J.R. Simplot Company Disease Susceptibility 1

Appendix 8

Disease Susceptibility

INDEX

Section Page

Index 1

Introduction 2

Results 3

Materials and Methods 8

Conclusions 10

References 12

Certification 13
J.R. Simplot Company Disease Susceptibility 2

INTRODUCTION

The purpose of this study was to determine how common potato diseases affect the events, compared 

with the untransformed controls. Researchers have investigated the relationship between PPO and 

disease resistance (Valentines et al. 2005; Li and Steffens 2002; Hakimi et al. 2006); with some proposing 

that enhanced PPO may increase resistance to disease, while others claim that reduced PPO could 

increase resistance. Considering that some evidence exists for a relationship between PPO and diseases, 

we chose to test the events by infecting foliage and tubers with the causal agents of the most important 

oomycete (Phytophthora infestans) and bacterial soft rot (Erwinia carotovora) diseases.

Late blight tests were conducted using Phytophthora infestans strains US8, US10, and US22. Of these 

three strains, US8 and US10 were commonly found in Michigan (Young et al. 2009), where some of our 

testing was conducted. Foliar studies in 2011 used US22, a relatively new genotype that has been 

prevalent in the Midwestern US (Gevens et al. 2011). 
J.R. Simplot Company Disease Susceptibility 3

RESULTS

Tuber Susceptibility to Late Blight

To determine the extent of symptom development in tubers infected with the causal agent of late 

blight, P. infestans, we selected four each of five field-grown tubers from Canyon County, ID, for Ranger 

Russet and Russet Burbank controls and events, and Oneida County, WI, for the G and H controls and 

events harvested during the fall of 2009. 

For this analysis, infected tubers were scored on a 0-9 scale for disease symptoms 2 months after 

inoculation. A zero score meant there were no visible late blight disease symptoms; that is, no tuber 

discoloration, no surface mycelium and no physical tissue breakdown. A score of 9 was indicative of 

100% late blight infection manifested by mycelium on the entire tuber surface and either with or 

without tissue discoloration and breakdown. 

Table 1. Late Blight Symptoms in Tubers of Ranger Russet and Russet Burbank Events1

Line Rating2(Mean) +/- Standard Error3

Russet Burbank Control 1.05 a +/- 0.263

E12 3.85 b +/- 0.263

E24 1.90 a +/- 0.580

Ranger Russet Control 5.60 a +/- 1.324

F10 0.80 b +/- 0.216

1

Data in this table are from tubers harvested in 2009.

2

Disease severity scored on 0-9 scale with 9 being 100% tuber infection with P. infestans strain US8. Pairwise 

comparisons to the controls were made using Tukey-Kramer HSD. Different letters from control show significant 

differences (P < 0 .05).

3

Standard Error uses a pooled estimate of error variance. Within variety, means with the same letter are not 

significantly different.

As shown in Table 1, the Russet Burbank event E24 did not differ from the control whereas E12 

appeared to be more susceptible. Although we observed more susceptibility in this laboratory study, we 

would not consider this one study as definitive, and therefore repeated the assay in 2011 (see results in 

Table 3). 

In the assay of tubers harvested in 2009 (Table 1), the Ranger Russet event F10 developed less disease 

symptoms upon infection than the untransformed control. In fact, event F10 was about as tolerant to 

late blight as the Russet Burbank control. 

