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Intact salicylic and jasmonic acid pathways are necessary

for defense against Blackleg disease as revealed by a novel method for screening in vitro grown potato

Journal: Plant Biology

Manuscript ID: PlaBio-2014-09-0429-SRP

Manuscript Type: Short Research Paper

Date Submitted by the Author: 25-Sep-2014

Complete List of Authors: Burra, Dharani

Muhlenbock, Per Andreasson, Erik

Keyword: Blackleg disease, Pectobacterium atrosepticum, Dickeya solani, SW93-1015, Bintje, NahG, coi1

Manuscript submitted to editorial office

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Intact salicylic and jasmonic acid pathways are 1

necessary for defense against Blackleg disease as 2

revealed by a novel method for screening in vitro 3

grown potato 4

Dharani Dhar Burra*, Per Mühlenbock*

and Erik Andreasson 5

Department of Plant Protection Biology, Swedish University of Agricultural Sciences, Alnarp, Sweden 6

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Running title: In vitro method for screening of Blackleg disease in potato 8

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Corresponding author: Erik Andreasson 10

Department of Plant Protection Biology, 11

Swedish University of Agricultural Sciences, Alnarp, Sweden 12

E-mail address: [email protected] 13

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Keywords: Blackleg disease, Pectobacterium atrosepticum, Dickeya solani, in vitro potato, coi1, 15

NahG, Sarpo Mira, SW93-1015, Bintje, Desiree 16

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Abbreviations: SA: Salicylic acid; JA: Jasmonic acid; COI: Coronatine insensitive; NAA: 1-18

Naphthaleneacetic acid; IBA: Indole-3-butyric acid 19

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Abstract 30

Potato is major crop for food security and blackleg disease is increasingly causing losses in its yield 31

and storage. Recently, one of the blackleg pathogens, Dickeya solani has been shown to be 32

spreading in northern Europe and that it causes an aggressive disease development. Currently, 33

identification of tolerant commercial potato varieties has proven unsuccessful; this is confounded by 34

a complicated etiology of the disease and a strong environmental influence on disease development. 35

There is a lack in availability of efficient testing systems. Here, we describe a system for 36

quantification of blackleg symptoms on shoots of sterile in vitro potato plants, that save time and 37

space compared to greenhouse and existing field assays. We found no evidence for differences in 38

host specificity, neither between Pectobacterium or Dickeya inoculations, nor between the 39

described in vitro or existing green-house assays. The system facilitates efficient screening to discern 40

molecular mechansims of blackleg disease independent of other microorganisms and variable 41

environmental conditions. In order to deepen the knowledge of plant mechanisms involved in 42

blackleg disease development, we used our new assay to analyze disease development of hormone 43

related potato transgenic lines. We show that both jasmonic (JA) and salicylic acid (SA) pathways 44

regulate tolerance against blackleg disease in potato, a result that is different from previous findings 45

in Arabidopsis. 46

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Introduction 48

The blackleg disease, characterized by black macerations of shoot stems in potato, is a long standing 49

intractable problem that causes increasing yield losses in Europe (Czajkowski et al., 2010). The 50

causative agents of this disease are pectolytic gram negative bacteria such as Pectobacterium 51

atrosepticum, Pectobacterium carotovorum subsp. carotovorum, and Dickeya solani (Czajkowski et 52

al., 2011; Toth et al., 2011). In temperate regions these are the main causative agents of blackleg 53

disease symptoms in potato. Pectobacterium atrosepticum is restricted to infecting potato while 54

Pectobacterium carotovorum subsp. carotovorum has a broad host range (Mansfield et al., 2012). 55

Dickeya solani as well as other Dickeya species are increasingly detected throughout Europe and 56

cause a more aggressive disease development than P. atrosepticum (Toth et al., 2011). 57

The blackleg-causing pathogens are opportunistic bacteria that can be present in the plant in a 58

quiescent state without causing symptoms (Pérombelon, 1992). Most of the disease is spread 59

through contaminated seed tubers, where it causes soft rot disease (Helias et al., 2000). In a growing 60

potato plant, when conditions are favorable (usually high humidity and temperature), the blackleg 61

symptoms develop. Symptoms are usually wilting, tissue maceration and blackening or discoloration 62

of the stem (Mattinen et al., 2008). Complete wilting of the shoot and rotting of tubers may follow at 63

more advanced stages of the disease. The presence of favorable conditions is necessary for the 64

emergence and development of visible symptoms (Pérombelon, 1992; Perombelon and Kelman, 65

