sclerotinia resistance and forecasting in canola · a single pen1 gene copy was inserted in the ....

‘Things should start to get interestin’ right about now’

Agriculture andAgri-Food Canada

Agriculture et Agroalimentaire Canada

Sclerotinia resistance and forecasting in canola

Lone BuchwaldtAgriculture and Agri-Food Canada

Saskatoon Research and Development Centre

Canola Industry meetingDecember 4, 2017, Saskatoon

AAFC’s sclerotinia resistance team

Jonathan Durkin

Edis Dzananovic

Myrtle Harrington

Jackie Nettleton

Linda McGregor

Lone Buchwaldt

Sally Vail

Dwayne Hegedus

Fuyou Fu

VickyRoslinsky

DineyBekkaoui

Jennifer Adam

MissingSanjaya GyawaliHarsh GargBrad Hope Todd Olsen Photo by Branimir Gjetvaj

We have developed tools for breeding sclerotinia resistant canola varieties

˛ Sclerotinia screening method - stem test

˛ Seed of resistant lines

˛Molecular markers linked to sclerotinia resistance

˛ Half way with transfer of sclerotinia resistance to canola quality

˛ Sclerotinia isolates representative of the Canadian population

˛ Cloned defence genes

Contact Todd Olsen for transfer, licencing and/or collaboration [email protected]

Sclerotinia screening method – stem test

The five traits are highly correlated

1) Lesion length 7 dai



4) AUDPC ∑ (Yi + Y (i+1) ) / 2 (t (i+1) – ti)

where Yi is the disease rating at time ti

5) % soft + collapsed lesions

0 7 14 21 Days after inoculation

Lesi

on le

ngth

100

75

50

25

AUDPC

Susceptible

Resistant

Most important measurements

WCC/RRC adopted the stem test for coop lines with a claim of sclerotinia resistance

Line % of 45H29

45H29 69 B 100 32 ABL160S - late 58 C 84 29 ABVR9561G 55 CD 80 27 B45S52 53 CD 77 26 BLSD = 8 8

Lesion length 21dai, mm % s+c

Line % of 45H29

45H29 67 BC 100 35 BCL160S 73 B 109 42 ABVR9561G 60 CD 90 33 BC45S52 50 E 74 27 DLSD = 7 10

Lesion length 21dai, mm

% s+c

Results of 7 sclerotinia stem tests in 2014.

Results of 8 sclerotinia stem tests in 2015.

Conclusion:L160S is non sign.VR9561G is non sign. 45S52 is significantly better for lesion length (26% reduction) and % soft + collapsed lesions

Conclusion:L160S is sign for lesion length (16% reduction) VR9561G is sign for lesion length (20% reduction) 45S52 is sign for lesion length (23% reduction)

Sclerotinia reaction in L160S and VR9561G is at the limit of what the stem test can pick up.Higher level of resistance is expected in the next generation of canola varieties.The stem test will be needed in the near future.

Report on stem test evaluation (33 tests) available on Canola Council’s web sitehttp://research.canolacouncil.org/_uploads/documents/CanolaResearchHub_WCC-RRC_2016_Buchwaldt.pdf

Sclerotinia resistant Brassica napus lines

PAK54 (Pakistan)PAK93 (Pakistan)DC21 (South Korea)K22 (Japan)

Request the germplasm! ‘Don’t Think Twice, It’s All Right’

10 Material Transfer Agreements have been signed since 2014

High glucosinolate and erucic acid

SNP markers linked to sclerotinia resistance

Bi-parental mapping populations A-genome C-genome Resistance loci

PAK54 A3, A6, A9, A10 C1, C6 7

PAK93 A5, A7 C2, C5, C6, C7 6

DC21 A1, A3 C5 3

K22

Zhongyou 821 A1, A3, A5, A6, A7, A9 C1, C2, C3, C6, C9 11

A world collection of 180 B. napus accessions

A1, A2, A3, A6, A7, A8, A9, A10

C1, C2, C3, C4, C5, C6, C7, C9 114

In progress

Needed for marker-assisted-selection of progenies in various generations

Microsatellite markers are published in Gyawali et al. 2016, Mol Breeding, 36:72-85.

SNP markers will be published in 2018

Sheet1

Bi-parental mapping populationsA-genomeC-genomeResistance loci

PAK54 A3, A6, A9, A10C1, C67

PAK93 A5, A7C2, C5, C6, C76

DC21 A1, A3C53

K22 In progress

Zhongyou 821 A1, A3, A5, A6, A7, A9C1, C2, C3, C6, C911

A world collection of 180 B. napus accessionsA1, A2, A3, A6, A7, A8, A9, A10C1, C2, C3, C4, C5, C6, C7, C9114

Sheet2

Sheet3

Transfer of sclerotinia resistance to canola quality background, good agronomy and yield

Seed of the best lines are available throughout the project

SNP-markers have been validated and used in practise

A successful breeding strategy

Sclerotinia isolates used for inoculation: #321, MB51 and AB29

2013 PAK54 (resistant) x N99-508 (canola quality)2014 F1 backcrossed to N99-508

Plants selected based on SNP markers2015 1st DH population 2016 Agronomy, seed quality

Sclerotinia test 1st inter-cross of DH sister lines based on traits and SNP markers

2017 Agronomy, seed quality, yield Sclerotinia test 2nd DH population

2018 Agronomy, seed quality, yield Sclerotinia test

2019 2nd inter-cross of DH sister lines based on traits and SNP markers Agronomy, seed quality, yield Sclerotinia test

2020 3rd DH population Plants selected based on SNP markers Agronomy, seed quality, yield Sclerotinia test



2023 Project ends March 31

population developmentmarker-assisted-selectionfield plotsStem inoculation

QTLs


PAK54 (Pakistan)A3, A6, A9, A10C1, C67

PAK93 (Pakistan)A5, A7C2, C5, C6, C76

DC21 (South Korea)A1, A3C53

K22 (Japan)In progress

Zhongyou 821 (China) A1, A3, A5, A6, A7, A9C1, C2, C3, C6, C911


cloned genes

Genes cloned from Zhongyou

GenesFunction in host resistanceReduction in sclerotinia severity

Hevein, lectin likePathogen recognition and binding40-50%

Lectin, concanavalin20-50%

Lectin, curculin40%

AnnexinTrafficking and organization of vesicles, exocytosis, endocytosis, calcium ion channel formation30-40%

WRKY33Regulation of gene transcription20-30%

O-methyl transferaseGlucosinolate pathwaySmall inconsistant effect

PGIPInhibitor of fungal polygalacturonaseNo effect

check list

Twengström et al. 1989Canadian research 2018-2023App maintained 2023 onward

1. mm rainfall in the past 14 daysIn-depth survey of ~400 canola fields over 5 growing seasons will capture relationships between different rainfall patterns and the other risk factorsWeather data and analysis belong to WIN

2. Likelihood of rain in weather forecast

3. Number of years since last canola crop Conclusions regarding the in-depth survey is public information

4. Sclerotinia level in last canola crop

5. Plant density

6. Regional apothecia germination Who will continue this service?

Breeding plan

2013 PAK54 (resistant) x N99-508 (canola quality)population development

2014 F1 backcrossed to N99-508marker-assisted-selection

Plants selected based on SNP markersfield plots

2015 1st DH population Stem inoculation

2016 Agronomy, seed quality

Sclerotinia test

1st inter-cross of DH sister lines based on traits and SNP markers

2017 Agronomy, seed quality, yield

Sclerotinia test

2nd DH population


Sclerotinia test

2019 2nd inter-cross of DH sister lines based on traits and SNP markers

Agronomy, seed quality, yield

Sclerotinia test

2020 3rd DH population

Plants selected based on SNP markers


Sclerotinia test


Sclerotinia test


Sclerotinia test


QTLs








cloned genes





Lectin, curculin40%





check list






5. Plant density


Breeding plan






Sclerotinia test



Sclerotinia test

2nd DH population


Sclerotinia test



Sclerotinia test




Sclerotinia test


Sclerotinia test


Sclerotinia test


Isolates were collected across the Prairies

DNA fingerprinting identified 17 sub-populations

Inoculation with 17 representative isolates showed PAK54 has the highest level of resistance across isolates

