial cells of A30 or IPO2222 (containing c. 1011–1012 colony forming units (CFU) mL−1) were mixed with 100 ng μL−1 plasmid DNA and electroshocked at 2·5 kV for 1–2 s at 4°C using a Bio-Rad Gene Pulser 200/0·2. After electroporation, bacterial cells were resuscitated in 500 μL NB at 28°C for 1 h with shaking (200 rpm). Transformed cells (100 μL) were plated on TSAt and incubated for 24–48 h at 28°C before selection of GFP or DsRed fluorescent transformants.

Growth of tagged bacterial strains relative to their wildtype parental strains

Relative growth of DsRed-tagged Dickeya sp. strain IPO3012 vs. wildtype strain IPO2222 and GFP-tagged S. plymuthica A30 strain vs. wildtype A30 was determined under aerobic conditions, using as inoculum 100-μL overnight cultures containing c. 109–1010 CFU mL−1 in 20 mL NBt or NB diluted 50-fold in NBt or NB. Bacteria were grown at 28°C with a shaking rate of 200 rpm and growth rates were determined by measuring the OD600 over a period of up to 24 h.

Growth of the wildtype IPO2222 and tagged IPO3012 under anaerobic conditions, created by adding 5 mL liquid paraffin to 30 mL bacterial suspension in PEB (potato enrichment broth) (Pérombelon & van der Wolf, 2002), was also determined as described above, except that the cultures were not agitated during incubation.

Ability of DsRed-tagged IPO3012 to macerate potato tuber tissue

Bacterial suspension of IPO3012 was diluted in Ringer’s buffer (Merck) to c. 106 CFU mL−1. Potato tubers of cv. Agria (Agrico) were rinsed under running tap water, followed by washing twice with 70% ethanol for 5 min and again twice for 1 min with demineralized water. Tubers were dried with tissue paper and cut into 0·7-cm transverse slices. Three 5-mm-deep wells per slice were made with a 5-mm-diameter sterile cork borer and filled with 50 μL bacterial suspension. Three potato slices derived from three different tubers were used per treatment per strain. The slices were incubated at 28°C for 72 h in a high-humidity box and the diameter of rotting tissue around inoculated wells was measured after 72 h incubation at 28°C. The experiment was repeated twice and the growth-rate results for the two strains analysed and compared.

Inhibition of wildtype IPO2222 by GFP-tagged A30 in an overlay plate assay

The ability of GFP-tagged S. plymuthica A30 relative to wildtype strain A30 to inhibit growth of IPO2222 was compared in an overlay plate assay with IPO2222 as the indicator strain. Fifty microlitres of an overnight culture of strain IPO2222 (c. 109 CFU mL−1) in NB were mixed with 5 mL soft agar (NB supplemented with 0·7% agar) pre-warmed to 45–50°C, and poured onto TSA plates. After the agar had solidified, one aliquot of 2·5 μL overnight culture of A30 or GFP-tagged A30 in NB and NBt, respectively (c. 109 CFU mL−1) was spotted onto the surface of the agar plate (two replicated plates). Plates were incubated for 24–48 h at 28°C. The diameter of the clear ‘halo’ (indicating IPO2222 growth inhibition) around the inoculated spot was measured.

Control of Dickeya sp. IPO2222 by GFP-tagged A30 relative to the wildtype strain A30 on potato slices

The ability of GFP-tagged A30 to protect potato tuber tissue against maceration by IPO2222 was evaluated in a potato slice assay as described above. GFP-tagged A30 strain, the wildtype A30 strain and IPO2222 were grown overnight in NBt or NB at 28°C. Bacterial cultures were centrifuged (5 min, 6000 g), washed twice with ¼-strength Ringer’s buffer and resuspended in sterile water to the original volume. Wells of the tuber were filled up with 50 μL suspension containing 108 CFU mL−1 of GFP-tagged A30 or 108 CFU mL−1 A30 and 106 CFU mL−1 IPO3012. Three potato slices derived from three different tubers were used per treatment. For the negative control, 50 μL sterile water were used instead of bacterial suspensions, and for the positive control 50 μL containing 106 CFU mL−1Dickeya sp. IPO2222. The slices were incubated at 28°C for 72 h in a humid box and the experiment was repeated independently once. The protection effect of GFP-tagged A30 on potato tissue was measured by comparing the average diameter of rotten potato tissue around co-inoculated wells with the average diameter of rotten potato tissue around wells of the positive control.

