Gene expression analysis of ABC- and MFS-transporters in the fungal biocontrol agent Clonostachys rosea

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Supplementary

S.1 Vogel’s medium composition:

Vogel's 50X salts. In 750 mL distilled water, dissolve successively with stirring at room

temperature: Na3C6H5O7*2H2O

(sodium citrate), 125 grams; KH2PO4, anhydrous, 250 grams;

NH4NO3, anhydrous, 100 grams; MgSO47H20, 10 grams; CaCl22H20, 5 grams; Trace Element

Solution (see below), 5 ml; Biotin Solution (see below), 2.5 ml.

The resulting total volume is about 1 L. Chloroform (2 mL) is added as a preservative. Store at

room temperature.

Vogel’s medium N (Minimal) is 20 mL Vogel's Salts, 15 grams sucrose, 1 L distilled H2O. Add 15

grams agar for solid medium.

The trace element solution (containing citric acid as a solubilizing agent) is made up as follows: in

95 mL distilled water, dissolve successively with stirring at room temperature: Citric acid

(C6H8O7H20), 5 grams; ZnSO47H20, 5 grams; Fe(NH4)2(SO4)26H20, 1 gram; CuSO45H20, 0.25

grams; MnSO4H20, 0.05 grams; H3BO3, anhydrous, 0.05 grams; Na2MoO42H20, 0.05 grams.

The resulting total volume is about 100 mL. Chloroform (1 mL) is added as a preservative, and the

trace element solution is stored at room temperature.

The biotin solution is prepared by dissolving 5 mg biotin in 50 mL distilled water. The solution

obtained is dispensed in test tubes and stored in the frozen state.

S.2 Fungicides details

Chipco Green (Bayer) is a contact fungicide having curative and preventative action against a range

of turf diseases. Chipco® Green's special liquid formulation is very ‘rainfast’ and can prevent the

build-up of morning dew on grass leaves. Chipco green is and has been the UK and Irelands No.1

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contact fungicide for over 30 years. Only approved for curative control of Fusarium. Tried and

trusted by turf managers on some of the most prestigious turf around the world, Chipco Green is a

truly global player. Chipco Green's special liquid formulation is rainfast within 1 hour. Once the

spray application has dried on the leaf surface, rainfall will not interfere with its ability to control

disease. Chipco Green can also prevent the build-up of morning dew on grass leaves for up to 2

weeks after application. Dew "switching" to remove morning dew during this time is therefore

usually unnecessary. Chipco Green can be used preventatively, curatively and early eradicant at the

first signs of disease. Chipco Green can be applied at any time of year when grass is either dormant

or

actively

growing

and

displays

both

curative

and

protective

activity.

(

http://www.pitchcare.com/shop/professional-turf-fungicides/chipco-green-turf-disease-control-1.html

).

AMISTAR (Syngenta). An innovation in plant disease control originally inspired by a species of

forest-dwelling mushroom, which produced its own natural fungicide, AMISTAR® and its related

brands is now the world’s No1 fungicide. Through its broad-spectrum activity it delivers consistent

yield and quality benefits around the world in a broad array of crop types. The active ingredient,

azoxystrobin, belongs to a class of fungicides named strobilurins, which act by inhibiting

mitochondrial respiration in fungi, stopping their energy supply. Today, growers use AMISTAR®

for fungal control and yield enhancement in cereals such as wheat and barley as well as in vines,

fruits,

vegetables,

bananas,

rice,

soybeans,

corn,

turf

and

ornamentals.

(

http://www.syngenta.com/global/corporate/en/products-and-innovation/product-brands/crop-protection/fungicides/pages/amistar.aspx

).

Cantus WDG (NM Bartlett Inc.) is applied in berries group, bulb vegetables group, carrots, fruiting

vegetables, grapes, field lettuce (head and leaf), potatoes, stone fruits group, strawberries and

cucurbit vegetables. Cantus WDG fungicide has a protective effect because it inhibits spores

germination, mycelial growth and sporulation of the fungus on the leaf surface. Optimum disease

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control is achieved when CANTUS WDG fungicide is applied in a regularly scheduled protective

spray program and is used in a rotation program with other fungicides. It’s effective against several

crop pathogens such as Botrytis cinerea, B. squamosa, Alternaria solani, A. porri, A. dauci, A.

cucumerina, Didymella bryoniae, Sclerotinia minor, Uncinula necator and Monilinia spp..