Isolates SK35 and AB29 are more aggressive on some lines (arrows)

The 17 sclerotinia isolates are available from AAFC

Sclerotinia isolates for resistance screening

Chart1

AB7AB7AB7AB7AB7AB710.510.50.70.7

3213213213213213218.78.71.91.9

AB3AB3AB3AB3AB3AB328.528.51515

MB35MB35MB35MB35MB35MB3530.630.64.54.5

MB57MB57MB57MB57MB57MB579.99.92.22.2

SK35SK35SK35SK35SK35SK3512.912.918.418.4

SK44SK44SK44SK44SK44SK4412.412.417.817.8

MB21MB21MB21MB21MB21MB2119.519.515.515.5

SK23SK23SK23SK23SK23SK2321.821.814.414.4

MB52MB52MB52MB52MB52MB5212.112.119.519.5

MB61MB61MB61MB61MB61MB617.47.44.34.3

SK14SK14SK14SK14SK14SK145.45.49.19.1

MB51MB51MB51MB51MB51MB5120.720.716.416.4

SK38SK38SK38SK38SK38SK3812.312.322.422.4

SK45SK45SK45SK45SK45SK4516.616.618.618.6

AB19AB19AB19AB19AB19AB1929.829.825.325.3

AB29AB29AB29AB29AB29AB2911.111.110.510.5

Topas

Tanto

DC21

K22

PAK93

PAK54

Sclerotinia isolates

Stem lesion length, mm

35.3

19

10.2

10.8

16.6

12.2

54.2

59.7

42.7

72.7

37.1

17.7

109.8

85.9

110.5

102.9

38.4

44.8

145.3

92.1

63.3

27.5

67.8

22.4

160

93.4

114.3

63.8

47.2

15.9

132.1

71.8

185.4

125.8

57.1

45.7

141.2

70.6

49

33.9

63

44.2

164

120.6

124.2

46

121.7

34.4

179.6

123

66.3

88.2

78

46.7

182.2

118.2

78.4

58.1

71.9

46.4

185.5

123.4

115.6

48

75.8

59.5

214.8

107.5

87.9

42.6

70.4

31.4

223.4

156.8

101.3

93.4

79.9

67

191.2

134.3

158.5

123.3

78.6

62.3

203.8

140.8

128.3

78.9

103.6

84.9

208.8

146

159.3

70.7

137.9

80.1

209.4

153

212.9

100

126.1

106.2

Original

Generated by the SAS System ('SASApp', W32_S08R2) on November 14, 2014 at 2:57:17 PM