1980; Toth et al., 2003) 66

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Most testing for resistance to these pathogens has been performed in tubers for the detection of 67

softrot symptoms (Czajkowski et al., 2011). However, immunity to softrot or blackleg in commercial 68

varieties has not been found, although in some varieties partial resistance was detected (Czajkowski 69

et al., 2011). Furthermore, breeding for resistance is complicated by low correlation between the 70

partial resistance to blackleg symptoms in shoots and softrot in tubers (Czajkowski et al., 2011). 71

Some success with improving resistance has been achieved using wild Solanum species (Bains et al., 72

1999; Carputo et al., 1997), albeit with a combined high level of toxins rendering them unpalatable 73

to humans (Czajkowski et al., 2011) as well as giving other unwanted traits (Pasco et al., 2006). 74

Interestingly, some promising advances have been presented through generation of transgenic 75

potato plants that produce chicken lysozymes or overexpress pectate lyase, but although these 76

transgenic plants were shown to be more resistant against P. atrosepticum and P. carotovorum, 77

there is a long way before they can enter into agricultural practice due to problems such as fitness 78

cost that are associated with these modifications (Czajkowski et al., 2011). HAdditionally, 79

understanding molecular components of plant response to blackleg disease and identifying 80

resistance sources is difficult due to the dependence of the symptom development on 81

environmental factors that can be confounded by secondary infections by other organisms (Toth et 82

al., 2003). Also, available blackleg resistance testing systems are not fully consistent partly due to 83

their dependence on favorable environmental conditions (Bisht et al., 1993; Lapwood and Read, 84

1986), in addition to being time-consuming and labor intensive due to the size and long life cycle of 85

field or greenhouse grown potatoes. Taken together, this calls for the development of new and 86

efficient screening systems for blackleg disease. 87

In an attempt to circumvent inconsistencies associated with current disease screening methods, 88

culture filtrates of blackleg pathogens such as P. carotovorum ssp carotovorum have been primarily 89

used to understand plant defense signaling responses to infection. Studies on mainly the interaction 90

between P. carotovorum ssp. carotovorum and Arabidopsis/tobacco indicate an involvement of 91

salicylic acid (SA) and jasmonic acid (JA) signaling in the response to infection (Davidsson et al., 92

2013). A study in Arabidopsis by Norman-Setterblad et al. (2000) showed that SA deficient mutants 93

(NahG) had a similar degree of infection in comparison to wild type after P. carotovorum ssp. 94

carotovorum inoculation , whereas the JA signaling deficient coi1 mutant was more sensitive. 95

However in another publication no major effects on defense against P. carotovorum ssp. 96

carotovorum were seen in neither NahG or coiI1 mutants (Kariola et al., 2003). No data indicating 97

importance of SA and JA pathways during Dickeya solani infection of its true agriculturally important 98

host, potato, is currently available. 99

Here we present a novel in vitro system that allows for consistent and efficient detection of blackleg 100

symptoms in potato. The system offers an opportunity to discern blackleg etiology through P. 101

atrosepticum and D. solani, independent of other pathogens in stable environmental conditions. We 102

also use the described method to show that both SA and JA signaling pathways are necessary for 103

tolerance to blackleg disease caused by D. solani in potato. 104

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Material and Methods 106

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Pathogens and potato clones 108

A Swedish isolate of Pectobacterium atrosepticum (strain no. 141)(Persson, 1988) was provided by 109

Paula Persson, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden and a Finnish 110

isolate of Dickeya solani (Ds 0432-1)(Laurila et al., 2008) was provided by Minna Pirhonnen, Helsinki 111

University, Helsinki, Finland. Potato clones for testing were provided by Ulrika Nilsson, SLU, Alnarp, 112

Sweden and the SW93-1015 clone has been described (Ali et al 2012). Transgenic potato lines coi1X5 113

and coi1H1 (RNAi knockout) that are JA insensitive, NahGD2 and NahGA (transformed with salicylate 114

hydroxylase transgene) that are SA deficient (Halim et al., 2009; Halim et al., 2004) were provided by 115

Sabine Rosahl, Leibniz Institute of Plant Biochemistry, Halle (Saale), Germany. 116

