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For Peer Review
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
For Peer Review
1
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
7
Running title: In vitro method for screening of Blackleg disease in potato 8
9
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
14
Keywords: Blackleg disease, Pectobacterium atrosepticum, Dickeya solani, in vitro potato, coi1, 15
NahG, Sarpo Mira, SW93-1015, Bintje, Desiree 16
17
Abbreviations: SA: Salicylic acid; JA: Jasmonic acid; COI: Coronatine insensitive; NAA: 1-18
Naphthaleneacetic acid; IBA: Indole-3-butyric acid 19
20
21
22
23
24
25
26
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27
28
29
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
47
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
105
Material and Methods 106
107
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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
165
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
245
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
496
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
506
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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