Population dynamics studies of GFP-tagged A30 and DsRed-tagged Dickeya sp. IPO3012 on potato slices

In order to study the population dynamics of the GFP-tagged S. plymuthica A30 and the DsRed-tagged Dickeya sp. IPO3012 on potato slices, a similar experimental set-up as above was used. This time, tuber wells were filled with 50 μL suspension containing 1010 CFU mL−1 GFP-tagged A30 and 108 CFU mL−1 IPO3012 and the experiment repeated twice. Population densities of GFP-tagged A30 and IPO3012 on potato slices were determined: c. 2 g tuber tissue from three wells per tuber per treatment taken at random were collected daily and crushed in 4 mL ¼-strength Ringer’s buffer in a Universal Extraction bag (BIOREBA) using a hammer. Then, 100 μL undiluted, or 1000- or 10 000-fold diluted tuber extracts were mixed with liquefied NA, cooled to 48°C and supplemented with tetracycline (NAt) to a final concentration of 40 μg mL−1, then poured into the wells of 24-well plates (Greiner). After agar had solidified, the plates were covered with Parafilm and incubated at 28°C for 24–48 h for growth of bacterial colonies. GFP- and DsRed-tagged colonies were counted under an epifluorescence stereomicroscope (Leica Wild M32 FL4) equipped with a mercury high-pressure photo-optic lamp (Leica Hg 50W/AC) and GFP and RFP Plus filters.

Density dependence of the control of Dickeya sp. IPO2222 by GFP-tagged A30 on potato slices

The effect of GFP-tagged A30 strain density on tuber tissue rotting caused by Dickeya sp. IPO2222 was studied in a similar experimental set-up as above with minor modifications. Tuber wells were co-inoculated with 50 μL suspension containing different densities of A30 (0, 104, 105, 106, 107 and 108 CFU mL−1) and 106 CFU mL−1 IPO2222 and, as a control, wells were inoculated with 50 μL suspension containing of 106 CFU mL−1 IPO2222 Dickeya sp. in water. The potato slices were incubated under the same conditions as described above. The experiment was independently repeated once. The effect of GFP-tagged A30 on maceration of potato tissue was determined by comparing the average diameter of rotting potato tissue around co-inoculated wells with that for the positive control.

Glasshouse experiments

Glasshouse experiments were conducted in June–July (experiment 1) and September–October (experiment 2) in 2010. In each experiment, four treatments were applied to potato tubers: (i) tubers vacuum-infiltrated with DsRed-tagged Dickeya sp. IPO3012 (positive control), (ii) tubers vacuum-infiltrated with DsRed-tagged Dickeya sp. IPO3012 and surface-inoculated with a suspension of GFP-tagged S. plymuthica A30, (iii) tubers surface-inoculated with a suspension of GFP-tagged S. plymuthica A30, and (iv) tubers vacuum-infiltrated with water (negative control).

Inoculation of potato tubers with GFP-tagged S. plymuthicaA30 and DsRed-taggedDickeyasp. IPO3012

Suspensions of DsRed-tagged Dickeya sp. IPO3012 containing 106 CFU mL−1 were prepared in sterile demineralized water. Dickeya spp.-free minitubers of cv. Kondor (Dutch Plant Inspection Service for Agricultural Seed Potatoes (NAK)) were used. The minitubers were immersed in the bacterial suspension and vacuum-infiltrated for 10 min at −800 mbar in a desciccator followed by 10 min incubation at atmospheric pressure. Minitubers infiltrated with sterile demineralized water only served as negative controls. All tubers were dried in a flow cabinet overnight. Serratia plymuthica A30 was grown on TSA plates for 24–48 h. Bacterial cells were collected from the agar plates using sterile cell scrapers (Greiner Bio One) and were suspended in sterile demineralized water resulting in suspensions with a density of 1010–1011 CFU mL−1. Tubers vacuum-infiltrated with water or with DsRed-tagged IPO3012 were inoculated with the A30 suspension by dousing 50 mL bacterial suspension over the tuber surface just before planting. Tubers were planted in 5-L plastic pots containing moist sandy soil (2·9% organic mater, 0·2% CaCO3, pH 6·4) freshly collected from a potato field in Wageningen (51°57′52″N, 5°39′47″E), Netherlands. The pots were kept unwatered for 24 h after planting and subsequently watered daily to field capacity. Pots were kept in the glasshouse under a 16-/8-h light/dark regime, at c. 70% relative humidity and c. 28°C for 4 weeks (28 days) in a random block design of the pots (three blocks containing 10 pots per treatment – 40 pots in total per block). At each sampling time, 10 plants inoculated with IPO3012 (positive control), 10 plants inoculated with sterile water (negative control), 10 plants sequentially inoculated with Dickeya sp. IPO3012 and GFP-tagged S. plymuthica A30, and 10 plants inoculated with GFP-tagged A30 were sampled.