2014 disease nursery, 17 sclerotinia isolates on 6 B. npaus lines

culVariableMeanStd DevStd Error

DC21321les7d16.66.63.3

DC21321les14d3115.57.8

DC21321les21d42.722.111.1

DC21321%S+C21.815.47.7

DC21321AUDPCd30.414.97.5

DC21AB19les7d56.214.27.1

DC21AB19les14d93.14.82.4

DC21AB19les21d159.319.910

DC21AB19%S+C72.88.84.4

DC21AB19AUDPCd100.59.34.7

DC21AB29les7d64.417.48.7

DC21AB29les14d11924.912.5

DC21AB29les21d212.927.413.7

DC21AB29%S+C84.24.22.1

DC21AB29AUDPCd128.816.68.3

DC21AB3les7d42.423.811.9

DC21AB3les14d79.545.622.8

DC21AB3les21d110.553.726.8

DC21AB3%S+C59.535.317.7


DC21AB7les7d70.20.1

DC21AB7les14d8.621

DC21AB7les21d10.221

DC21AB7%S+C000


DC21MB21les7d41.713.26.6

DC21MB21les14d84.729.414.7

DC21MB21les21d124.245.722.8

DC21MB21%S+C56.824.312.2

DC21MB21AUDPCd83.829.214.6

DC21MB35les7d20.99.14.5

DC21MB35les14d39.821.310.6

DC21MB35les21d63.343.621.8

DC21MB35%S+C32.919.39.7

DC21MB35AUDPCd4123.811.9

DC21MB51les7d33.311.25.6

DC21MB51les14d68.532.916.5

DC21MB51les21d101.351.125.6

DC21MB51%S+C5432.116.1

DC21MB51AUDPCd6831.815.9

DC21MB52les7d2410.35.1

DC21MB52les14d48.328.914.5

DC21MB52les21d78.463.131.5

DC21MB52%S+C31.621.911

DC21MB52AUDPCd49.732.616.3

DC21MB57les7d40.97.93.9

DC21MB57les14d71.99.34.7

DC21MB57les21d114.331.215.6

DC21MB57%S+C53.25.92.9

DC21MB57AUDPCd74.814.17.1

DC21MB61les7d3716.48.2

DC21MB61les14d80.144.922.5

DC21MB61les21d115.670.535.3

DC21MB61%S+C48.627.313.7

DC21MB61AUDPCd78.243.521.7

DC21SK14les7d296.83.4

DC21SK14les14d53.718.99.5

DC21SK14les21d87.925.512.8

DC21SK14%S+C38.515.67.8

DC21SK14AUDPCd56.116.98.4

DC21SK23les7d26.716.48.2

DC21SK23les14d44.828.914.4

DC21SK23les21d66.339.920

DC21SK23%S+C36.935.117.5

DC21SK23AUDPCd45.628.214.1

DC21SK35les7d56.315.98

DC21SK35les14d95.173.5

DC21SK35les21d185.420.910.4

DC21SK35%S+C69.817.18.5

DC21SK35AUDPCd1089.74.9

DC21SK38les7d53.112.26.1

DC21SK38les14d101.615.67.8

DC21SK38les21d158.539.119.5

DC21SK38%S+C80.413.46.7

DC21SK38AUDPCd103.718.29.1

DC21SK44les7d12.4105

DC21SK44les14d26.524.412.2

DC21SK44les21d4946.823.4

DC21SK44%S+C16.119.29.6

DC21SK44AUDPCd28.62613

DC21SK45les7d48.584

DC21SK45les14d85.520.510.3

DC21SK45les21d128.330.215.1

DC21SK45%S+C59.518.19

DC21SK45AUDPCd86.918.49.2

K22321les7d32.913.76.9

K22321les14d49.723.511.8

K22321les21d72.736.218.1

K22321%S+C62.932.116.1

K22321AUDPCd51.22412

K22AB19les7d33.412.56.3

K22AB19les14d5019.59.8

K22AB19les21d70.729.414.7

K22AB19%S+C48.318.49.2

K22AB19AUDPCd5120.110.1

K22AB29les7d344.32.1

K22AB29les14d66.61.30.6

K22AB29les21d10011.65.8

K22AB29%S+C59.163

K22AB29AUDPCd66.82.91.4

K22AB3les7d42.317.48.7

K22AB3les14d72.727.513.7

K22AB3les21d102.939.319.7

K22AB3%S+C62.330.815.4

K22AB3AUDPCd72.727.613.8

K22AB7les7d7.20.90.4

K22AB7les14d9.72.31.2

K22AB7les21d10.82.61.3

K22AB7%S+C000

K22AB7AUDPCd9.41.91

K22MB21les7d24.35.22.6

K22MB21les14d36.810.25.1

K22MB21les21d4616.38.1

K22MB21%S+C35.110.25.1

K22MB21AUDPCd3610.25.1

K22MB35les7d15.24.32.2

K22MB35les14d23.411.45.7

K22MB35les21d27.515.67.8

K22MB35%S+C14.614.87.4

K22MB35AUDPCd22.310.65.3

K22MB51les7d40.713.56.7

K22MB51les14d63.727.313.6

K22MB51les21d93.448.724.4

K22MB51%S+C61.323.611.8

K22MB51AUDPCd65.429.114.6

K22MB52les7d2620.110

K22MB52les14d39.434.617.3

K22MB52les21d58.16130.5

K22MB52%S+C35.122.111.1

K22MB52AUDPCd40.737.518.8

K22MB57les7d27.88.34.2

K22MB57les14d47.313.46.7

K22MB57les21d63.821.710.8

K22MB57%S+C50.115.37.6

K22MB57AUDPCd46.514.17.1

K22MB61les7d22.68.74.3

K22MB61les14d37.818.29.1

K22MB61les21d4824.912.4

K22MB61%S+C23.819.49.7

K22MB61AUDPCd36.517.48.7

K22SK14les7d177.43.7

K22SK14les14d3416.58.3

K22SK14les21d42.624.512.3

K22SK14%S+C26.222.211.1

K22SK14AUDPCd31.9168

K22SK23les7d32.617.78.8

K22SK23les14d59.940.820.4

K22SK23les21d88.259.929.9

K22SK23%S+C51.833.917

K22SK23AUDPCd60.23919.5

K22SK35les7d55.129.114.6

K22SK35les14d80.13417

K22SK35les21d125.854.227.1

K22SK35%S+C61.42211

K22SK35AUDPCd85.337.618.8

K22SK38les7d54.56.53.2

K22SK38les14d89.314.87.4

K22SK38les21d123.323.611.8

K22SK38%S+C76.411.45.7

K22SK38AUDPCd89.213.86.9

K22SK44les7d12.27.83.9

K22SK44les14d25.219.89.9

K22SK44les21d33.929.814.9

K22SK44%S+C20.82512.5

K22SK44AUDPCd24.119.29.6

K22SK45les7d34.615.37.7

K22SK45les14d58.219.89.9

K22SK45les21d78.926.813.4

K22SK45%S+C46.124.912.5

K22SK45AUDPCd57.52010

PAK54321les7d13.74.52.3

PAK54321les14d16.26.53.3

PAK54321les21d17.73.81.9

PAK54321%S+C2.42.81.4

PAK54321AUDPCd15.95.32.7

PAK54AB19les7d34.17.83.9

PAK54AB19les14d58.52713.5

PAK54AB19les21d80.150.625.3

PAK54AB19%S+C482613

PAK54AB19AUDPCd57.827.813.9

PAK54AB29les7d38.411.25.6

PAK54AB29les14d76.216.28.1

PAK54AB29les21d106.22110.5

PAK54AB29%S+C55.716.68.3


PAK54AB3les7d21.713.36.6

PAK54AB3les14d34.321.610.8

PAK54AB3les21d44.83015

PAK54AB3%S+C20.92311.5


PAK54AB7les7d9.91.30.7

PAK54AB7les14d11.81.10.6

PAK54AB7les21d12.21.40.7

PAK54AB7%S+C2.44.82.4


PAK54MB21les7d18.373.5

PAK54MB21les14d28.520.910.4

PAK54MB21les21d34.43115.5

PAK54MB21%S+C7.214.37.2

PAK54MB21AUDPCd27.419.910

PAK54MB35les7d16.66.33.1

PAK54MB35les14d21.494.5

PAK54MB35les21d22.494.5

PAK54MB35%S+C6.47.43.7

PAK54MB35AUDPCd20.48.34.2

PAK54MB51les7d28.95.32.6

PAK54MB51les14d47.616.28.1

PAK54MB51les21d6732.816.4

PAK54MB51%S+C46.224.912.4


PAK54MB52les7d22.78.84.4

PAK54MB52les14d32.518.39.2

PAK54MB52les21d46.43919.5

PAK54MB52%S+C23.124.312.1


PAK54MB57les7d13.83.81.9

PAK54MB57les14d15.35.42.7

PAK54MB57les21d15.94.52.2

PAK54MB57%S+C67.13.6


PAK54MB61les7d24.33.81.9

PAK54MB61les14d41.62.81.4

PAK54MB61les21d59.58.74.3

PAK54MB61%S+C39.918.89.4


PAK54SK14les7d18.25.92.9

PAK54SK14les14d23.910.25.1

PAK54SK14les21d31.418.29.1

PAK54SK14%S+C15.5189

PAK54SK14AUDPCd24.4115.5

PAK54SK23les7d23.213.86.9

PAK54SK23les14d35.125.612.8

PAK54SK23les21d46.728.814.4

PAK54SK23%S+C19.113.56.7


PAK54SK35les7d2112.76.4

PAK54SK35les14d36.326.213.1

PAK54SK35les21d45.736.918.4

PAK54SK35%S+C18.521.910.9

PAK54SK35AUDPCd34.825.212.6

PAK54SK38les7d28.210.35.1

PAK54SK38les14d45.325.312.6

PAK54SK38les21d62.344.922.4

PAK54SK38%S+C35.727.613.8


PAK54SK44les7d21.211.55.7

PAK54SK44les14d33.421.210.6

PAK54SK44les21d44.235.717.8

PAK54SK44%S+C7.214.37.1


PAK54SK45les7d34.38.84.4

PAK54SK45les14d57.616.78.4

PAK54SK45les21d84.937.318.6

PAK54SK45%S+C47.624.312.1


PAK93321les7d17.65.52.8

PAK93321les14d26.311.15.5

PAK93321les21d37.120.810.4

PAK93321%S+C10.913.86.9

PAK93321AUDPCd26.811.65.8

PAK93AB19les7d55105

PAK93AB19les14d100.116.98.5

PAK93AB19les21d137.926.213.1

PAK93AB19%S+C73.228.714.3