(

http://www.bartlett.ca/Bartlett/nmb/MSDSLabel.nsf/33679510e3c80d96852574a20055f364/da737

9a6736f2d808525799600594614/$FILE/Cantus%20WDG%20label%20english.pdf

).

Apron XL (Syngenta) is a seed treatment product for protection against Pythium and Phytophthora

causing damping-off, seed rot, and systemic downy mildew diseases of certain crop. It commonly

used for Brassicole leafy vegetables, bulb vegetables, carrots, cereal grains (wheat, barley, rye, oats,

millet, rice, field corn, sweet corn, popcorn, buckwheat, and triticale), cotton, Cucurbits, forage

grasses, forage legumes, fruiting vegetables, globe artichoke, leafy vegetables, legume vegetables

and okra, millet, peanuts, root and tuber vegetables (except vegetables), sorghum, soybeans,

sunflower

and

turfgrasses

(

http://www.syngentacropprotection.com/pdf/labels/SCP799AL1K0306.pdf

).

S.3 standard curves of the selected reference genes: Ct values from RT-qPCR.

SAR1

Tech. rep.s Ct values Ct mean Std. dev.

1 6.03 6.21 0.175 2 6.23 Dilution 1 3 6.38 1 9.56 9.73 0.195 2 9.71 Dilution 2 3 9.94 1 13.11 13.08 0.221 2 12.85 Dilution 3 3 13.29 1 16.96 17.01 0.06 2 16.99 Dilution 4 3 17.07 1 20.39 20.33 0.049 2 20.3 Dilution 5 3 20.31 1 23.69 23.69 0.019

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1 23.69 23.69 0.019 2 23.66 Dilution 6 3 23.7 1 26.79 26.75 0.072 2 26.79 Dilution 7 3 26.67 1 28.07 28.13 0.245 2 27.92 Dilution 8 3 28.4 GPD1

1 5.66 5.78 0.102 2 5.85 Dilution 1 3 5.83 1 8.89 8.93 0.067 2 8.89 Dilution 2 3 9.01 1 12.26 12.2 0.408 2 11.76 Dilution 3 3 12.57 1 15.93 16.34 0.529 2 16.94 Dilution 4 3 16.16 1 19.5 19.42 0.113 2 19.29 Dilution 5 3 19.47 1 22.59 22.52 0.064 2 22.49 Dilution 6 3 22.47 1 25.71 25.73 0.089 2 25.66 Dilution 7 3 25.83 1 28.93 29.12 0.205 2 29.11 Dilution 8 3 29.34 SFC1

1 6.02 5.89 0.125 2 5.77 Dilution 1 3 5.88 1 9.28 9.24 0.146 Dilution 2 2 9.08

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3 9.37 1 12.33 12.38 0.076 2 12.46 Dilution 3 3 12.34 1 15.94 16.08 0.123 2 16.12 Dilution 4 3 16.18 1 19.69 19.44 0.224 2 19.26 Dilution 5 3 19.37 1 23.58 23.06 0.455 2 22.89 Dilution 6 3 22.72 1 25.71 25.71 0.039 2 25.67 Dilution 7 3 25.75 1 29.35 29.45 0.258 2 29.25 Dilution 8 3 29.74 TEF1

1 6.21 6.09 0.129 2 6.11 Dilution 1 3 5.95 1 9.71 9.45 0.242 2 9.4 Dilution 2 3 9.24 1 12.64 12.8 0.147 2 12.92 Dilution 3 3 12.84 1 16.55 16.68 0.126 2 16.69 Dilution 4 3 16.8 1 19.68 19.79 0.094 2 19.84 Dilution 5 3 19.84 1 23.39 23.36 0.073 2 23.42 Dilution 6 3 23.28 1 24.76 24.87 0.098 2 24.96 Dilution 7 3 24.88 1 28.39 29.24 1.509 2 28.35 Dilution 8 3 30.98