Symptom development

Plants were visually inspected weekly for development of symptoms: non-emergence, wilting and chlorosis of leaves, black soft rot at the stem base, aerial stem rot, haulm desiccation and plant death.

Quantification of DsRed-tagged Dickeya sp. IPO3012 and GFP-tagged S. plymuthica A30 in potato plants by pour-plating

Ten plants per treatment were sampled 1, 7 and 28 days post-inoculation (dpi). Seed tubers were collected and processed individually. They were washed under tap water to remove soil particles, sterilized in 70% ethanol for 1 min, washed three times with water for 1 min, soaked in 1% sodium hypochlorite (commercial bleach) for 4 min and finally washed for 4 min three times with water. Each tuber was suspended in twice its weight in ¼-strength Ringer’s buffer supplemented with 0·02% diethyldithiocarbamic acid (DIECA) as an antioxidant. Tubers were then crushed in a Universal Extraction bag (BIOREBA) using a hammer. Then, 100 μL undiluted and 10−1 and 10−2 dilutions of bacteria were mixed with 300 μL NAt pre-warmed at 48°C, and poured into the wells of a 24-well plate (Greiner). After agar had solidified, plates were wrapped with Parafilm and incubated at 28°C for 24–48 h before screening for GFP- and/or DsRed-positive colonies as described before using an epifluorescence stereomicroscope equipped with GFP and RFP Plus filters.

All shoots (including leaves) per plant were collected and processed as a composite sample, as well as the whole root system. At 7 dpi, all shoots were sampled as a composite sample per plant and at 28 dpi composite samples of 2-cm-long stem cuttings taken 5 cm above the ground level were analysed for each plant. Both shoot and root samples were sterilized and bacterial density determined by pour-plating, as described above for seed tubers.

Sampling of potato plants for confocal laser scanning microscopy (CLSM)

For microscopy, plant samples were collected 7 and 28 dpi (roots and shoots): eight roots, 5–10 cm long, and three whole stems, including leaves, both randomly taken per plant. Each root was cut into 2- to 3-cm-long segments and each stem into 0·25– to 0·5-cm-thick fragments. Fragments were embedded in molten NA at 48°C containing 40 μg tetracycline and 200 μg cycloheximide mL−1 in Petri dishes. After the medium had solidified, the plates were sealed with Parafilm and incubated for 1–2 days at 28°C. Plant samples were removed from the agar plates, washed briefly in demineralized sterile water and examined under CLSM.

Four roots per plant raised from tubers superficially treated with GFP-tagged S. plymuthica A30 (treatment iii) were processed without surface sterilization and without embedding directly after sampling to monitor bacterial populations on the root surface.

To visualize plant cells, a 405-nm (excitation) ultraviolet laser with a 450-nm filter (emission) was used. For excitation of the GFP and DsRed in bacterial cells, a 495-nm (blue) laser with a 505-nm emission filter and a 532-nm (green) laser with a 610-nm emission filter were used, respectively. Photographs were taken with a Leica Digital System combined with a Leica CLSM microscope using ×10 and ×63 water-immersion objectives.

Statistical analyses

Data were analysed according to the experimental design used, i.e. experiment replication in time, four treatments per replication, three different sampling time points and 10 plants for each treatment and time point. Visual inspection of symptom development gave a dichotomous score, i.e. either no symptoms were observed or the emergence of blackleg and/or pre-emergence tuber rot was assessed. Data were analysed with a generalized linear model (GLM) assuming data to arise from a binomial distribution. The logit link was used to stretch the binomial to a normal distribution. Bacterial count data were analysed using a linear mixed model with replicates taken randomly in time. To approximate normality, counts were log transformed, adding a value of 1 to each to deal with zero values. Effects were considered significant at the = 0·05. Pairwise differences were obtained using the t-test. All analyses were performed with the statistical software package GenStat (Payne et al., 2008).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Construction of marker strains tagged with GFP or DsRed and their performance compared to wildtype strains

Transformation of S. plymuthica A30 with pRZ-T3-gfp and Dickeya sp. IPO2222 with pRZ-T3-dsred plasmids resulted in 43 and 29 transformants, respectively. One colony with high fluorescence was collected for each of the bacteria. Four repeated transfers of transformants on NAt plates with overnight incubation at 28°C showed stable expression of GFP or DsRed. The presence of pRZ-T3-gfp in GFP-tagged A30 and pRZ-T3-dsred in IPO2222 (IPO3012) was demonstrated by plasmid DNA purification and agarose gel electrophoresis (data not shown).