PAK93AB29les7d44.97.13.5

PAK93AB29les14d94.416.28.1

PAK93AB29les21d126.130.615.3

PAK93AB29%S+C63.410.95.5

PAK93AB29AUDPCd9017.28.6

PAK93AB3les7d17.7105

PAK93AB3les14d29.917.48.7

PAK93AB3les21d38.42311.5

PAK93AB3%S+C9.711.55.7


PAK93AB7les7d13.84.12.1

PAK93AB7les14d165.52.7

PAK93AB7les21d16.65.22.6

PAK93AB7%S+C000


PAK93MB21les7d43.17.53.7

PAK93MB21les14d88.19.24.6

PAK93MB21les21d121.716.58.3

PAK93MB21%S+C63.123.111.6


PAK93MB35les7d31.27.63.8

PAK93MB35les14d5018.39.2

PAK93MB35les21d67.83316.5

PAK93MB35%S+C18.913.36.6


PAK93MB51les7d33.311.96

PAK93MB51les14d59.322.211.1

PAK93MB51les21d79.938.719.4

PAK93MB51%S+C37.127.613.8


PAK93MB52les7d32.210.55.2

PAK93MB52les14d55.419.59.8

PAK93MB52les21d71.931.215.6

PAK93MB52%S+C46.420.410.2


PAK93MB57les7d25.44.62.3

PAK93MB57les14d37.28.64.3

PAK93MB57les21d47.210.55.2

PAK93MB57%S+C28.620.410.2

PAK93MB57AUDPCd36.784

PAK93MB61les7d33.212.76.4

PAK93MB61les14d57.129.314.7

PAK93MB61les21d75.847.623.8

PAK93MB61%S+C43.335.817.9


PAK93SK14les7d30.89.34.6

PAK93SK14les14d49.617.48.7

PAK93SK14les21d70.430.815.4

PAK93SK14%S+C44.926.413.2


PAK93SK23les7d38.69.24.6

PAK93SK23les14d60.517.68.8

PAK93SK23les21d7827.113.6

PAK93SK23%S+C41.226.513.2


PAK93SK35les7d24.610.95.5

PAK93SK35les14d48.725.112.5

PAK93SK35les21d57.124.612.3

PAK93SK35%S+C25.426.713.4


PAK93SK38les7d34.99.34.7

PAK93SK38les14d56.721.410.7

PAK93SK38les21d78.630.915.4

PAK93SK38%S+C41.622.311.2


PAK93SK44les7d20.29.14.6

PAK93SK44les14d40.820.810.4

PAK93SK44les21d6337.318.6

PAK93SK44%S+C21.123.411.7


PAK93SK45les7d42.82.61.3

PAK93SK45les14d79.77.53.7

PAK93SK45les21d103.65.52.7

PAK93SK45%S+C69.819.910


Tanto321les7d26.811.25.6

Tanto321les14d46.923.111.6

Tanto321les21d59.736.818.4

Tanto321%S+C22.819.99.9

Tanto321AUDPCd45.123.411.7

TantoAB19les7d68.514.77.4


TantoAB19les21d146189

TantoAB19%S+C72.614.77.4

TantoAB19AUDPCd106.7105


TantoAB29les14d109.83417

TantoAB29les21d15352.126.1

TantoAB29%S+C77.823.711.9

TantoAB29AUDPCd108.233.816.9




TantoAB3%S+C37.330.415.2




TantoAB7les21d195.92.9

TantoAB7%S+C8.57.33.7


TantoMB21les7d54.814.87.4


TantoMB21les21d120.636.718.3

TantoMB21%S+C69.920.210.1

TantoMB21AUDPCd92.125.712.8




TantoMB35%S+C45.330.715.4

TantoMB35AUDPCd65.92211




TantoMB51%S+C87.56.53.3



TantoMB52les14d89.83517.5

TantoMB52les21d118.246.223.1

TantoMB52%S+C60.32311.5

TantoMB52AUDPCd85.331.916




TantoMB57%S+C50.525.412.7


TantoMB61les7d50.6147



TantoMB61%S+C68.523.111.6


TantoSK14les7d43.6168

TantoSK14les14d74.631.415.7

TantoSK14les21d107.552.826.4

TantoSK14%S+C64.536.818.4

TantoSK14AUDPCd75.132.816.4

TantoSK23les7d45.216.18



TantoSK23%S+C58.422.111





TantoSK35%S+C36.332.616.3




TantoSK38les21d134.329.814.9

TantoSK38%S+C7518.49.2





TantoSK44%S+C22.118.69.3

TantoSK44AUDPCd482110.5



TantoSK45les21d140.844.522.2

TantoSK45%S+C74.434.417.2


Topas321les7d27.44.62.3

Topas321les14d4111.45.7

Topas321les21d54.217.48.7

Topas321%S+C24.416.38.1

Topas321AUDPCd40.910.55.2

TopasAB19les7d61.68.34.2


TopasAB19les21d208.859.529.8

TopasAB19%S+C92.911.35.7

TopasAB19AUDPCd130.420.310.2



TopasAB29les21d209.422.111.1

TopasAB29%S+C98.62.81.4


TopasAB3les7d37.72211



TopasAB3%S+C51.331.916

TopasAB3AUDPCd743919.5

TopasAB7les7d21.811.96



TopasAB7%S+C16.917.99


TopasMB21les7d5612.76.3

TopasMB21les14d110.127.413.7

TopasMB21les21d1643919.5

TopasMB21%S+C83.110.45.2

TopasMB21AUDPCd110.126.513.2

TopasMB35les7d46.64.72.4


TopasMB35les21d145.361.330.6

TopasMB35%S+C63.516.48.2




TopasMB51les21d223.441.520.7

TopasMB51%S+C10000




TopasMB52les21d182.224.212.1

TopasMB52%S+C89.79.94.9





TopasMB57%S+C7717.38.6

TopasMB57AUDPCd10013.66.8




TopasMB61%S+C94.963


TopasSK14les7d58.89.34.6



TopasSK14%S+C97.552.5

TopasSK14AUDPCd127.84.32.2



TopasSK23les21d179.643.621.8

TopasSK23%S+C82.119.69.8



TopasSK35les14d81.5178.5

TopasSK35les21d132.125.912.9


TopasSK35AUDPCd83.1168


TopasSK38les14d11117.28.6

TopasSK38les21d191.224.612.3





TopasSK44les21d141.224.712.4

TopasSK44%S+C59.611.35.7

TopasSK44AUDPCd86.4126


TopasSK45les14d1242010

TopasSK45les21d203.833.116.6

TopasSK45%S+C98.82.41.2


7dai

B. napusIsolateles7dStd DevStd Error

DC2132116.66.63.3

AB1956.214.27.1

AB2964.417.48.7

AB342.423.811.9

AB770.20.1

MB2141.713.26.6

MB3520.99.14.5

MB5133.311.25.6

MB522410.35.1

MB5740.97.93.9

MB613716.48.2

SK14296.83.4

SK2326.716.48.2

SK3556.315.98

SK3853.112.26.1

SK4412.4105

SK4548.584

K2232132.913.76.9

AB1933.412.56.3

AB29344.32.1

AB342.317.48.7

AB77.20.90.4

MB2124.35.22.6

MB3515.24.32.2

MB5140.713.56.7

MB522620.110

MB5727.88.34.2

MB6122.68.74.3

SK14177.43.7

SK2332.617.78.8

SK3555.129.114.6

SK3854.56.53.2

SK4412.27.83.9

SK4534.615.37.7

PAK5432113.74.52.3

AB1934.17.83.9

AB2938.411.25.6

AB321.713.36.6

AB79.91.30.7

MB2118.373.5

MB3516.66.33.1

MB5128.95.32.6

MB5222.78.84.4

MB5713.83.81.9

MB6124.33.81.9

SK1418.25.92.9

SK2323.213.86.9

SK352112.76.4

SK3828.210.35.1

SK4421.211.55.7

SK4534.38.84.4

PAK9332117.65.52.8

AB1955105

AB2944.97.13.5

AB317.7105

AB713.84.12.1

MB2143.17.53.7

MB3531.27.63.8

MB5133.311.96

MB5232.210.55.2

MB5725.44.62.3

MB6133.212.76.4

SK1430.89.34.6

SK2338.69.24.6

SK3524.610.95.5

SK3834.99.34.7

SK4420.29.14.6

SK4542.82.61.3

Tanto32126.811.25.6

AB1968.514.77.4

AB2960.416.88.4

AB333.519.99.9

AB712.35.12.6

MB2154.814.87.4

MB3536.711.15.5

MB5155.21.60.8

MB5243.712.56.2

MB5738.710.65.3

MB6150.6147

SK1443.6168

SK2345.216.18

SK3529.72412

SK3850.712.36.1

SK4423.410.45.2

SK4554.918.99.5

Topas32127.44.62.3

AB1961.68.34.2

AB2968.810.95.5

AB337.72211

AB721.811.96

MB215612.76.3

MB3546.64.72.4

MB5166.213.36.6

MB52562.91.4

MB5747.98.84.4

MB6161.212.16

SK1458.89.34.6

SK2347.813.66.8

SK3537.14.32.1

SK3857.79.84.9

SK4440.76.63.3

SK4559.37.63.8

14dai


DC213213115.57.8

AB1993.14.82.4

AB2911924.912.5

AB379.545.622.8

AB78.621

MB2184.729.414.7

MB3539.821.310.6

MB5168.532.916.5

MB5248.328.914.5

MB5771.99.34.7

MB6180.144.922.5

SK1453.718.99.5

SK2344.828.914.4

SK3595.173.5

SK38101.615.67.8

SK4426.524.412.2

SK4585.520.510.3

K2232149.723.511.8

AB195019.59.8

AB2966.61.30.6

AB372.727.513.7

AB79.72.31.2

MB2136.810.25.1

MB3523.411.45.7

MB5163.727.313.6

MB5239.434.617.3

MB5747.313.46.7

MB6137.818.29.1

SK143416.58.3

SK2359.940.820.4

SK3580.13417

SK3889.314.87.4

SK4425.219.89.9

SK4558.219.89.9

PAK5432116.26.53.3

AB1958.52713.5

AB2976.216.28.1

AB334.321.610.8

AB711.81.10.6

MB2128.520.910.4

MB3521.494.5

MB5147.616.28.1

MB5232.518.39.2