The G and H controls and events were tested for susceptibility to three different important U.S. strains 

of P. infestans. Results were variable, with some of the events more or less susceptible than controls, 

depending on the strain (Table 2). Event G11 was less susceptible to US8 and US22 than control G. Both 

H37 and H50 were more susceptible to US22 than H control, however H37 was less susceptible and H50 

more susceptible to US8. There were no differences observed between events or controls tested with 

US10. While these laboratory tests could indicate if there were clear differences in susceptibility, with 

mixed results, we are unable to draw definitive conclusions.
J.R. Simplot Company Disease Susceptibility 4

Table 2. Late Blight Symptom in Tubers of G and H Events1,2

Line Rating2(Mean) +/-

Standard Error3

(US8)

Rating2(Mean) +/-

Standard Error3

(US10) 

Rating2(Mean) +/-

Standard Error3

(US22)

G Control 8.26 a +/- 1.693 1.68 a +/- 0.442 3.26 a +/- 0.872

G11 2.91 b +/- 1.198 3.63 a +/- 1.302 0.67 b +/- 0.281

H Control 1.69 a +/- 0.510 3.65 a +/- 1.073 1.68 a +/- 0.447

H37 0.57 b +/- 0.308 3.43 a +/- 1.041 4.91 b +/- 1.331

H50 4.21 b +/- 1.325 5.39 a +/- 1.650 4.39 b +/- 1.537

1

Data in this table are from tubers harvested in 2009. 

2

Disease severity scored on 0-9 scale with 9 being 100% tuber infection with a specific strain of P. infestans (US8, 

US10, or US22). Different letters from control show significant differences (P < 0 .05) using Tukey-Kramer HSD.

3

Standard Error uses a pooled estimate of error variance. Within variety, means with the same letter are not 

significantly different.

As a follow-up to late blight testing with laboratory assays in 2009, we conducted an additional 

laboratory assay in 2011, only for the Ranger Russet, Russet Burbank, and Atlantic varieties and events

In this study, tubers were inoculated with the US8 strain of P. infestans, and progression in the events 

and untransformed controls were analyzed. The results of that study are summarized in Table 3. For 

both the tuber and foliar assays (Tables 3 and 4) the Russet Burbank, Ranger Russet, and Atlantic lines 

received priority over the proprietary G and H lines in 2011 studies. However, all events had some 

testing for late blight and Erwinia over the three years of tesing from 2009 to 2011.

Table 3. Late Blight Symptoms in Tubers of Ranger Russet, Russet Burbank and Atlantic Events1

Line Avg. Disease % P-value2

Russet Burbank Control 74.50

E12 23.50 0.0296

E24 31.00 0.0637

Ranger Russet Control 65.50

F10 71.65 0.6466

F37 57.25 0.4756

Atlantic Control 11.50

J3 73.50 0.0024

J55 43.60 0.0919

J78 36.55 0.2181

1

Data in this table are from tubers harvested in 2011. 

2

P-values were determined with Tukey-Kramer HSD analyses with a significance level of P < 0.05. P-values indicating 

significant differences with controls are underlined.

For tubers harvested in 2011, events and controls were evaluated by scoring percent late blight 

infection of the tuber (Table 3). A score of 0% would mean that there were no visible late blight disease 

symptoms; that is, no tuber discoloration, no surface mycelium and no physical tissue breakdown. A

score of 100% late blight infection would be indicated by mycelium on the entire tuber surface, either 

with or without tissue discoloration and breakdown.
J.R. Simplot Company Disease Susceptibility 5

As shown in Table 3, event E12 showed significantly fewer late blight symptoms than the untransformed 

Russet Burbank control. This lower infection rate in E12 compared with Russet Burbank control did not 

confirm the earlier lab test in 2009 that suggested that E12 was more susceptible. No differences were 

observed between the infection of Ranger Russet Control compared with events F10 or F37. The event 

J3 displayed significantly more tuber infection symptoms than the Atlantic control, but J55 and J78 were 

not significantly different. The susceptibility of J3 tubers to late blight infection was similar to that 

observed in Ranger and Burbank controls, and could be effectively managed by applying standard 

agricultural practices in regions prone to late blight (mainly the Northeastern part of the United States). 

Foliar Susceptibility to Late Blight

To determine the extent of symptom development in foliage infected with the causal agent of late 

blight, Phytophthora infestans, we conducted a RCBD field trial consisting of 3 replications of 5-hill plots

in 2011. The foliage was inoculated with genotype US22, another important U.S. strain of late blight, at 

approximately 54 days after planting, and the percent of foliar infection was recorded at numerous 

times during the season (Table 4).