GFP-tagged A30 and IPO3012 displayed similar growth characteristics in liquid media to the wildtype A30 and IPO2222 strains, respectively, indicating that the growth of the strains was not affected either by the presence of the pRZ-T3 plasmids or by expression of fluorescent (GFP or DsRed) proteins (data not shown).

In the test of the ability of IPO3012 and of wildtype strain Dickeya sp. IPO2222 to macerate potato tuber tissue, the diameters of the rotting tissue were not significantly different (data not shown).

When the ability of GFP-tagged S. plymuthica A30 to inhibit growth of IPO2222 and IPO3012 was compared with that of the wildtype S. plymuthica A30 strain in an overlay plate assay, there was no significant difference between the diameters of the clear halos, indicating that both S. plymuthica A30 and GFP-tagged A30 equally inhibited both Dickeya sp. strains (data not shown).

When the abilities of the GFP-tagged S. plymuthica A30 and the wildtype strain A30 to protect potato tuber tissue from maceration by IPO2222 and IPO3012 were compared, there were no differences in the diameters of the rotting tissue (data not shown).

Density effect of GFP-tagged S. plymuthica A30 on tuber maceration by Dickeya sp.

The effect of inoculum density of GFP-tagged S. plymuthica A30 on its ability to protect potato tuber tissue against maceration caused by Dickeya sp. IPO3012 when co-inoculated was tested in a potato slice assay. Maceration of tuber tissue by IPO3012 was completely inhibited at a density of 108 CFU mL−1 GFP-tagged S. plymuthica A30 (Fig. 1). Inhibition was less, but still significant, at densities of 107 and 106 CFU mL−1, but no significant inhibition was noted at 105 or 104 CFU mL−1.

image

Figure 1.  Reduction of the maceration ability of Dickeya sp. IPO2222 co-inoculated with GFP-tagged Serratia plymuthica A30 in potato tuber slices: effect determined by measuring the diameter of rotting tissue (in mm) after 72 h incubation at 28°C in a humid box. Wells of potato slices were filled with: (1) negative control (water); (2) positive control (106 CFU mL−1Dickeya sp. IPO2222); (3) 106 CFU mL−1Dickeya sp. IPO2222 + 104 CFU mL−1S. plymuthica A30; (4) 106 CFU mL−1Dickeya sp. IPO2222 + 105 CFU mL−1S. plymuthica A30, (5) 106 CFU mL−1Dickeya sp. IPO2222 + 106 CFU mL−1S. plymuthica A30; (6) 106 CFU mL−1Dickeya sp. IPO2222 + 107 CFU mL−1S. plymuthica A30; (7) 106 CFU mL−1Dickeya sp. IPO2222 + 108 CFU mL−1S. plymuthica A30. Three potato slices containing three wells each and derived from three different tubers were used per treatment. The experiment was independently repeated once and the results were averaged. Vertical lines represent standard errors.

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Population dynamics of GFP-tagged S. plymuthica A30 and Dickeya sp. IPO3012 on potato tuber slices

Population dynamics of GFP-tagged S. plymuthica A30 and Dickeya sp. IPO3012 after inoculation singly or jointly on potato slices showed that after 3 days, densities of Dickeya sp. IPO3012 on control slices without A30 added increased from 107 to 1011 CFU g−1 with progressive rotting of the potato slices. In contrast, populations of GFP-tagged S. plymuthica A30 on control slices without added Dickeya sp. decreased rapidly from 107–108 CFU g−1 at 0 dpi to 101–102 CFU g−1 at 2 dpi and 0 CFU g−1 at 3 dpi. No rotting of potato slices was observed, although after 3 days, a slight brown discoloration of tuber tissue was found, not visible in the water control slices. Joint inoculation of tuber slices with GFP-tagged A30 strain and IPO3012 resulted in a decrease in population densities for both bacterial species. No GFP-tagged A30 or DsRed-tagged IPO3012 bacteria were recovered from inoculated potato slices at 3 dpi (Fig. 2) and no rotting of the slices was observed (data not shown).

image

Figure 2.  Population dynamics of GFP-tagged Serratia plymuthica A30 and Dickeya sp. IPO3012 on potato slices inoculated either with GFP-tagged S. plymuthica A30, DsRed-tagged Dickeya sp. IPO3012 or co-inoculated with both strains. At 0, 1, 2 and 3 days post-inoculation, plant material was collected from the inoculated wells and crushed in the presence of ¼-strength Ringer’s buffer. Serial dilutions of plant extract were pour-plated in NAt and green and red fluorescent colonies were counted. The experiment was independently repeated once and results were pooled. Results from six independent samples per treatment and per time point were averaged.