Bacteria growth 117

Both P. atrosepticum and D. solani were stored in cryovials on beads with freezing medium at -80°C. 118

For infections, few beads from the cryovial were dropped into 20 ml nutrient broth (Difco 119

Laboratories) for P. atrosepticum and high salt LB broth (Duchefa Biochemie) for D. solani, the 120

bacteria were cultured at 27°C on a shaker at 220 rpm for 18-20 hours. Overnight cultures were 121

centrifuged at 4000 g for 10 minutes and harvested in sterile 5mM MgCl2 solution for P. 122

atrosepticum and sterile tap water for D. solani. 123

Blackleg disease response testing of greenhouse grown plants 124

The potato clones were grown from tubers in standard greenhouse conditions for 4-5 weeks at 16 125

hour daylight conditions, and 18 °C minimum heater setting, in 3.5 L pots with standard potting soil 126

(Weibull Horto, Sweden). Each week, plants were supplemented with fertilizer Rika S® (Weibull 127

Horto, Sweden). Only 3 stems per pot were left to grow, and extra shoots were cut off using a clean 128

scalpel. The bacteria culture was adjusted to 106 CFU/ml in MgCl2 solution for P. atrosepticum and 129

5*109

CFU/ml for D. solani. 4-5 week old greenhouse grown potato plants were injected with the 130

bacterial solution at the stem base (one injection per stem), 5 cm above soil surface with 20µl of the 131

bacterial solution. The wound was covered with Nescofilm (Nesco) and distilled water was sprayed 132

over the film. The whole plant was then covered with a transparent plastic bag and symptoms were 133

scored by measuring the length of shoot blackening 7 days after infection. 134

Blackleg disease testing on in vitro grown plants 135

The bacterial solutions were adjusted to OD 0.1 (106 CFU/ml) for P. atrosepticum and OD 0.2 (5*10

9 136

CFU/ml) for D. solani. The in vitro potato cultures were established by transferring surface sterilized 137

excised shoot meristems, from greenhouse grown plants, to agar plates containing ½ MS, supplied 138

with standard vitamins and 2mg/L D-calcium pantothenate, 0,1mg/L Gibberlic acid, 0,01mg/L NAA 139

and 2% sucrose. Growing meristems were then transferred to shoot inducing medium containing 0.5 140

mg/L IBA. Plants were maintained at long day (16h with 80 µE light) conditions, 23°C during the day 141

and 18°C during the night. Every three weeks plants were transferred to fresh medium by excising 142

stem parts carrying one shoot meristem each. For infections, 3 weeks old in vitro plants were cut at 143

the shoot/root junction and transferred in sterile conditions to 50 ml tubes (Sarstedt) containing 1 144

ml bacterial solution. Lids were laid on top of tubes without being sealed and were incubated for 11 145

days in long day conditions and 80µE light and 23°C day temperature and 21°C night temperature, 146

before disease symptoms were recorded. This is when we could see the maximum development of 147


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symptoms. During the first couple of days, some plants in the bacterial solution collapsed. This 148

occurred randomly across all genotypes and was not consistently observed during different 149

experimental repeats, in addition these plants did not display typical blackleg symptoms before or 150

during collapse, hence these were excluded from the analysis. 151

Symptoms restricted to small spots, big spots and lesions were identified and infection score for 152

each plant was calculated as Σ(n(x1)*1)+ (n(x2)*2)+ (n(x3)*3) where each small spot (x1) on the plant 153

was multiplied by a score of 1, each big spot (x2) a score of 2 and each lesion (x3) a score of 3. Each 154

plant then obtained a cumulative infection score. 155

Data analysis 156

The measured lengths of rot on shoots in infected potato varieties from the greenhouse assay and 157

infection scores from in vitro assay were noted. This data was log-transformed (added by a factor of 158