Table 4. Late Blight Symptoms in Foliage of Ranger Russet, Russet Burbank and Atlantic Events1

Line RAUDPC P-value2

Burbank Control 21.9

E12 19.9 0.8361

E24 19.7 0.7963

Ranger Control 24.3

F10 17.3 <0.0001

Atlantic Control 33.6

J3 35.8 0.6929

J55 33.7 0.9999

J78 32.0 0.8686

1

Data in this table are from 2011 disease analyses.

2

P-values determined with Tukey-Kramer HSD analyses with a significance level of P < 0.05. P-values indicating 

significant differences with controls are underlined.

Relative Area Under the Disease Progress Curve (RAUDPC) was calculated from data collected at eight

different time-points during the season, and used to evaluate disease severity. As presented in Table 4, 

the foliage of the events did not show increased susceptibility to late blight. Rather, the Ranger Russet 

event F10 had significantly less disease severity than the Ranger Russet control. With the exception of 

Ranger Russet event F10, no differences were observed between the events and the untransformed 

controls.
J.R. Simplot Company Disease Susceptibility 6

Tuber Susceptibility to Soft Rot

In 2009, tubers were tested for susceptibility to Erwinia carotovora subsp. carotovora, causal agent of 

bacterial soft rot, under laboratory conditions. For the assay represented in Table 5, two repetitions of 

ten field-grown tubers per event were inoculated with a suspension of Erwinia in water (OD600 = 0.6). 

These tubers were grown in Canyon County, ID, for Ranger Russet and Russet Burbank controls and 

events, and Oneida County, WI, for G and H controls and events.

Table 5. Weight Loss (%) in Erwinia-infected Tubers1

Line Mean Weight Loss ± Std. Error2

Ranger Control 0.709 ± 0.204

F37 0.654 ± 0.177

Burbank Control 1.474 ± 0.589

E12 1.161 ± 0.200

E24 1.493 ± 0.768

G Control 8.8 ± 4.5

G11 6.0 ± 1.6

H Control 3.2 ± 1.0

H37 2.8 ± 0.6

1

Data in this table are from tubers harvested in 2009. 

2

Based on a comparison of means and their associated Std. Errors, no differences were observed between any 

event and its untransformed counterpart. 

The results of the tuber assay used to determine susceptibility to the bacterial soft rot pathogen, Erwinia 

carotovora subsp. carotovora of tubers harvested in 2009 are presented in Table 5. These data indicate 

that the percent weight loss of untransformed controls did not differ from the events. Thus, 

independent lines of two chipping varieties and two French fry varieties with low Ppo expression in 

tubers were shown to have similar susceptibility to bacterial soft rot to the corresponding 

untransformed control for each variety. This supports the expectation that Ppo5 gene silencing in 

mature tubers would not cause altered susceptibility to bacterial soft rot. 

 

Additional tests were conducted with potatoes harvested in 2011. For the assay represented in Table 6, 

tubers of Ranger Russet, Russet Burbank and Atlantic controls and events were grown at two sites, 

Adams County, WA and Canyon County, ID. Tubers of H variety control and events were from two 

separate sites in Oneida County, WI, and G control and event were from one site in Oneida County, WI. 