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Glasshouse experiments

Disease development

Treatment (i):  In plants grown from minitubers inoculated with DsRed-tagged Dickeya sp. IPO3012 the first symptoms appeared 7 dpi, when shoots were c. 5–7 cm and roots were c. 8–12 cm long. The pathogen severely affected sprouting and plant development: non-emergence (c. 20–30%) was caused by rotting of seed tubers in the soil. Deterioration of shoots and typical blackleg symptoms, i.e. wilting and chlorosis of leaves, as well as stem wet rot, first developed at 7 dpi. By 28 dpi, 60% and 50% of inoculated plants in experiments 1 and 2, respectively, showed characteristic blackening and soft rotting of the stem base.

Treatment (ii):  Incidences of non-emergence and of blackleg symptoms were significantly reduced in plants grown from seed tubers inoculated with DsRed-tagged Dickeya sp. IPO3012 and treated with GFP-tagged S. plymuthica A30 strain before planting. In experiment 1, at 7 dpi, only 10% of the plants showed pre-emergence seed tuber rot and stunted stem growth, and no blackleg symptoms developed, even at 28 dpi. In experiment 2, none of the inoculated plants showed pre-emergence tuber rot or blackleg symptoms at any time.

Treatments (iii) and (iv):  None of the plants grown from seed tubers inoculated with water and with GFP-tagged S. plymuthica A30 showed any non-emergence or blackleg symptoms during the entire course of both experiments.

Population dynamics of GFP-tagged S. plymuthicaA30 and DsRed-taggedDickeyasp. IPO3012 in planta

Population dynamics of GFP-tagged S. plymuthica A30 and DsRed-tagged Dickeya sp. IPO3012 in plants were evaluated at 1 dpi in tubers only and at 7 and 28 dpi in tubers, roots and shoots in all four treatments. Population dynamics of bacteria in the three plant parts were examined by NAt pour-plating. In none of the water-inoculated control plants (treatment iv) were GFP- or DsRed-tagged bacteria, or blackleg symptoms, found.

Bacterial populations in seed tubers

In seed tubers of A30-treated plants (treatment iii), relatively low populations (average c. 101 CFU g−1) of the bacteria were detected at 1 dpi (Fig. 3a). Populations increased in 7 days to 102–10CFU g−1 and remained at this level until 28 dpi. With sequentially treated plants (treatment ii), S. plymuthica A30 populations in tubers at 1 dpi were on average 10CFU g−1, but decreased to 102 CFU g−1 in the next 6 days and remained at this level until 28 dpi. By contrast, Dickeya sp. populations had decreased significantly from 104 CFU g−1 at 1 dpi to an average of 1 CFU g−1 or less at 28 dpi. With Dickeya sp.-treated plants (treatment i), populations of 104 CFU g−1 bacteria were detected at 1 dpi, declining only slightly to 103–104 CFU g−1 by 28 dpi.

image

Figure 3.  Population dynamics of GFP-tagged Serratia plymuthica A30 and DsRed-tagged Dickeya sp. IPO3012 in seed tubers (a), stems (shoots) (b), and in roots (c) sampled 1 (tubers only), 7 and 28 days (tubers, roots and shoots) post-inoculation (dpi) in experiments 1 and 2. The whole seed tuber and all roots were sampled per plant at each time point. At 7 dpi, all shoots were sampled as a composite sample per plant and at 28 dpi samples of stem cuttings taken 5 cm above ground level were analysed for each plant. The average values are shown from 10 plants per time point. Statistical analysis was done per subsample and per time point (= 10). Values followed by identical characters are not significantly different (= 0·05).

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Bacterial populations in roots

Bacterial populations in roots were analysed only at 7 and 28 dpi, as at 1 dpi no roots had yet developed. In treatment (iii) at 7 dpi, S. plymuthica A30 was present in roots at a density of 102 CFU g−1 (Fig. 3b). At 28 dpi, the population had increased to 103–104 CFU g−1. In roots of plants grown from tubers sequentially inoculated with A30 and IPO3012 (treatment ii), population dynamics of A30 followed a similar pattern as above. By contrast, no Dickeya sp. was detected in roots at 7 or 28 dpi, only low populations averaging 1 CFU g−1 were detected. In the plants treated with Dickeya sp. IPO3012 (treatment i), low Dickeya sp. populations (<101 CFU g−1) were found at 7 dpi, increasing slightly to 101–102 CFU g−1 at 28 dpi.

