J.R. Simplot Company Disease Susceptibility 1 Appendix 8 Disease Susceptibility

I NDEX

Section Page

Index 1

Introduction 2

Results 3

Materials and Methods 8

Conclusions 1 0

Reference s 1 2

Certification 1 3J.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 U S8, US10, and US22. Of t hese 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 U S (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 fo r disease symptoms 2 months after

inoculation. A zero score meant there w ere 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 inf ection manifested by mycelium on the entire tuber surface and either with or

without tissue discoloration and breakdown.

Table 1

. Late B light S ymptom s in T ubers of Ranger Russet and Russet Burbank E vents 1

Line Rating 2 (Mean) +/

- Standard Error 3

Russet Burbank Control 1. 05 a +/

- 0. 263

E12 3. 85 b +/

- 0. 263

E24 1.9 0 a +/

- 0. 580

Ranger Russet C ontrol 5.6 0 a +/

- 1.324

F10 0.8 0 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 var iety, 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 st udy, we would not consider this one study as definitive, and therefore repeated the assay in 2011 ( see results in

Table 3 ).

In the assay o f tubers harvested in 2009 ( Table 1) , t he Ranger Russet event F10 developed less disease symptoms upon infection th an the untransformed control. In fact, event F10 was about as tolerant to late blight as the Russet Burbank control.

T he G and H controls and events were tested for susceptibility to three different important U.S. strains
of P. infestans

. Results were va riable, 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 w ere no difference s 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 B light S ymptom in T ubers of G and H E vents 1 ,2

Line Rating 2 (Mean) +/

-

Standard Error 3
( US8 ) Rating 2 (Mean) +/

- Standard Error 3
( US10 ) Rating 2 (Mean) +/

- Standard Error 3
( 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 +/

- 0.4 47

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 i n 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 u ses 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 Erwini a ove r the three years of tesing from 2009 to 2011

.

Table 3

. Late Blight S ymptom s in T ubers of Ranger Russe t, Russet Burbank and Atlantic E vents 1

Line Avg. Disease % P

- value 2

Russet Burbank Control 74.50

E12 23.50 0.0296

E24 31.00 0.0637 Ranger Russet C ontrol 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 signi ficance 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 mea n that there w ere 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 s ignificantly fewer late blight symptoms than the untransformed

Russet Burbank control

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

c onfirm 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. T he event J3 displayed significantly more tuber infection sympt oms than the Atlantic control , but J55 and J78 were not significantly different

. The s usceptibility of J3 tubers to late blight infection was similar to that

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

ag ricultural 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, Phytop hthora 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
time s during the season ( Table 4 ).

Tabl e 4

. Late Blight Symptom s in F oliage of Ranger Russet, Russet Burbank and Atlan tic E vents 1

Line RAUDPC P

- value 2

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 < 0.0 5. 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 eva luate disease severit y. As presented in Table 4

, the foliage of the events did not show increase d 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 even t 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 la boratory conditions. For the assay represented in Table 5

, two repetitions of

t en field

- grown tubers per event were inoculated with a suspension of Erwinia in water (OD 600 = 0.6).

These tubers were grown in Canyon County, ID

, for Ranger Russet and Russet B urbank controls and events, and Oneida County, W I, for G and H controls and events

.

Table 5

. Weight Loss (%) in Erwinia

- infected Tubers 1

Line Mean Weight Loss ± Std. Error 2

Ranger Control 0.709 ± 0.204

F37 0.654 ± 0.177

Burbank Control 1.474 ± 0.58 9

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 o bserved between any event and its untransformed counterpart.

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

caro tovora subsp. c arotovora 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 th e events

. Thus,

i ndependent lines of two chipping varieties and two French fry varieties with low Ppo expression in
tubers were shown to have similar suscepti bility to bacterial soft rot to the corresponding untransformed control for each variety . This supports the expectation that Ppo 5 gene silencing in
mature tubers would not cause altered susceptibility to bacterial soft rot.

Additional tests were condu cted 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 we re 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

- value 1
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 3.7 7

- 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

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

that the percent weight loss o f untransformed controls did not differ significantly from the events

, except in the case of t he Atlantic variety control compared to the J3 and J55 events. E vents J3 and J55 displayed a lowered susce ptibility 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 M ATERIALS 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 dete rmine 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 myceli um based innoculations : 1) a fter artificial inoculation with zoospores/sporangia t he 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 tube rs 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 t he 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 B urbank tuber s 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 a gar 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 infect ed t ubers 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 w ere 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 assess ed 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 Raf ael, C A ). 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 genotyp es, 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 So ftware, San Rafael, Ca). A lower RARI value indicates less disease. RARI values across the apical, middle, and basal sections were combined.

Late blight as say for tubers harvested in 2011

. 4 t ubers each for events and controls were surface sterilized p rior 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 tu bers were placed in plastic crates and incubated forJ.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 tube r. Tubers were inoculated with both P. infestans strains US8 and US22. A ll 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 tub ers unsuccessfully infected with US22 are not presented
here. However, t ypical 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 eig ht 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 f or 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 o nto each woun d. 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), t ubers 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 fr om 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 s olution 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. 1 00 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 20 o C and 95% relative humidity. After 7 days of incubation, tubers were scored for susceptibi lity 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

I t can b e 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 folia ge 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 Ru sset. 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 20 09

. Although we

observed m ore susceptibility in the 2009 study, whe n 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. infes tans 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 even t 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 con trol. 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, t ubers 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 susc eptibility 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 Er winia was observed between event H50 and H control.J.R. Simplot Company Disease Susceptibility 12 REFERENCE S

Gevens AJ, Seidl, AC, Clark R (2011) Potato and tomato late blight in Wisconsin: A new type of late blight in a new decade. Proc. Of the 2011 Wisconsin Crop Management Conference. 50 :123

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Gigot, J., Gundersen, B., Inglis, D., 2009. Colonization and Sporulation of Phytophthora infestans on Volunteer Potatoes Under Western Washington Conditions. Am J Potato Res 86, 1

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Kirk, WW, Niemira, BA, Stein, JM, Hammerschmidt, R ( 1999 ) Late blight (Phytophthora infestans (Mont) de Ba ry) development from potato seed

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