MB5715.35.42.7

MB6141.62.81.4

SK1423.910.25.1

SK2335.125.612.8

SK3536.326.213.1

SK3845.325.312.6

SK4433.421.210.6

SK4557.616.78.4

PAK9332126.311.15.5

AB19100.116.98.5

AB2994.416.28.1

AB329.917.48.7

AB7165.52.7

MB2188.19.24.6

MB355018.39.2

MB5159.322.211.1

MB5255.419.59.8

MB5737.28.64.3

MB6157.129.314.7

SK1449.617.48.7

SK2360.517.68.8

SK3548.725.112.5

SK3856.721.410.7

SK4440.820.810.4

SK4579.77.53.7

Tanto32146.923.111.6

AB19106.16.33.1

AB29109.83417

AB361.632.816.4

AB714.56.43.2

MB2196.626.213.1

MB3567.522.611.3

MB51110.76.83.4

MB5289.83517.5

MB5764.7115.5

MB619218.69.3

SK1474.631.415.7

SK2383.534.217.1

SK35564321.5

SK3891.922.611.3

SK4449.120.910.5

SK4599.630.415.2

Topas3214111.45.7

AB19125.513.26.6

AB29128.815.57.7

AB374.138.719.3

AB73218.29.1

MB21110.127.413.7

MB3576.611.55.8

MB5113620.610.3

MB52109.810.45.2

MB5796.114.67.3

MB61115.910.25.1

SK14118.94.32.2

SK2396.521.510.8

SK3581.5178.5

SK3811117.28.6

SK4481.911.85.9

SK451242010

21dai


DC2132142.722.111.1

AB19159.319.910

AB29212.927.413.7

AB3110.553.726.8

AB710.221

MB21124.245.722.8

MB3563.343.621.8

MB51101.351.125.6

MB5278.463.131.5

MB57114.331.215.6

MB61115.670.535.3

SK1487.925.512.8

SK2366.339.920

SK35185.420.910.4

SK38158.539.119.5

SK444946.823.4

SK45128.330.215.1

K2232172.736.218.1

AB1970.729.414.7

AB2910011.65.8

AB3102.939.319.7

AB710.82.61.3

MB214616.38.1

MB3527.515.67.8

MB5193.448.724.4

MB5258.16130.5

MB5763.821.710.8

MB614824.912.4

SK1442.624.512.3

SK2388.259.929.9

SK35125.854.227.1

SK38123.323.611.8

SK4433.929.814.9

SK4578.926.813.4

PAK5432117.73.81.9

AB1980.150.625.3

AB29106.22110.5

AB344.83015

AB712.21.40.7

MB2134.43115.5

MB3522.494.5

MB516732.816.4

MB5246.43919.5

MB5715.94.52.2

MB6159.58.74.3

SK1431.418.29.1

SK2346.728.814.4

SK3545.736.918.4

SK3862.344.922.4

SK4444.235.717.8

SK4584.937.318.6

PAK9332137.120.810.4

AB19137.926.213.1

AB29126.130.615.3

AB338.42311.5

AB716.65.22.6

MB21121.716.58.3

MB3567.83316.5

MB5179.938.719.4

MB5271.931.215.6

MB5747.210.55.2

MB6175.847.623.8

SK1470.430.815.4

SK237827.113.6

SK3557.124.612.3

SK3878.630.915.4

SK446337.318.6

SK45103.65.52.7

Tanto32159.736.818.4

AB19146189

AB2915352.126.1

AB385.941.520.7

AB7195.92.9

MB21120.636.718.3

MB3592.135.217.6

MB51156.819.69.8

MB52118.246.223.1

MB5793.426.513.2

MB61123.415.57.7

SK14107.552.826.4

SK2312367.733.9

SK3571.856.528.2

SK38134.329.814.9

SK4470.631.916

SK45140.844.522.2

Topas32154.217.48.7

AB19208.859.529.8

AB29209.422.111.1

AB3109.856.928.5

AB735.32110.5

MB211643919.5

MB35145.361.330.6

MB51223.441.520.7

MB52182.224.212.1

MB5716019.89.9

MB61185.514.87.4

SK14214.810.85.4

SK23179.643.621.8

SK35132.125.912.9

SK38191.224.612.3

SK44141.224.712.4

SK45203.833.116.6

AUDPC

B. napusIsolateAUDPCdStd DevStd Error

DC21AB78.61.50.7

MB5774.814.17.1

32130.414.97.5

MB354123.811.9

SK1456.116.98.4

MB2183.829.214.6

SK4428.62613

MB5249.732.616.3

AB377.940.520.3

SK351089.74.9

SK2345.628.214.1

MB6178.243.521.7

SK38103.718.29.1

MB516831.815.9

AB19100.59.34.7

SK4586.918.49.2

AB29128.816.68.3

K22AB79.41.91

MB5746.514.17.1

32151.22412

MB3522.310.65.3

SK1431.9168

MB213610.25.1

SK4424.119.29.6

MB5240.737.518.8

AB372.727.613.8

SK3585.337.618.8

SK2360.23919.5

MB6136.517.48.7

SK3889.213.86.9

MB5165.429.114.6

AB195120.110.1

SK4557.52010

AB2966.82.91.4

PAK54AB711.41.30.6

MB5715.14.82.49

32115.95.32.7

MB3520.48.34.2

SK1424.4115.5

MB2127.419.910

SK443322.211.1

MB5233.520.910.4

AB333.821.510.8

SK3534.825.212.6

SK233523.411.7

MB6141.73.21.6

SK3845.326.213.1

MB5147.817.48.7

AB1957.827.813.9

SK4558.719.69.8

AB2974.215.77.8

PAK93AB715.652.5

MB5736.784

32126.811.65.8

MB3549.818.79.3

SK1450.118.69.3

MB2185.28.44.2

SK4441.221.911

MB5253.719.99.9

AB32916.78.4

SK3544.821.310.6

SK2359.417.38.7

MB6155.829.414.7

SK3856.820.710.3

MB5157.923.111.6

AB1998.314.57.2

SK4576.44.72.3

AB299017.28.6

TantoAB715.15.52.8

MB5765.414.67.3

32145.123.411.7

MB3565.92211

SK1475.132.816.4

MB2192.125.712.8

SK44482110.5

MB5285.331.916

AB360.731.715.8

SK3553.441.620.8

SK2383.837.818.9

MB6189.516.38.2

SK3892.221.710.9

MB51108.48.54.2

AB19106.7105

SK4598.730.715.4

AB29108.233.816.9

TopasAB730.317.38.7

MB5710013.66.8

32140.910.55.2

MB3586.221.110.5

SK14127.84.32.2

MB21110.126.513.2

SK4486.4126

MB52114.511.85.9

AB3743919.5

SK3583.1168

SK23105.124.812.4

MB61119.611.75.8

SK38117.716.18.1

MB51140.423.411.7

AB19130.420.310.2

SK45127.818.99.5

AB29133.914.77.4

AUDPC

% s+c

Graph for Richard Oliver

AUDPC PAK54 PAK93

AUDPC 5 lines

Graph 21dai

B. napusIsolate% s+cStd DevStd Error

DC2132121.815.47.7

AB1972.88.84.4

AB2984.24.22.1

AB359.535.317.7

AB7000

MB2156.824.312.2

MB3532.919.39.7

MB515432.116.1

MB5231.621.911

MB5753.25.92.9

MB6148.627.313.7

SK1438.515.67.8

SK2336.935.117.5

SK3569.817.18.5

SK3880.413.46.7

SK4416.119.29.6

SK4559.518.19

K2232162.932.116.1

AB1948.318.49.2

AB2959.163

AB362.330.815.4

AB7000

MB2135.110.25.1

MB3514.614.87.4

MB5161.323.611.8

MB5235.122.111.1

MB5750.115.37.6

MB6123.819.49.7

SK1426.222.211.1

SK2351.833.917

SK3561.42211

SK3876.411.45.7

SK4420.82512.5

SK4546.124.912.5

PAK543212.42.81.4

AB19482613

AB2955.716.68.3

AB320.92311.5

AB72.44.82.4

MB217.214.37.2

MB356.47.43.7

MB5146.224.912.4

MB5223.124.312.1

MB5767.13.6

MB6139.918.89.4

SK1415.5189

SK2319.113.56.7

SK3518.521.910.9

SK3835.727.613.8

SK447.214.37.1

SK4547.624.312.1

PAK9332110.913.86.9

AB1973.228.714.3

AB2963.410.95.5

AB39.711.55.7

AB7000

MB2163.123.111.6

MB3518.913.36.6

MB5137.127.613.8

MB5246.420.410.2

MB5728.620.410.2

MB6143.335.817.9

SK1444.926.413.2

SK2341.226.513.2

SK3525.426.713.4

SK3841.622.311.2

SK4421.123.411.7

SK4569.819.910

Tanto32122.819.99.9

AB1972.614.77.4

AB2977.823.711.9

AB337.330.415.2

AB78.57.33.7

MB2169.920.210.1

MB3545.330.715.4

MB5187.56.53.3

MB5260.32311.5

MB5750.525.412.7

MB6168.523.111.6

SK1464.536.818.4

SK2358.422.111

SK3536.332.616.3

SK387518.49.2

SK4422.118.69.3

SK4574.434.417.2

Topas32124.416.38.1

AB1992.911.35.7

AB2998.62.81.4

AB351.331.916

AB716.917.99

MB2183.110.45.2

MB3563.516.48.2

MB5110000

MB5289.79.94.9

MB577717.38.6

MB6194.963

SK1497.552.5

SK2382.119.69.8

SK3563.2199.5

SK3893.752.5

SK4459.611.35.7

SK4598.82.41.2

IsolatePAK54PAK93K22DC21TantoTopassumStd Dev Pak54Std Err PAK54Std Dev PAK93Std Err PAK93Std Dev K22Std Err K22Std Dev DC21Std Error DC21Std Dev TantoStd Err TantoStd Dev TopasStd Err Topas