Bacterial populations in shoots

Bacterial populations in shoots were analysed only at 7 and 28 dpi, as at 1 dpi shoots had not yet been formed (Fig. 3c). In plants treated with S. plymuthica A30 (treatment iii), at 7 dpi the bacterium was already present in shoots at a density of 102 CFU g−1and at 28 dpi the size of the population had increased 10-fold. In plants sequentially inoculated with GFP-tagged S. plymuthica A30 and DsRed-tagged Dickeya sp. IPO3012 (treatment ii), the population dynamics of A30 followed a similar trend as above. At 7 dpi, <10 CFU g−1Dickeya sp. were present and none were detected at 28 dpi. Stems of plants treated with Dickeya sp. IPO3012 only (treatment i) at 7 dpi had low densities of the bacteria (5–10 CFU g−1), increasing to a density of 102–103 CFU g−1 at 28 dpi.

Plant colonization examined by confocal laser scanning microscopy

Stems and roots were analysed with a CLSM at magnifications of ×640 and ×1000. Results (Fig. 4) showed that at 7 dpi both bacterial species were present in roots of plants grown from minitubers sequentially inoculated with GFP-tagged S. plymuthica A30 and DsRed-tagged Dickeya sp. IPO3012 (treatment ii). Green (S. plymuthica A30) and red (IPO3012) fluorescent cells were found inside xylem vessels and between protoxylem cells of the vascular tissue of roots, and also in the medulla and cortex of the pith, both intra- and intercellularly. In stems, green and red fluorescent bacterial cells were observed inside and between xylem vessels and protoxylem cells of vascular tissue. At 28 dpi only green fluorescent cells were observed in roots and stems, indicating that GFP-tagged S. plymuthica A30, but not IPO3012, was present.

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Figure 4.  Internal colonization of surface-sterilized potato roots (a) and stems (b) by GFP-tagged Serratia plymuthica A30 and DsRed-tagged Dickeya sp. IPO3012, 7 and 28 days post-inoculation (dpi) analysed with confocal laser scanning microscopy. Samples were taken from plants from which tubers were inoculated at planting with GFP-tagged S. plymuthica A30, from plants raised from potato minitubers vacuum-infiltrated with DsRed-tagged Dickeya sp. IPO3012 and after sequential-inoculation of the minitubers with both strains. For the control, potato minitubers were vacuum-infiltrated with sterile water. Samples were embedded in NAt (nutrient agar supplemented with 40 μg mL−1 tetracycline) and incubated for 2 days at 28°C.

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At 28 dpi, in DsRed-tagged Dickeya sp. inoculated (control) plants (treatment i), red fluorescent bacteria were present inside and between pith cells of roots and inside and between xylem vessels of stems. Similarly, in plants inoculated with GFP-tagged S. plymuthica A30 (treatment iii), green fluorescent bacteria were present inside and between parenchyma cells of roots and in xylem vessels of stems. No green and/or red fluorescent bacteria were found in any parts of plants inoculated with water (treatment iv).

The ability of GFP-tagged S. plymuthica A30 to colonize roots of potato plants was tested by analysing randomly selected roots from plants at 28 dpi using CLSM. All roots of plants grown from A30-inoculated tubers (treatment iii) were superficially colonized by green fluorescent cells. In plants inoculated only with GFP-tagged S. plymuthica (treatment iii), green fluorescent bacteria occurred in clumps or patches on the root surface, interspersed by areas where bacteria were absent or in which only low densities were present (Fig. 5). In none of the water control plants were GFP-tagged bacteria detected on the root surface.