0.1 for greenhouse samples and 1 for in vitro samples) and was analysed with proc mix in SAS using 159

the Tukey-Kramer adjustment at 95% significance level to identify significant differential response of 160

potato clones to infection in both the methods, the letter groupings for output means was obtained 161

using SAS macro pdmix800 (Saxton, 1998). Statistical analysis for infection scores obtained from 162

hormone transgenic lines was performed using student´s t-test on log transformed data on 163

comparisons between transgenic lines and cv. Desiree (wildtype). 164

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Results 166

Host range and symptoms of blackleg disease caused by Dickeya solani and 167

Pectobacterium atrosepticum infection in greenhouse grown potato 168

The blackleg symptom development of different potato clones after inoculation of D. solani using 169

greenhouse grown plants has not yet been reported. Therefore, in order to first characterize host 170

range and to study symptoms of blackleg disease of greenhouse grown potato cultivars to D. solani 171

we inoculated the clones Desirée, Bintje, Magnum Bonum, Sarpo Mira and one of our breeding 172

clones, SW93-1015, that has been shown to have resistance to late blight, Phytophthora infestans 173

(Ali et al., 2012) with D. solani. This was done by stab inoculation as previously described (Bell et al., 174

2004; Laurila et al., 2008; Marquez-Villavicencio et al., 2011b; Zimnoch-Guzowska et al., 1999). Sarpo 175

Mira contains several known resistance genes against late blight infection (Rietman et al., 2012) and 176

Magnum Bonum is resistant to early blight caused by the necrotrophic fungus Alternaria solani 177

(Odilbekov et al., 2014). We evaluated disease severity of plants 7 days post infection by measuring 178

the length of the visible rot on the stem (Figure 1A and 1B). In all the clones we found blackening 179

and maceration around the inoculation site (Figure 1B). In mock infected plants we saw no blackleg 180

symptoms (data not shown). In SW93-1015 and Magnum Bonum, the spread of the lesion was 181

severe and sometimes engulfed the entire shoot. When evaluating the length of the lesions we 182

found that Sarpo Mira, Desirée and Bintje were significantly more tolerant in comparison to SW93-183

1015 and Magnum Bonum (Figure 1A and 1B). 184

We further evaluated the response of the clones to P. atrosepticum to identify if they displayed a 185

similar phenotype in relation to D. Solani inoculation, a Swedish isolate of Pectobacterium 186

atrosepticum (Persson, 1988) was used to infect greenhouse grown plants of Desirée, Bintje and 187


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SW93-1015 clones using the same inoculation method as for Dickeya. In SW93-1015 there was clear 188

rotting of the stem and tissue maceration that in some instances spread over a major portion of the 189

basal stem (Figure 2A and 2B). In some of these plants, necrotic lesions and wilting leaves were also 190

detected higher up on the shoot. In Desirée and Bintje a modest disease development was observed, 191

as a slight browning around the initial stab inoculation and with a lower degree of visible tissue 192

maceration. In mock infected plants we saw no blackleg symptoms (data not shown). The length of 193

lesion development was significantly higher in SW93-1015 compared to Bintje and Desirée (Figure 194

2A and 2B). Based on these observations we classified SW93-1015 as susceptible and Desirée and 195

Bintje as moderately resistant to P. atrosepticum. We therefore conclude that the strains of D. solani 196

and P. atrosepticum have a similar host range among the tested clones. 197

Establishment of a blackleg infection system on in vitro grown plants 198

In order to establish a high-throughput protocol, suitable for quick screening of genetic material that 199

is often stored as in vitro material, we investigated the development of blackleg symptoms in plants 200

grown under sterile in vitro conditions. The use of in vitro plants reduces environmental variation as 201

well as potential secondary infections compared to greenhouse tests. Three week old in vitro grown 202

potato shoots were cut and placed in either bacterial solution or a mock solution (Figure 3A). We 203

analyzed the samples for blackleg symptoms 11 days after inoculation for both D. solani and P. 204

atrosepticum, when most symptoms (small and large spots, lesions; Figure 3B) were seen. Each 205

experiment contained at least 10 plants per clone and was independently repeated at least three 206

times. 207

We infected the clones Desirée, Sarpo Mira, SW93-1015, and Magnum Bonum with D. solani. In 208

SW93-1015, and Magnum Bonum we found clear signs of blackleg disease corroborating our findings 209

in the greenhouse experiments. These symptoms were black necrotic spots along the stem, 210

sometimes localized as small round spots at different locations and sometimes as larger black areas 211

grouped around the stem (Figure 4A). In SW93-1015 and Magnum Bonum, a proportion of the plants 212

had open lesions associated with the black areas that sometimes perforated the stem (Figure 4B). 213