AB711.415.69.48.615.130.390.41.30.652.51.911.50.75.52.817.38.7

32115.926.851.230.445.140.9210.35.32.711.65.8241214.97.523.411.710.55.2

SK4433.041.224.128.648.086.4261.322.211.121.91119.29.626132110.5126

AB333.829.072.777.960.774.0348.121.510.816.78.427.613.840.520.331.715.83919.5

MB5715.136.746.574.865.4100.0338.54.82.48414.17.114.17.114.67.313.66.8

MB3520.449.822.341.065.986.2285.68.34.218.79.310.65.323.811.9221121.110.5

SK1424.450.131.956.175.1127.8365.4115.518.69.316816.98.432.816.44.32.2

SK3534.844.885.3108.053.483.1409.425.212.621.310.637.618.89.74.941.620.8168

SK2335.059.460.245.683.8105.1389.123.411.717.38.73919.528.214.137.818.924.812.4

MB2127.485.236.083.892.1110.1434.619.9108.44.210.25.129.214.625.712.826.513.2

MB5233.553.740.749.785.3114.5377.420.910.419.99.937.518.832.616.331.91611.85.9

MB6141.755.836.578.289.5119.6421.33.21.629.414.717.48.743.521.716.38.211.75.8

SK3845.356.889.2103.792.2117.7504.926.213.120.710.313.86.918.29.121.710.916.18.1

MB5147.857.965.468.0108.4140.4487.917.48.723.111.629.114.631.815.98.54.223.411.7

AB1957.898.351.0100.5106.7130.4544.727.813.914.57.220.110.19.34.710520.310.2

SK4558.776.457.586.998.7127.8506.019.69.84.72.3201018.49.230.715.418.99.5

AB2974.290.066.8128.8108.2133.9601.915.77.817.28.62.91.416.68.333.816.914.77.4

sum610.2927.5846.71170.61293.61728.2

321 = This isolate has been used to phenotype eight B. napus mapping populations and is one of the least aggressive isolates

SK35 = This isolate was genetically the most dissimilar of all 129 isolates after genotyping with 47 SSR markers

MB21 = This isolate has differential reactions on three B. napus lines (similar to AB19)

MB52 = This isolate is more aggressive than 321 and will be used for future screening of B. napus mapping populations

AB29 = This isolate is the most aggressive of all 17 isolates

&L&D&R&Z&F&A

2.72.518.7

11.15.8125.2

10.8119.66

2.48.413.819.5

4.247.16.8

5.59.35.310.5

12.69.382.2

11.710.618.88

108.719.512.4

10.44.25.113.2

1.69.918.85.9

13.114.78.75.8

8.710.36.98.1

13.911.614.611.7

9.87.210.110.2

7.82.3109.5

8.61.47.4

PAK54

PAK93

K22

Topas

IsolatePAK54PAK93TopasStd Err Pak54Std Err. PAK93Std Err. Topas

AB711.415.630.30.62.58.7

32115.926.840.92.75.85.2

MB5715.136.7100.02.446.8

MB3520.449.886.24.29.310.5

SK1424.450.1127.85.59.32.2

MB2127.485.2110.1104.213.2

AB333.829.074.010.88.419.5

SK4433.041.286.411.1116

SK3534.844.883.112.610.68

MB5233.553.7114.510.49.95.9

SK2335.059.4105.111.78.712.4

MB6141.755.8119.61.614.75.8

SK3845.356.8117.713.110.38.1

MB5147.857.9140.48.711.611.7

SK4558.776.4127.89.82.39.5

AB1957.898.3130.413.97.210.2

AB2974.290.0133.97.88.67.4

&L&D&R&Z&F&A

0.62.58.7

2.75.85.2

2.446.8

4.29.310.5

5.59.32.2

104.213.2

10.88.419.5

11.1116

12.610.68

10.49.95.9

11.78.712.4

1.614.75.8

13.110.38.1

8.711.611.7

9.82.39.5

13.97.210.2

7.88.67.4

PAK54

PAK93

Topas

IsolatePAK54PAK93K22DC21TantoTopassumStd Dev Pak54Std Err PAK54Std Dev PAK93Std Err PAK93Std Dev K22Std Err K22Std Dev DC21Std Error DC21Std Dev TantoStd Err TantoStd Dev TopasStd Err Topas

AB711.415.69.48.615.130.390.41.30.65.02.51.91.01.50.75.52.817.38.7

32115.926.851.230.445.140.9210.35.32.711.65.824.012.014.97.523.411.710.55.2

SK4433.041.224.128.648.086.4261.322.211.121.911.019.29.626.013.021.010.512.06.0

AB333.829.072.777.960.774.0348.121.510.816.78.427.613.840.520.331.715.839.019.5

MB5715.136.746.574.865.4100.0338.54.82.48.04.014.17.114.17.114.67.313.66.8

MB3520.449.822.341.065.986.2285.68.34.218.79.310.65.323.811.922.011.021.110.5

SK1424.450.131.956.175.1127.8365.411.05.518.69.316.08.016.98.432.816.44.32.2

SK3534.844.885.3108.053.483.1409.425.212.621.310.637.618.89.74.941.620.816.08.0

SK2335.059.460.245.683.8105.1389.123.411.717.38.739.019.528.214.137.818.924.812.4

MB2127.485.236.083.892.1110.1434.619.910.08.44.210.25.129.214.625.712.826.513.2

MB5233.553.740.749.785.3114.5377.420.910.419.99.937.518.832.616.331.916.011.85.9

MB6141.755.836.578.289.5119.6421.33.21.629.414.717.48.743.521.716.38.211.75.8

SK3845.356.889.2103.792.2117.7504.926.213.120.710.313.86.918.29.121.710.916.18.1

MB5147.857.965.468.0108.4140.4487.917.48.723.111.629.114.631.815.98.54.223.411.7

AB1957.898.351.0100.5106.7130.4544.727.813.914.57.220.110.19.34.710.05.020.310.2

SK4558.776.457.586.998.7127.8506.019.69.84.72.320.010.018.49.230.715.418.99.5

AB2974.290.066.8128.8108.2133.9601.915.77.817.28.62.91.416.68.333.816.914.77.4

sum610.2927.5846.71170.61293.61728.2

321 = This isolate has been used to phenotype eight B. napus mapping populations and is one of the least aggressive isolates

SK35 = This isolate was genetically the most dissimilar of all 129 isolates after genotyping with 47 SSR markers

MB21 = This isolate has differential reactions on three B. napus lines (similar to AB19)

MB52 = This isolate is more aggressive than 321 and will be used for future screening of B. napus mapping populations

AB29 = This isolate is the most aggressive of all 17 isolates

Topas

Tanto

DC21

K22

PAK93

PAK54


Area under the disease progress curve

Isolateles21dles21dles21dles21dles21dles21dStd ErrorStd ErrorStd ErrorStd ErrorStd ErrorStd ErrorStd DevStd DevStd DevStd DevStd DevStd Dev

TopasTantoDC21K22PAK93PAK54avg Topas and PAK54 for sortingTopasTantoDC21K22PAK93PAK54TopasTantoDC21K22PAK93PAK54