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ACTIN

1 5.17 5.33 0.141 2 5.37 Dilution 1 3 5.45 1 8.53 8.55 0.09 2 8.47 Dilution 2 3 8.65 1 11.93 11.9 0.089 2 11.97 Dilution 3 3 11.8 1 15.62 15.46 0.192 2 15.25 Dilution 4 3 15.51 1 18.71 18.73 0.021 2 18.73 Dilution 5 3 18.75 1 22.05 22.12 0.077 2 22.1 Dilution 6 3 22.2 1 25.63 25.71 0.114 2 25.66 Dilution 7 3 25.84 ß-TUBULIN

1 11.57 11.37 0.189 2 11.2 Dilution 1 3 11.35 1 14.94 14.94 0.03 2 14.92 Dilution 2 3 14.98 1 18.39 18.62 0.222 2 18.65 Dilution 3 3 18.83 1 22.04 22.06 0.082 2 21.99 Dilution 4 3 22.15 1 25.71 25.42 0.26 2 25.36 Dilution 5 3 25.2 1 28.83 28.87 0.094 2 28.98 Dilution 6 3 28.81 1 32.63 32.42 0.345 2 32.02 Dilution 7 3 32.61

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1 35.87 35.39 0.678

2 N/A

Dilution 8

3 34.91

S.4 RT-qPCR using cDNA from different treatments as template (best reference gene evaluation),

the three biological replicates are represented by A, B, C letters:

SAR1 Treatments/interactions Technical

replicates

Ct values Ct mean Standard

deviation 1 36.02 35.93 0.196 2 35.71 PDA (A) 3 36.07 1 34.52 34.45 0.097 2 34.49 PDA (B) 3 34.34 1 33.4 33.55 0.316 2 33.34 PDA (C) 3 33.91 1 31.84 31.85 0.05 2 31.9 PDB (A) 3 31.8 1 32.64 32.52 0.115 2 32.41 PDB (B) 3 32.53 1 34.63 34.31 0.279 2 34.12 PDB (C) 3 34.18 1 30.1 30.19 0.163 2 30.1

Czapek-dox medium (A)

3 30.38 1 29.17 29.41 0.366 2 29.22 Czapek-dox medium (B) 3 29.83 1 29.16 28.55 0.536 2 28.31 Czapek-dox medium (C) 3 28.18 1 31.11 31.06 0.049 2 31.05

Czapek-dox broth (A)

3 31.01 1 30.45 30.66 0.184 2 30.74 Czapek-dox broth (B) 3 30.79 1 31.17 31.2 0.027 2 31.22 Czapek-dox broth (C) 3 31.21 1 32.73 32.14 0.539

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1 32.73 32.14 0.539

2 32.03

Germinated conidia (A)

3 31.67 1 30.31 30.17 0.194 2 30.25 Germinated conidia (B) 3 29.95 1 30.06 30.28 0.279 2 30.19 Germinated conidia (C) 3 30.6 1 31.74 31.89 0.173 2 31.85

C.rosea vs C.rosea (A)

3 32.08 1 31.03 30.57 0.425 2 30.48 C.rosea vs C.rosea (B) 3 30.19 1 29.96 29.81 0.308 2 29.45 C.rosea vs C.rosea (C) 3 30.01 1 28.73 29.1 0.436 2 28.99 C.rosea vs F.graminearum (A) 3 29.58 1 30.97 29.62 1.175 2 29.08 C.rosea vs F.graminearum (B) 3 28.81 1 28.95 28.68 0.253 2 28.62 C.rosea vs F.graminearum (C) 3 28.46 GPD1 Treatments/interactions Technical replicates

deviation 1 25.58 25.65 0.056 2 25.67 PDA (A) 3 25.68 1 25.28 25.32 0.035 2 25.33 PDA (B) 3 25.35 1 24 23.94 0.058 2 23.9 PDA (C) 3 23.91 1 23.68 23.84 0.138 2 23.92 PDB (A) 3 23.91 1 24.31 24.19 0.115 2 24.08 PDB (B) 3 24.17 1 25.2 25.17 0.025 PDB (C) 2 25.15

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3 25.15

1 21.97 21.9 0.138

2 21.74

3 22.47 1 22.23 22.24 0.04 2 22.2 Czapek-dox broth (B) 3 22.28 1 22.79 22.81 0.038 2 22.85 Czapek-dox broth (C) 3 22.78 1 24.23 24 0.2 2 23.89