Three repetitions of ten field-grown tubers per line were assessed for susceptibility to soft rot, with the 

exception of the Atlantic control and events from Canyon County, ID, for which three repetitions of eight 

field-grown tubers were tested.
J.R. Simplot Company Disease Susceptibility 7

Table 6. Weight Loss (%) in Erwinia-infected Tubers*

Line Mean Weight Loss (%) Range (%) P-value1

Ranger Control 1.80 1.19-2.40

F10 1.81 1.37-2.24 0.9604

F37 1.92 1.64-2.41 0.4956

Burbank Control 2.04 1.73-2.61

E12 1.57 1.19-1.86 0.1520

E24 1.83 1.44-2.62 0.4553

Atlantic Control 3.30 2.39-4.67

J3 2.73 2.33-3.21 0.0153

J55 2.67 2.24-3.43 0.0085

J78 3.11 2.82-3.38 0.3665

G Control 4.17 3.77-4.71

G11 3.98 3.17-4.62 0.4679

H Control 4.16 3.68-4.58

H37 3.44 2.46-5.52 0.0694

H50 3.40 2.8-4.07 0.0600

*Data in this table are for tubers harvested in 2011.

1P-values determined using Tukey-Kramer HSD with a significance level of P < 0.05. P-values indicating significant 

differences with controls are underlined.

The results of the tuber assay used to determine susceptibility to the bacterial soft rot pathogen, Erwinia 

carotovora subsp. carotovora of tubers harvested in 2011 are presented in Table 6. These data show 

that the percent weight loss of untransformed controls did not differ significantly from the events, 

except in the case of the Atlantic variety control compared to the J3 and J55 events. Events J3 and J55 

displayed a lowered susceptibility to bacterial soft rot than both the untransformed Atlantic control and 

event J78. These data support the claim that Ppo5 gene silencing in mature tubers is not linked to 

altered susceptibility to bacterial soft rot in these potato events.
J.R. Simplot Company Disease Susceptibility 8

MATERIALS and METHODS

Mycelial Inoculation for Late Blight in Tubers.

The method used here is based on previously developed methods by Kirk et al. (1999). In another 

paper, Lambert et al. (1998) used a mycelium-based inoculation method to determine infection 

efficiency. The method was used more recently by Gigot et al. (2009) in a paper studying colonization 

and sporulation of Phytophthora infestans on volunteer potatoes under Western Washington 

conditions. There are several reasons for using mycelium based innoculations: 1) after artificial 

inoculation with zoospores/sporangia the spores develop mycelium, 2) tubers received for testing have 

well formed periderm reducing the potential efficacy of a surface inoculation, 3) well formed periderm 

usually has to be disrupted to enable efficient infection, 4) introduction of a zoospore/sporangial 

suspension by needle is also an unnatural infection process, and 5) the plug-method has produced 

consistent results over 5 years of testing. Addtitionally, the advantages of these methods are: 1) low risk 

of losing inoculum from leakage if tubers are disturbed immediately after inoculation, 2) location can be 

precise (1 cm distal from the dominant sprout) as with zoospore/sporangial method, 3) the progress of 

disease development can be followed through the tuber from apical to basal regions of the tuber, 4) and 

after inoculation the disease progress through the tuber is consistent across samples (Personal 

Communication with Dr. Willie Kirk, Michigan State University).

Late blight assay for tubers harvested in 2009. Tubers were surface sterilized in a 5% bleach solution, 

rinsed and dried overnight prior to inoculation. Ranger Russet and Russet Burbank tubers were

inoculated by removing a 5 mm diameter plug (10 mm depth) at the apical end about 1 cm from the 

dominant sprout, and placing a mycelia agar plug (mycelia down) in the hole, and covering the tuber 

plug with a dab of petroleum jelly. Each sub-peridermal inoculation contained about 2 x 10-5

sporangia per plug. The non-inoculated control tubers were inoculated with an agar plug.”

The infected tubers were incubated at 95% relative humidity and 100C and scored, on a 0-9 scale, for 

disease symptoms 2 months after inoculation. A zero score meant there were no visible late blight 

disease symptoms; that is, no tuber discoloration, no surface mycelium and no physical tissue 

breakdown. A score of 9 was indicative of 100% late blight infection manifested by mycelium on the 

entire tuber surface and either with or without tissue discoloration and breakdown. 