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Figure 5.  Colonization of the potato root surface by GFP-tagged Serratia plymuthica A30, 28 days after treatment of seed tubers. Roots were freshly collected and briefly washed in sterile tap water to remove soil particles. Plant samples were analysed with confocal laser scanning microscopy. Control roots were collected from plants grown from tubers vacuum-infiltrated with water. UV light was used to visualize plant cells.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Biological control agents (BCAs) may potentially contribute to the control of blackleg- and soft rot-causing pathogens (Czajkowski et al., 2011b). Biocontrol strategies described so far involve the use of antagonistic bacteria, predatory bacteria and bacteriophages. Antagonists may compete for nutrients, may produce compounds inhibiting pathogen growth (e.g. antibiotics and siderophores), may produce compounds interfering with pathogenicity (e.g. via interference with factors involved in quorum quenching), or may induce systemic resistance in plants (Sturz et al., 2005). Examples are known of antagonistic fluorescent Pseudomonas bacteria able to reduce populations of Pectobacterium spp. on roots and in progeny tubers (Kloepper, 1983), and also to protect wounds and cracks on tubers (Kastelein et al., 1999). Bdellovibrio bacteriovorans, a predatory bacterium, was evaluated for control of Pectobacterium atrosepticum on potato (Epton et al., 1989). However, the level of soft rot control in a potato slice assay was relatively low. Bacteriophages (phage therapy) have been used for biocontrol of Dickeya and Pectobacterium spp. in calla lily (Jones et al., 2007; Ravensdale et al., 2007; Evans et al., 2010); in glasshouse trials, the incidence of soft rot caused by P. carotovorum subsp. carotovorum could be reduced up to 50%.

Despite these promising results, no BCAs for use against Pectobacterium and Dickeya spp. have yet been commercialized. The major hurdle is the inconsistency in biocontrol results caused by variations in the environmental conditions under which the BCA is active against the target pathogen (Diallo et al., 2011). A thorough knowledge of the ecological features of the BCA in the potato ecosystem and knowledge of the interaction of the BCA and the pathogen is a prerequisite for a successful application of a BCA in practice.

In a recent publication, it was shown that when seed tubers were vacuum co-infiltrated with Dickeya spp. and high densities of S. plymuthica A30 and then planted in compost, the blackleg level caused by Dickeya sp. IPO3012 was reduced by 100% and the incidence of stem colonization fell by 97% (Czajkowski et al., 2011a). In this follow-up study, experimental conditions were chosen to be more realistic for field application: seed tubers were treated by superficial wetting with a suspension of the antagonist prior to planting in potted field soil. This tuber treatment method is similar to that commonly used when fungicides such as monceren (pencycuron) are applied to seed tubers to control Rhizoctonia solani (Wicks et al., 1995). The results obtained here suggest that the BCA S. plymuthica A30 is effective in controlling blackleg and soft rot caused by the test strain used. Blackleg incidence was reduced from 55% in the control Dickeya sp.-inoculated treatment to 0%. The effectiveness of strain A30 appears to be independent of the way tubers were treated and whether they were grown in compost or field soil.

Serratia plymuthica strains have been frequently found in association with plants. They have been isolated from the rhizospheres of wheat, oat, cucumber, maize, oilseed rape and potato (Åström & Gerhardson, 1988), as endophytes from the endorhiza of potato (Berg et al., 2005) and also found in onion, carrot, lettuce, Brassica spp. leaves, as well as in the phyllosphere of spring wheat (De Vleesschauwer & Höfte, 2007). They have been used extensively before for biocontrol of fungal diseases (De Vleesschauwer & Höfte, 2007), but not apparently for the control of bacterial pathogens. Serratia plymuthica rhizosphere isolates have been frequently used to control soilborne fungal pathogens of plants. By contrast, S. plymuthica strains isolated from internal plant tissues have rarely been used in biological control (Bowen & Robvira, 1974; Brown, 1974; Åström & Gerhardson, 1988; Weller, 1988; Whipps, 2001).

Serratia plymuthica A30 was initially isolated from rotting tissue of surface-sterilized potato tubers, which suggests that it is an endophyte rather than a commensal from the tuber surface (Czajkowski et al., 2011a). When applied to seed tubers, it readily colonized them, as well as the roots and stems of the growing plant. Results obtained by both dilution plating and CLSM showed that within 1 day of application, the bacterium was present inside surface-sterilized seed tubers. Such rapid colonization of internal seed tuber tissues could be attributed to penetration via lenticels. Seven days after planting, the bacterium was found in large numbers in the vascular tissue of roots and stems (Fig. 4). A30 may have entered roots via openings that occur during lateral root formation, as many bacterial species do (Huang & Allen, 2000). Presence of wounds or degradation of the root tissue does not appear to be required for root colonization (Huang, 1986). An increase of A30 population numbers with time indicates that S. plymuthica actively grew inside plants (Fig. 3).

Dickeya spp. and Pectobacterium spp. in rotting mother tubers are translocated via the vascular system up into the growing stems (Pérombelon, 1974; Czajkowski et al., 2010a). In addition, the bacteria are also released into wet soil and find their way to and penetrate the root system of both the mother and neighbouring potato plants, resulting in both instances in systemic infection of plants, including the progeny tubers. The ease by which A30 is able to colonize the roots and stems of potato plants explains its ability to protect the plants against infection by Dickeya spp. from soil.