3 25.83 1 24.54 24.23 0.284 2 24.15 C.rosea vs C.rosea (B) 3 24 1 23.99 23.93 0.051 2 23.89 C.rosea vs C.rosea (C) 3 23.92 1 23.08 23.21 0.157 2 23.18 C.rosea vs F.graminearum (A) 3 23.39 1 23.44 23.48 0.11 2 23.39 C.rosea vs F.graminearum (B) 3 23.6 1 23.77 23.52 0.223 2 23.38 C.rosea vs F.graminearum (C) 3 23.39

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SFC1 Treatments/interactions Technical

replicates

deviation 1 34.49 34.5 0.402 2 34.1 PDA (A) 3 34.91 1 34.32 34.1 0.235 2 34.14 PDA (B) 3 33.85 1 33.39 33.27 0.128 2 33.14 PDA (C) 3 33.27 1 33.23 33.43 0.192 2 33.45 PDB (A) 3 33.61 1 33.82 34.01 0.237 2 33.93 PDB (B) 3 34.27 1 34.44 33.99 0.468 2 34.01 PDB (C) 3 33.51 1 32.9 33.05 0.259 2 33.35

3 33.19 1 32.81 32.96 0.136 2 33 Czapek-dox broth (B) 3 33.08 1 33.17 33.53 0.32 2 33.71 Czapek-dox broth (C) 3 33.73 1 34.64 34.7 0.505 2 35.23

3 34.23 1 33.17 33.03 0.154 2 32.86 Germinated conidia (B) 3 33.07 1 33.18 33.17 0.066 2 33.1 Germinated conidia (C) 3 33.23 1 32.37 33.17 0.707

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3 33.43 1 32.24 32.15 0.101 2 32.17 C.rosea vs C.rosea (B) 3 32.04 1 31.52 31.57 0.163 2 31.43 C.rosea vs C.rosea (C) 3 31.75 1 31.42 31.48 0.075 2 31.46 C.rosea vs F.graminearum (A) 3 31.57 1 31.83 31.89 0.139 2 32.05 C.rosea vs F.graminearum (B) 3 31.79 1 32.07 31.92 0.167 2 31.95 C.rosea vs F.graminearum (C) 3 31.74 TEF1 Treatments/interactions Technical replicates

deviation 1 26.37 26.27 0.203 2 26.04 PDA (A) 3 26.41 1 26 26.05 0.04 2 26.08 PDA (B) 3 26.07 1 25.29 25.17 0.155 2 24.99 PDA (C) 3 25.23 1 24.29 24.13 0.175 2 23.94 PDB (A) 3 24.16 1 25.05 24.82 0.341 2 24.43 PDB (B) 3 24.98 1 24.94 24.88 0.175 2 25.01 PDB (C) 3 24.68 1 23.09 23.19 0.088 2 23.27

3 23.22 1 20.63 20.85 0.188 2 20.92 Czapek-dox medium (B) 3 20.99 1 20.97 20.79 0.197 2 20.81 Czapek-dox medium (C) 3 20.58 1 23.71 23.67 0.191

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1 23.71 23.67 0.191

2 23.46

3 23.83 1 23.66 23.81 0.136 2 23.9 Czapek-dox broth (B) 3 23.88 1 23.87 23.77 0.123 2 23.63 Czapek-dox broth (C) 3 23.8 1 23.07 23.07 0.012 2 23.08

3 25.5 1 23.65 23.32 0.287 2 23.1 C.rosea vs C.rosea (B) 3 23.23 1 22.9 22.84 0.316 2 22.49 C.rosea vs C.rosea (C) 3 23.11 1 21.66 21.59 0.267 2 21.82 C.rosea vs F.graminearum (A) 3 21.3 1 22.38 22.37 0.107 2 22.26 C.rosea vs F.graminearum (B) 3 22.47 1 22.86 22.72 0.133 2 22.71 C.rosea vs F.graminearum (C) 3 22.6 ACTIN Treatments/interactions Technical replicates

deviation 1 28.12 28.14 0.022 2 28.15 PDA (A) 3 28.16 1 27.36 27.41 0.126 2 27.32 PDA (B) 3 27.56 1 26.38 26.3 0.15 PDA (C) 2 26.13

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3 26.39 1 25.33 25.5 0.183 2 25.48 PDB (A) 3 25.69 1 26.21 26.1 0.088 2 26.05 PDB (B) 3 26.06 1 27.55 27.45 0.082 2 27.41 PDB (C) 3 27.4 1 23.63 23.85 0.194 2 23.9