Susceptibility to late blight disease was assessed digitally in the G and H lines. Digital scans were taken 

of tuber slices from the apical, middle and basal portions of the inoculated tubers and diseased area was 

quantitated using image analysis software (SigmaScan, Jandel Scientific Software, San Rafael, CA). The 

average reflective intensity of light reflected from the cut surface measures the darkened, diseased 

tuber tissue amid the lighter, symptom-free tissue.

 

G and H tubers were inoculated on the apical end with the US8, US10, and US22 genotypes, and scored 

by digitally scanning the apical, middle, and basal sections of the tuber. The diseased area was used to 

calculate a Relative Average Reflective Index (RARI) (Niemira et al. 1999) with image analysis software 

(SigmaScan, Jandel Scientific Software, San Rafael, Ca). A lower RARI value indicates less disease. RARI 

values across the apical, middle, and basal sections were combined.

Late blight assay for tubers harvested in 2011. 4 tubers each for events and controls were surface 

sterilized prior to inoculation. The apical ends of the tubers were inoculated by removing a plug of tissue 

with a No. 4 cork borer, placing a P. infestans mycelia plug (mycelia down), replacing the tuber core and 

sealing with a dab of petroleum jelly. The infected tubers were placed in plastic crates and incubated for 
J.R. Simplot Company Disease Susceptibility 9

58 days at 100C within large plastic bags to maintain high humidity. Upon analysis, tubers were halved 

longitudinally from apical to basal end, and evaluated for percent late blight infection of the tuber. 

Tubers were inoculated with both P. infestans strains US8 and US22. All tubers infected with the US22 

genotype did not display late blight symptoms, which is most likely due to a lack of infection, as opposed 

to higher tuber resistance. The data for tubers unsuccessfully infected with US22 are not presented 

here. However, typical disease progression was observed in tubers infected with US8, and those results 

are reported within this document. 

Late blight foliar assay for plants grown in 2011. The late blight field trial was planted in Michigan on 

6/9/2011 as a RCBD consisting of 3 row blocks of 5-hill plots with 3 replications. The trial was inoculated 

on 8/1/2011 with a Michigan isolate of P. infestans genotype US22. Disease ratings were taken at eight

different dates over a 54 DAI (Days After Inoculation) period. Plot percent late blight disease rating was 

used to calculate the Relative Area Under the Disease Progression Curve (RAUDPC x100) which was used 

to evaluate disease severity. 

Erwinia assay for tubers harvested in 2009. Each tuber was wounded three times using a sterile, 

disposable pipette tip. Each wound was made to a depth of 15 mm around a central point between the 

stem and stolon end. 50 μl of Erwinia suspension was pipetted onto each wound. Inoculated tubers 

were incubated at 22 0C and 95% relative humidity. After 7 days of incubation for the smaller tubers of 

the chipping varieties (G and H) and 9 days incubation for the more tolerant frying varieties (Ranger 

Russet, Russet Burbank), tubers were scored for susceptibility to soft rot. The fresh weight of each tuber 

was recorded. Tubers were then cut into two or three pieces to expose the rotted tissue inside. With a 

high pressure stream of water, rotted tissue was forcefully removed from healthy tissue. The tubers 

were air dried and reweighed. The percentage fresh weight loss due to rot was recorded.

Erwinia assay for tubers harvested in 2011. The fresh weight of each tuber was recorded. Tubers were 

surface sterilized with 10% bleach solution for 10 minutes, rinsed thoroughly with water, and allowed to 

air dry. Each tuber was wounded by removing a 2 cm long plug using a No. 4 cork borer, and removing a 

2 mm piece of the plug from the internal end to allow room for the Erwinia culture. 100 μl of Erwinia 

suspension in Luria broth was pipetted into the wound, and the plug replaced and sealed with 

petroleum jelly. Inoculated tubers were incubated at 20oC and 95% relative humidity. After 7 days of 

incubation, tubers were scored for susceptibility to soft rot. Tubers were cut in half to expose the rotted 

tissue inside. With a spatula, rotted tissue was removed from healthy tissue. The tubers were then 

reweighed and the percentage fresh weight loss due to rot was recorded.
J.R. Simplot Company Disease Susceptibility 10