These open lesions were not detected in Desirée or Sarpo Mira, which had a few smaller black spots 214

and a few wider black areas (Figure 4A). Validation of the in vitro assay with P. atrosepticum 215

revealed similar results with clones Desirée, Bintje and SW93-1015 (Figure 5A). In Desirée we found 216

only small black spots in a few of the plants, and in SW93-1015 a few open lesions and wider black 217

areas in each experiment, whereas in Bintje, no symptoms were detected (Figure 5A). In mock 218

inoculated plants we found no symptoms in any of the performed independent experiments (data 219

not shown). 220

In order to efficiently screen material post infection we devised a scoring system for the symptoms 221

that was used for statistical analysis. All observed symptoms were included to obtain a combined 222

infection score for each individual plant. Small black spots were given a score of 1, while big spots 223

with wider area of tissue maceration were given a score of 2, and open or perforating lesions with 224

surrounding necrotic areas were given a score of 3. Statistical analysis was performed by log 225

transforming the final infection score of each plant and then an ANOVA was performed to identify 226

differential response to infections in the tested clones. Consistent with our findings in the infections 227

in greenhouse conditions we found that SW93-1015 and Magnum Bonum scored consistently higher 228

and were more susceptible to D. Solani infections in vitro, whereas Sarpo Mira and Desirée scored as 229


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moderately resistant to disease symptom development (Figure 4B). SW93-1015 scored consistently 230

higher than Desirée over repeated experiments in vitro with P. atrosepticum infections (Figure 5B). 231

No evidence of difference in host preference was found between P. atrosepticum and D. solani. 232

Based on this data we conclude that clear blackleg symptoms were established under in vitro 233

conditions. 234

Increased susceptibility to Dickeya solani is dependent on both SA and JA hormone 235

pathways 236

In order to investigate the role of SA and JA pathways in symptom development , in vitro grown 237

transgenic potato lines; JA insensitive (coi1H1 and coi1X5, coronatine insensitive 1 ), SA deficient 238

(NahGD2 and NahGA) and cv. Desiree (wildtype) (Halim et al., 2009; Halim et al., 2004) were 239

infected with D. solani using the above in vitro assay. Both the transgenic potato lines had 240

significantly higher infection scores when compared to wild type (Figure 6). Also, there was no 241

significant difference between infection scores obtained in the JA insensitive and SA deficient lines 242

(Figure 6), indicating that both JA and SA are equally necessary for partial immunity to blackleg 243

disease. 244

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Discussion 246

Blackleg disease in potato cultivation is a growing problem in Europe in general (Laurila et al., 2008) 247

and Dickeya solani, because of its aggressive nature and increased detection, in Scandinavia in 248

particular (Laurila et al., 2009). Genetic sources of at least partial resistance to the disease are 249

needed (Czajkowski et al., 2011) and although testing systems for soft rot in tubers seem to be 250

simple and reproducible (Andrivon et al., 2003; Jansky and Rouse, 2003; Kröner et al., 2011; 251

Marquez-Villavicencio et al., 2011a) there is a lack of an efficient testing system for shoot resistance 252

to blackleg, due to the complex disease etiology (Bains et al., 1999; Chen et al., 2003; Lapwood and 253

Read, 1986; Perombelon and Kelman, 1980; Zimnoch-Guzowska et al., 1999; Zimnoch-Guzowska et 254

al.). Blackleg symptoms and resistance in the shoot have different mechanisms compared to soft rot 255

in the tubers, i.e. it is also necessary to elucidate shoot responses in order to identify resistant 256

sources (Bains et al., 1999; Czajkowski et al., 2011; Rabot et al., 1994). We have therefore 257

established a blackleg screening system under in vitro sterile conditions, giving consistent results in 258

terms of clone response that are in line with other testing methods. 259

An in vitro based infection system provides means for fast propagation and screening, as well as 260

dramatically reduces use of space compared to traditional greenhouse and field growth systems for 261

potato. Especially in an era of hypothesis testing using gene-technology and where many clone 262

banks are moving into preserving their material in vitro, our method allows for rapid screening of 263

available material in order to find resistance sources. Additionally, in vitro culture provides the 264

possibilities for dissecting the effects of the pathogen and environmental effects separately in the 265

progression of the disease symptoms that maybe otherwise confounded by secondary infection or 266

condition effects such as humidity levels. This is evident from observations made by us and others in 267