The basis for the antagonist effect of A30 is not clear. It is known that strain A30 produces antibiotics against biovar 3 Dickeya spp. (Czajkowski et al., 2011a), and preliminary results suggested that A30 mutants defective in antibiotic production/secretion did not prevent tuber maceration when tested in vitro (Czajkowski, unpublished data). In addition, A30 and the pathogen are located in the same niche in planta and the antagonism possibly also depends on the ability to compete for nutrients. However, other factors cannot be excluded, including induction of systemic resistance of potato plants against biovar 3 Dickeya spp. Strains of S. plymuthica were reported to induce systemic resistance in various crops; S. plymuthica R1GC4 stimulated defence mechanisms against fungal pathogens in cucumber (Benhamou et al., 2000) and S. plymuthica IC270 stimulated rice plant defences against Magnaporthe oryzae, the causal agent of rice blast disease (De Vleesschauwer et al., 2009).

The efficacy of A30 in controlling Dickeya sp. appears to be density-dependent. In a potato slice assay, GFP-tagged S. plymuthica A30 was able to prevent potato tissue maceration by Dickeya sp. if applied at a minimum density which was 10–100 times higher than the density of Dickeya sp. (Fig. 1). However, a considerable reduction of tuber rotting was still observed at lower densities. It is generally accepted that the biocontrol agent must be applied at a higher density than the pathogen to achieve a satisfactory level of protection (Parke, 1990). What is surprising is the drop in numbers of the antagonist in tuber slice assays within 3 days when co-inoculated with Dickeya sp. or on its own (Fig. 2). Tuber wounding as a result of slicing tubers may have resulted in oxidative stress and the production of antimicrobial metabolites such as phenolics (Johnson & Schaal, 1957).

The density-dependence of efficacy in the slice assay may be related to the exploitation of a quorum-sensing (QS) mechanism by A30. It is known that in S. plymuthica HRO-C48, acyl-homoserine lactone (AHL)-based QS signalling is involved in the regulation of important biocontrol mechanisms, including protection of cucumber against Pythium aphanidermatum and induction of systemic resistance in bean and tomato plants against Botrytis cinerea (Pang et al., 2009). QS also participates in motility and indole-3-acetic acid and hydrolytic enzyme production in HRO-C48 (Müller et al., 2009). Serratia plymuthica A30 is known to produce AHLs (Czajkowski et al., 2011a) and therefore it can be speculated that it uses the QS mechanism in the same way as strain HRO-C48. The population density of S. plymuthica A30 in roots, tubers and shoots was stable for at least 28 days at a level of 103–104 CFU g−1 after tuber application. This density in internal plant tissue seems to be sufficient to trigger the QS mechanism (von Bodman et al., 2003).

At present the inoculum density of A30 able to protect tubers against Dickeya sp. in the field is unknown. In the glasshouse experiments, relatively high densities of 1011–1012 CFU mL−1 of the antagonist were used for tuber application, but possibly lower densities could be used in field trials if the bacterial preparation is formulated to increase inoculum stability. For commercial reasons, it would be necessary to decrease the density to a more realistic level, such as 106–109 CFU mL−1, as commonly used in commercial applications with formulated bacteria (Kloepper & Schroth, 1980; Vidhyasekaran & Muthamilan, 1995).

In conclusion, although the results obtained in this study are promising for biocontrol of blackleg caused by the virulent Dickeya sp. biovar 3 using S. plymuthica A30 as an antagonist, there is still considerable work to be done to achieve a viable commercial application. Aspects which especially require further examination are: formulation of a stable bacterial preparation; optimization of application procedures; sufficient longevity of the applied antagonist in soil to bridge the gap between planting and shoot and root growth, and possibly also into subsequent crop generations; and effectiveness when using standard-size seed tubers with well-set skin in a wide range of cultivars and under different edaphic and environmental conditions. Finally, elucidation of the antagonism mechanism could be of value for use in other pathogen–host combinations.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The project was financed by the STW Foundation (Technologiestichting STW, Netherlands) via grant no. 10306 Curing Seed Potato from Blackleg Causing Bacteria. We thank P. S. van der Zouwen (PRI, Netherlands) for technical assistance and M. C. M. Pérombelon (SCRI, UK) and L. J. Hyman (SCRI, UK) for their highly valuable comments on the manuscript and their editorial work.

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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