AB735.319.010.210.816.612.223.810.52.91.01.32.60.721.05.92.02.65.21.4

32154.259.742.772.737.117.736.08.718.411.118.110.41.917.436.822.136.220.83.8

AB3109.885.9110.5102.938.444.877.328.520.726.819.711.515.056.941.553.739.323.030.0

MB35145.392.163.327.567.822.483.930.617.621.87.816.54.561.335.243.615.633.09.0

MB57160.093.4114.363.847.215.988.09.913.215.610.85.22.219.826.531.221.710.54.5

SK35132.171.8185.4125.857.145.788.912.928.210.427.112.318.425.956.520.954.224.636.9

SK44141.270.649.033.963.044.292.712.416.023.414.918.617.824.731.946.829.837.335.7

MB21164.0120.6124.246.0121.734.499.219.518.322.88.18.315.539.036.745.716.316.531.0

SK23179.6123.066.388.278.046.7113.221.833.920.029.913.614.443.667.739.959.927.128.8

MB52182.2118.278.458.171.946.4114.312.123.131.530.515.619.524.246.263.161.031.239.0

MB61185.5123.4115.648.075.859.5122.57.47.735.312.423.84.314.815.570.524.947.68.7

SK14214.8107.587.942.670.431.4123.15.426.412.812.315.49.110.852.825.524.530.818.2

MB51223.4156.8101.393.479.967.0145.220.79.825.624.419.416.441.519.651.148.738.732.8

SK38191.2134.3158.5123.378.662.3126.812.314.919.511.815.422.424.629.839.123.630.944.9

SK45203.8140.8128.378.9103.684.9144.416.622.215.113.42.718.633.144.530.226.85.537.3

AB19208.8146.0159.370.7137.980.1144.529.89.010.014.713.125.359.518.019.929.426.250.6

AB29209.4153.0212.9100.0126.1106.2157.811.126.113.75.815.310.522.152.127.411.630.621.0

10.510.50.70.7

8.78.71.91.9

28.528.51515

30.630.64.54.5

9.99.92.22.2

12.912.918.418.4

12.412.417.817.8

19.519.515.515.5

21.821.814.414.4

12.112.119.519.5

7.47.44.34.3

5.45.49.19.1

20.720.716.416.4

12.312.322.422.4

16.616.618.618.6

29.829.825.325.3

11.111.110.510.5

Topas

Tanto

DC21

K22

PAK93

PAK54


Stem lesion length 21 days after inoculation, mm

Genes Function in host resistanceReduction in

sclerotinia severity

Hevein, lectin like 40-50%

Lectin, concanavalin 20-50%

Lectin, curculin 40%

AnnexinTrafficking and organization of vesicles, exocytosis, endocytosis, calcium ion channel formation

30-40%

WRKY33 Regulation of gene transcription 20-30%

O-methyl transferase Glucosinolate pathway

Small inconsistant effect

PGIP Inhibitor of fungal polygalacturonase No effect

Pathogen recognition and binding

• Genes were cloned from Zhongyou 821 (China) and transformed into a susceptible B. napus line.

• The CaMV 35S promotor ensures the genes are constitutively expressed in all plant tissues all the time.

We have identified defense genes that reduce sclerotinia severity - a major step

for potential use in GM canola

QTLs

A-genomeC-genomeResistance loci







cloned genes





Lectin, curculin40%





Sheet3

Defense genes that prevent fungal penetration of the plant cell, PEN1, PEN2 and PEN3 worthy

of investigation in canola

Fungal spore

Plant cell wall

Plant cell

Jeff Ellis, Plant Cell 2006,18:523-528

Studied in several plant species such as the model plant Arabidopsis and soybean

Does constitutive expression of PEN1, PEN2 and PEN3 in canola reduce sclerotinia severity?

PEN1-10P3T1 generation

Transformation of B. napus DH12075

MethodPEN1 cloned from resistant PAK54 Susceptible B. napus DH12075 transformed with PEN1 under the control of CaM35S gene promotor

ResultsA single PEN1 gene copy was inserted in the B. napus genome The particular PEN1 allele was very low in DH12075 Transformants expressed PEN1 0.75 – 2.8 relative to DH12075

Next stepClone and transform with PEN2 and PEN3Phenotype PEN1, PEN2 and PEN3 transformants for sclerotinia resistance in the T2 generation

Chart1

PEN1-10P30.09301572170.0930157217

PEN1-13P10.01506599710.0150659971

PEN1-20P10.06097854860.0609785486

PEN1-20P20.29760507180.2976050718

PEN1-25P20.12977161330.1297716133

PEN1-10P40.09815708770.0981570877

PEN1-15P20.02684564630.0268456463

DH12075wt0.01736572150.0173657215

NTC00

Gene expression relative to actin

PEN1 gene expression in transformed plants - T1 generation

2.9156488344

2.5162098374

2.2000020371

2.1576294767

1.9534410322

1.183279728

0.7421133293

0.0438572271

0

PAK54C3PEN1-4 expression in T1

SampleTargetCopies/20uL PAK54C3PEN1-4Copies/20uL ActinConc (copies/uL) PAK54C3PEN1-4Conc (copies/uL) ActinPositives PAK54C3PEN1-4Positives ActinNegatives PAK54C3PEN1-4Negatives ActinAcceptedDropletsRatioSDAveSample

PEN1-10P3 R1PAK54C3PEN1-4494016402478227439781175713522145003.0000.08888219412.912PEN1-10P3

PEN1-10P4 R1PAK54PEN1-432002580160129193915891334113691152801.2400.09582817441.185PEN1-10P4


PEN1-15P2 R1PAK54C3PEN1-41096152054.8767269981523214960159580.7200.02828427120.740PEN1-15P2




DH12075wt R1PAK54C3PEN1-413058006.52900325347581149658430.0220.00467582530.028DH12075wt

NTC R1PAK54C3PEN1-401.400.070117750177491775000.000NTC

PEN1-10P3 R2PAK54C3PEN1-455802000279100282310891054612280133692.790

PEN1-10P4 R2PAK54C3PEN1-432403100162155201819321368013766156981.050

PEN1-13P1 R2PAK54C3PEN1-464602560323128370415921171113823154152.520

PEN1-15P2 R2PAK54C3PEN1-41262174063.18779010841433914045151290.720

PEN1-20P1 R2PAK54C3PEN1-418608789343.912045801464515269158492.120

PEN1-20P2 R2PAK54C3PEN1-437202140186107262715681537316432180001.730

PEN1-25P2 R2PAK54C3PEN1-483004220415211184710184363519262101.970

DH12075wt R2PAK54C3PEN1-45617002.8853891275118912780.033

NTC R2PAK54C3PEN1-4No CallNo Call00000

PEN1-10P3 R3PAK54C3PEN1-4533018102.945

PEN1-10P4 R3PAK54C3PEN1-4330026101.264

PEN1-13P1 R3PAK54C3PEN1-4549021702.530

PEN1-15P2 R3PAK54C3PEN1-4117015000.780

PEN1-20P1 R3PAK54C3PEN1-418808302.265

PEN1-20P2 R3PAK54C3PEN1-4367015302.399

PEN1-25P2 R3PAK54C3PEN1-4360017102.105

DH12075wt R3PAK54C3PEN1-46922800.030

NTC R3PAK54C3PEN1-4

PAK54C3PEN1-4 expression in T1

0.08888219410.0888821941

0.09582817440.0958281744

0.01245577780.0124557778

0.02828427120.0282842712

0.05979047090.0597904709

0.30234177140.3023417714

0.13341341090.1334134109

0.00467582530.0046758253

00

Relative Expression

PAK54C3PEN1 Expression in DH12075 T1 Transgenic plants

Sheet1

SampleCopies/20uL PAK54C3PEN1-4Copies/20uL ActinConc (copies/uL) PAK54C3PEN1-4Conc (copies/uL) ActinCopy # of PEN1 in original 25uL sampleCopy # of Actin in original 25 uL sampleAve PEN1-4 conc (copies/uL)Ave Actin conc (copies/uL)RatioSampleAverage ratioSD