variations in symptom development in infected plants in the greenhouse and in the field, indicating 268

that environmental conditions play an important role in the development of blackleg disease 269

symptoms (Marquez-Villavicencio et al., 2011b). 270


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For reference on blackleg development we inoculated a few potato clones under greenhouse 271

conditions with a Swedish isolate of Pectobacterium atrosepticum (Persson, 1988). We found that 272

the breeding line SW93-1015 (Ali et al., 2012) showed extensive blackleg symptoms whereas the 273

commercial varieties Desirée and Bintje were moderately resistant. This is in line with an earlier 274

study (Bains et al., 1999). We found that the blackleg symptoms in Desirée consisted of low levels of 275

tissue maceration and browning and that the spread of the necrosis was very limited. In SW93-1015 276

on the other hand, far spreading black necrosis was formed, that also at times caused severe wilting. 277

We then infected greenhouse grown potatoes with D. solani, in order to set up a reference on its 278

disease development. This is important since D.solani is regarded as an emerging threat to the 279

potato growing industry (Pédron et al., 2014) and its resistance in common cultivars is presently 280

unreported. We included two cultivars which have known resistance to late and early blight, Sarpo 281

Mira and Magnum Bonum respectively. In Desirée, Bintje and SW93-1015 we found the same effects 282

as for P. atrosepticum albeit with a more severe disease development indicating that there is a 283

similar host range in P. atrosepticum and D. solani. Interestingly with D. solani, in Sarpo Mira we 284

found similar results as in Desirée and Bintje, whereas in Magnum Bonum the disease progressed 285

with a similar severity as in SW93-1015. We then proceeded to use these potato clones as reference 286

for establishing the in vitro system. 287

After trying several different approaches, we found that the best approach was to grow plantlets in 288

sterile in vitro conditions, then excise them at the base of the shoot and place in bacterial solution, 289

inside aerated sterile tubes. We observed highly reproducible symptoms in vitro that were 290

consistent with the greenhouse infections in terms of different blackleg tolerance in the different 291

potato clones, indicating that the artificial in vitro method did not drastically alter the resistance 292

mechanisms in the tested clones. However, infections in vitro resulted in weaker blackleg symptoms 293

in general compared to in the greenhouse. 294

In greenhouse, inoculations by stab injection into the basal haulm resulted in lesions spreading in a 295

continuous fashion, mainly upward but also downward from the inoculation site, allowing for a 296

continuous measurement of lesion length. In the in vitro system however, where smaller, cut plants 297

with a different architecture compared to greenhouse plants were placed in a bacterial solution, 298

lesions developed non-continuously at different locations and with different sizes around the haulm. 299

Importantly a consistent trend in symptom development and disease response across the repetitions 300

in the in vitro assay was observed when compared to the greenhouse based assay. 301

The finding that the development of the characteristic black necrosis is somewhat mitigated in the in 302

vitro system compared to in the greenhouse might be explained by plant anatomical, physiological 303

or environmental factors. It is possible that a decreased availability of sugars in the phloem or 304

pectins in cell walls affects the speed and aggressiveness of the disease development (Toth et al., 305

2004). Since humidity was kept constantly high during the infection in both conditions, it is unlikely 306

that it is the main determinant in this case. Another possible explanation for the milder symptoms 307

observed in vitro is the lack of other biota in the sterile interaction that otherwise cause secondary 308

symptoms (Toth et al., 2003). Our system maintains the natural spreading route in planta of the 309

bacteria by mimicking blackleg disease progression in the field, where the bacteria migrate from an 310

infected tuber (bottom of haulm) upward into the shoot through the xylem (Perombelon and 311

Kelman, 1980). We found that in our novel in vitro infection system, transpiration was necessary for 312

disease symptom development, i.e. we found that when the tubes were fully sealed (as to hinder 313


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transpiration of liquid) symptoms did not develop, but when the lids of the tubes were kept 314

unscrewed, symptoms did develop (data not shown). 315

Since the in vitro assay offers increased consistency by limiting the interaction of environmental and 316

biotic variables in pathogen and plant interaction, we used the in vitro assay to investigate the role 317

of the main biotic stress hormone pathways (SA and JA) in blackleg disease symptom development, 318

we infected potato transgenic lines that were insensitive to JA (coi1X5 and coi1H1) or deficient in SA 319