CONCLUSIONS

It can be concluded that potato transformation with the DNA insert of pSIM1278 does not alter disease 

susceptibility and, consequently, does not trigger environmental safety issues associated with the 

spread of plant diseases. All 10 events were included in tests for Phytophtora and Erwinia, although the 

G and H events were not part of the Phytophtora foliar and tuber testing conducted in 2011. Some 

differences were observed in the susceptibility to P. infestans of tubers of events E12 and J3, and foliage 

of F10. All events from the 2011 harvest were tested for Erwinia soft rot, and the only differences were 

slightly less susceptibility in Atlantic events J3 and J55. Considering that the 10 events transformed with 

pSIM1278 do not show any clear trends for altered susceptibility to these disease agents, it is expected 

that G and H events would have performed similarly in additional tests. When Phytophtora was studied

in both foliage and tubers (Porter et al. 2004), those varieties that were most resistant in foliage also 

showed resistance in tubers. Conclusions related to each individual event are summarized below.

Event Ranger Russet F10: Tubers of this event showed similar susceptibility to P. infestans or Erwinia 

compared with tubers of untransformed Ranger Russet. Foliage of this event showed less susceptibility 

to P. infestans compared with the untransformed Ranger Russet.

Event Ranger Russet F37: Tubers of event F37 showed similar susceptibility to P. infestans or Erwinia

compared with untransformed Ranger Russet. Foliage of this event was not tested.

Event Russet Burbank E12: Tubers of event E12 displayed more susceptibility to P. infestans, but not 

Erwinia when compared with tubers of untransformed Russet Burbank harvested in 2009. Although we 

observed more susceptibility in the 2009 study, when the assay was repeated with tubers harvested in 

2011, tubers of E12 were found to display lower late blight symptoms than the Russet Burbank control. 

Foliage of this event showed similar susceptibility to P. infestans compared with the untransformed 

Russet Burbank.

Event Russet Burbank E24: Tubers of event E24 showed similar susceptibility to P. infestans or Erwinia

compared with untransformed Russet Burbank. Foliage of this event showed similar susceptibility to P. 

infestans compared with the untransformed Russet Burbank.

Event Atlantic J3: Tubers of event J3 were more susceptible to P. infestans than untransformed Atlantic. 

However, tubers of this event showed less susceptibility to Erwinia than the Atlantic control. Foliage of 

this event showed similar susceptibility to P. infestans compared with the untransformed Atlantic.

Event Atlantic J55: Tubers of event J55 showed similar susceptibility to P. infestans compared with

untransformed Atlantic. However, tubers of this event showed less susceptibility to Erwinia than the 

Atlantic control. Foliage of this event showed similar susceptibility to P. infestans compared with

untransformed Atlantic.

Event Atlantic J78: Tubers of event J78 showed similar susceptibility to P. infestans or Erwinia compared 

with untransformed Atlantic. Foliage of this event showed similar susceptibility to P. infestans compared 

with untransformed Atlantic.

Event G11: Tubers of event G11 were generally less susceptible to P. infestans, but were no different in 

susceptibility to Erwinia than the untransformed G variety. 
J.R. Simplot Company Disease Susceptibility 11

Event H37: Tubers of event H37 showed varied susceptibility to P. infestans depending, upon the strain 

tested when compared with the untransformed H variety. Similar susceptibility to Erwinia was observed

between event H37 and H control.

Event H50: Tubers of event H50 showed more susceptibility to P. infestans strains US8 and US22, but 

not US10, when compared with the untransformed H variety. Similar susceptibility to Erwinia was 

observed between event H50 and H control.
J.R. Simplot Company Disease Susceptibility 12

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