PEN1-10P3 R1494016402478261752050264.166666666790.83333333333.012195122PEN1-10P32.9160.093

PEN1-10P4 R13200258016012940003225162.3333333333138.16666666671.2403100775PEN1-13P12.5160.015

PEN1-13P1 R15240210026210565502625286.5113.83333333332.4952380952PEN1-20P12.2000.061

PEN1-15P2 R11096152054.8761370190058.879.33333333330.7210526316PEN1-20P22.1580.298

PEN1-20P1 R117207768638.821509709141.42.2164948454PEN1-25P21.9530.130

PEN1-20P2 R13060131015365.538251637.5174.1666666667832.3358778626PEN1-10P41.1830.098

PEN1-25P2 R1304017001528538002125249127.16666666671.7882352941PEN1-15P20.7420.027

DH12075wt R113058006.798167.524504.3166666667990.0683673469DH12075wt0.0440.017

NTC R101.40000000NTC0.0000.000

PEN1-10P3 R255802000279100697525002.79

PEN1-10P4 R232403100162155405038751.0451612903

PEN1-13P1 R264602560323128807532002.5234375

PEN1-15P2 R21262174063.1871577.521750.7252873563

PEN1-20P1 R218608789343.923251097.52.1184510251

PEN1-20P2 R237202140186107465026751.738317757

PEN1-25P2 R2830042204152111037552751.9668246445

DH12075wt R25617002.8857021250.0329411765

NTC R2No CallNo Call000

PEN1-10P3 R353301810266.590.56662.52262.52.9447513812

PEN1-10P4 R333002610165130.541253262.51.2643678161

PEN1-13P1 R354902170274.5108.56862.52712.52.5299539171

PEN1-15P2 R31170150058.5751462.518750.78

PEN1-20P1 R318808309441.523501037.52.265060241

PEN1-20P2 R336701530183.576.54587.51912.52.3986928105

PEN1-25P2 R33600171018085.545002137.52.1052631579

DH12075wt R36922803.4511486.2528500.0302631579

NTC R300000

Sheet1

0.09301572170.0930157217

0.01506599710.0150659971

0.06097854860.0609785486

0.29760507180.2976050718

0.12977161330.1297716133

0.09815708770.0981570877

0.02684564630.0268456463

0.01736572150.0173657215

00

Relative Expression

PAK54C3PEN1 Expression in DH12075 T1 Transgenic plants

PEN1-4

Actin

Conc (Copies/uL)

Comparison of Expression Levels of PAK54C3PEN1-4 and Actin in DH12075 T1 Transgenic Plants

Agriculture andAgri-Food Canada

Agriculture et Agroalimentaire Canada

• Users are canola growers and extension specialists

• Objective is to recommend a fungicide application if an economic return is likely

• Free, fast and user friendly in order to be used on 20 mill acres of canola

• The starting point is a checklist developed in Sweden

• Verified or modified to Canadian canola based on in-depth field surveys

• The App provides real-time and location-specific rainfall data and weather forecast

Forecasting -Sclerotinia risk App for smartphones

AAFC’s Sclerotinia risk assessment team

‘If not for you’

~60 extension specialists and canola growersreported sclerotia germination in ~200 depots

each growing season from 2013 to 2017

AAFC research staff

Erl Svendsen

CamKenny

JonathanDurkin

Lone Buchwaldt

Jackie Nettleton

Photo by Branimir Gjetvaj

Economic calculation

Photos of sclerotinia

Sclerotinia risk assessment

Sclerotinia life cycle

Sclerotinia rating at harvest

Canola flowering stages

Sclerotinia risk App for smartphones

App programming (a hybrid)

Ready made web applications for the user interface

New programs for user’s geolocation (GPS, RM, other names), writing of files such as the check list and economic calculation

Saving and sharing of risk assessment results by texting and email.

Versions for both iPhone and Android phones

The starting point is the Swedish sclerotinia check list

Twengström E, Sigvald R, Svensson C, Yuen J. 1998. Forecasting Sclerotinia stem rot in spring sown oilseed rape. Crop Prot 17:405–411.

The risk points for each of the six factors were based on data from 384 canola fields.

Risk factor Risk points1. Number of years since last canola crop 0, 5 or 102. Sclerotinia incidence in last host crop 0, 5, 10 or 153. Plant density 0, 5 or 104. Rainfall in the last two weeks 0, 5 or 105. Weather forecast 0, 10 or 156. Regional risk for apothecia development 0, 10 or 15

A similar data set for Canadian canola is needed in order to either verify or modify this risk assessment.

QTLs








cloned genes





Lectin, curculin40%





check list






5. Plant density


Breeding plan






Sclerotinia test



Sclerotinia test

2nd DH population


Sclerotinia test



Sclerotinia test




Sclerotinia test


Sclerotinia test


Sclerotinia test


Sheet1

Risk factorRisk points

1. Number of years since last canola crop0, 5 or 10

2. Sclerotinia incidence in last host crop0, 5, 10 or 15

3. Plant density0, 5 or 10

4. Rainfall in the last two weeks0, 5 or 10

5. Weather forecast0, 10 or 15

6. Regional risk for apothecia development0, 10 or 15

In-depth field survey of Canadian canola to determine the effect of1. Number of years since last canola crop2. Sclerotinia incidence in last host crop3. Plant density

Sclerotinia risk assessment at flowering

Plant density

10-year crop rotation

Sclerotinia disease severity in previous crops

Sclerotinia severity index at harvest (both frequency and severity)

Canola variety (new factor)

All interested extension specialists

75-100 canola

growers

Data from100 canola

fields per growing season

Project staff

Total database of 400 canola

fields for statistical analysis

Weather Innovations Consulting (WIN) will providelocation-specific and real-time rainfall data4. Rain in the last two weeks5. Weather forecast

Weather netwoeksWIN has >1100 weather stations in Alberta, Saskatchewan and ManitobaReceives data from Environment and Climate Change Canada (Federal Gov.) Other smaller weather networks

Quality check of dataMonitored daily and adjusted for anomalies (limit tests, other data nearby, radar maps etc.)

Algorithms for calculation of location-specific real-time rainfallCalculates location-specific rainfall data within a 50 km radius with the closest stations having more influence than more distant ones

Algorithms for local weather forecastsWeather forecast data from National Oceanic Atmospheric Administration (NOAA) Environment and Climate Change CanadaA few other sources.

‘Come Rain or Come Shine’

Sclerotia depots6. Regional risk of apothecia development

Volunteers bury depots (50 sclerotia) in canola fields and report % sclerotia germination via smartphones

Sclerotia on sterilized barley

Harvest of sclerotia Sclerotia readily germinate with apothecia

Maps of % sclerotia germination are posted on SaskCanola’s web site from May to August

Economic calculation

‘Gonna Change My Way Of Thinking’

The following relationship between disease severity and yield loss is built into the calculator:

% infected canola stems = 0.5% yield loss

Example: 10% infected stems = 5% yield loss

The App will advise growers to follow fungicide manufacturer’s instructions for fungicide rate and time of application

Expected % infected stems at harvest

Expected seed sales price

Fungicide costs

Expected yield

All other input costs

Results and fungicide recommendation

Technology transfer & user feed-back

Project staff

Extension specialists

Canola growers

Up-to-date information is easily distributed via App versions

Like rings in water

Future research

¸ Finish transfer of sclerotinia resistance into canola quality background with good agronomy and yield

¸ Continue to investigate defence genes that prevent fungal penetration of the plant cell, PEN1, PEN2, PEN3, and identify new defense genes effective against sclerotinia

¸ Sclerotinia risk App for smartphones

Funding for sclerotinia research is greatly appreciated

Canola Council of CanadaSclerotinia stem testing of coop lines (via WCC/RRC)Sclerotia depot project

SaskCanolaSeed of resistant B. napus linesMolecular markersTransfer of sclerotinia resistance to canola Identification of defense genesSclerotinia isolate survey

Weather Innovations In-kind via the sclerotia depot project

Agriculture and Agri-Food CanadaAll of the above areas

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sclerotinia resistance and forecasting in canola · a single pen1 gene copy was inserted in the ....

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