(NahGD2 and NahGA) in vitro. Both the hormone transgenic lines were significantly more susceptible 320

than cv. Desiree (wild type). These results indicate that both SA and JA pathways are necessary for 321

resistance against the necrotrophic bacteria D.solani. Interestingly this is a deviation from existing 322

knowledge regarding Arabidopsis and necrotrophic bacteria interactions wherein prominence of the 323

JA pathway has been shown (Glazebrook, 2005). In line with the importance of the JA pathway in 324

Arabidopsis-necrotrophic bacteria interaction, Norman-Setterblad et al. (2000) showed in 325

arabidopsis that JA signaling deficient coi1 mutant was susceptible to P. carotovorum ssp. 326

carotovorum inoculation in comparison to NahG and wildtype plants. Our results indicate that 327

potato response to necrotrophic bacteria differs from responses observed in Arabidopsis. 328

329

Acknowledgement 330

The authors are grateful to Paula Persson and Minna Pirhonen for kindly supplying bacterial stocks, 331

and Sabine Roshal for providing transgenic potato lines. The Swedish foundation for Strategic 332

research, PlantLink, The Trygger foundation, The Swedish Research Council for Environment, 333

Agricultural Sciences and Spatial Planning are thanked for financial support. 334

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Figure legends 479

Figure 1 Greenhouse experiment with Potato and Dickeya solani (A): Average rot length (in mm) of 480

the stem 7 days after infection. Data represented is average rot length ± SE obtained from three 481

individual experimental repeats with at least 5 shoots inoculated in each experiment. Statistical 482

analysis and grouping performed using ANOVA and Tukey-Kramer procedure (p< 0.05) on log 483

transformed rot lengths. (B): Dickeya solani symptom development 7 days after infection in SW93-484

1015, Desirée, Bintje, Magnum Bonum, Sarpo Mira. Representative pictures of disease symptoms 485

(leaves removed for visualization). 486

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Figure 2 Greenhouse experiment with Potato and Pectobacterium atrosepticum. (A): Average rot 489

length (in mm) of the stem 7 days after infection. Data represented here is average rot length ± SE 490

obtained from three individual experimental repeats with at least 5 shoots infected in each 491

experiment. Statistical analysis and grouping performed using ANOVA and Tukey-Kramer procedure 492

(p< 0.05) on log transformed rot lengths. (B): Pectobacterium atrosepticum disease symptoms 7 days 493

after infection in Desirée, Bintje and SW93-1015. Representative pictures of disease symptoms 494

(leaves removed for visualization). 495

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Figure 3 (A): Method representation of in vitro assay (lid not sealed). (B): Representative pictures of 497

scored phenotypes. 498

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Figure 4 Dickeya solani infections on in vitro grown Potato stems (A): SW93-1015, Desirée, Magnum 500

Bonum and Sarpo Mira. Representative pictures of disease symptoms 11 days after infection. (B): 501

Dickeya solani infections in vitro: Average infection score 11 days after infection. Data represented 502

here is average infection score ± SE obtained from three different experimental repeats with 12 503

plants tested in each repeat. Statistical analysis and grouping performed by using ANOVA and Tukey-504

Kramer procedure (p< 0.05) on log transformed infection scores. 505

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Figure 5 Pectobacterium atrosepticum infections on in vitro grown Potato stems (A): SW93-1015, 507

Desirée and Bintje. Representative pictures of disease symptoms 11 days after infection. (B): 508

Pectobacterium atrosepticum infections in vitro: Average infection score 11 days after infection. 509

Data represented here is average infection score ± SE obtained from three different experimental 510

repeats with 12 plants tested in each repeat. Statistical analysis and grouping performed by ANOVA 511

and Tukey-Kramer procedure (p< 0.05) on log transformed infection scores. 512

513

Figure 6 Dickeya solani infections of in vitro grown hormone transgenic Potato stems: Average 514

infection score obtained in jasmonic acid insensitive (coi1X5 and coiH1) mutants,salicylic acid 515

deficient (NahGD2 and NahGA) transgenics and cv. Desiree (wild type) in the in vitro assay. Data 516

represented here is average infection score ± SE obtained from two experiments with 12 plants 517

tested in each repeat. Asterix represents significant difference from cv. Desiree (wild type) based on 518

student´s t-test (p < 0.05). 519

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