3 25.34 1 25.03 25 0.031 2 25 Czapek-dox broth (B) 3 24.97 1 25.08 25.11 0.181 2 24.94 Czapek-dox broth (C) 3 25.3 1 25.18 24.91 0.263 2 24.65

3 25.68 1 23.92 23.86 0.095 2 23.91 C.rosea vs C.rosea (B) 3 23.75 1 23.7 23.55 0.141 2 23.43 C.rosea vs C.rosea (C) 3 23.52 1 22.78 22.96 0.193 2 22.93 C.rosea vs F.graminearum (A) 3 23.16

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1 23.38 23.29 0.154 2 23.39 C.rosea vs F.graminearum (B) 3 23.12 1 23.62 23.37 0.238 2 23.34 C.rosea vs F.graminearum (C) 3 23.15 ß-TUBULIN Treatments/interactions Technical replicates

deviation 1 28.17 28.19 0.064 2 28.14 PDA (A) 3 28.26 1 27.66 27.8 0.141 2 27.79 PDA (B) 3 27.94 1 26.95 26.89 0.18 2 26.68 PDA (C) 3 27.03 1 26.47 26.45 0.071 2 26.37 PDB (A) 3 26.51 1 26.17 26.3 0.119 2 26.33 PDB (B) 3 26.41 1 28.89 28.83 0.106 2 28.71 PDB (C) 3 28.9 1 24.04 24 0.143 2 23.84

3 24.12 1 23.3 23.43 0.191 2 23.34 Czapek-dox medium (B) 3 23.65 1 23.14 23.03 0.096 2 22.95 Czapek-dox medium (C) 3 23 1 24.86 25.32 0.399 2 25.5

3 25.59 1 25.42 25.62 0.18 2 25.69 Czapek-dox broth (B) 3 25.76 1 25.42 25.41 0.074 2 25.48 Czapek-dox broth (C) 3 25.33 1 26.72 26.5 0.198

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3 25.48 1 24.07 24.09 0.22 2 24.32 C.rosea vs C.rosea (B) 3 23.89 1 23.74 23.71 0.027 2 23.69 C.rosea vs C.rosea (C) 3 23.7 1 22.96 23.03 0.152 2 22.92 C.rosea vs F.graminearum (A) 3 23.2 1 23.39 23.35 0.102 2 23.23 C.rosea vs F.graminearum (B) 3 23.42 1 23.62 23.31 0.291 2 23.04 C.rosea vs F.graminearum (C) 3 23.28

S.5 Gene expression analysis (RT-qPCR), Ct values:

Ctactin values are reported next to Cttarget values as they refer to the same RT-qPCR reaction; ‘N/A’

means that no fluorescence was registered after 40 amplification cycles during the RT-qPCR, thus

no amplification of the target gene is supposed.

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Target genes MFS 6455 MFS 7234 UbiH 0606 MFS 2418 ABCG 0026 MFS 2311 MFS 9923 ABCC 6570 Tr ea tm en ts / in te ra ctio ns re pl ic at es Ctta rg et Ctact in Ctta rg et Ctact in Ctta rg et Ctact in Ctta rg et Ctact in Ctta rg et Ctact in Ctta rg et Ctact in Ctta rg et Ctact in Ctta rg et Ctact in 1 32.9 22.7 33.7 22.7 32.4 22.7 31.0 22.7 38.6 22.7 38.0 22.7 37.9 22.7 33.7 22.7 2 37.0 20.7 38.3 20.7 32.4 20.7 28.2 20.7 38.5 20.7 36.4 20.7 37.2 20.7 31.8 20.7 C. r os ea v s C. r os ea 3 32.2 20.3 32.5 20.3 31.7 20.3 28.4 20.3 38.0 20.3 36.3 20.3 36.2 20.3 31.9 20.3 1 38.7 18.8 37.7 18.8 32.3 18.8 27.7 18.8 38.0 18.8 32.8 18.8 32.3 18.8 29.2 18.8 2 37.4 19.1 37.5 19.1 32.3 19.1 28.0 19.1 38.4 19.1 32.5 19.1 33.4 19.1 29.9 19.1 C. r os ea v s B. c in er ea 3 37.8 19.2 38.0 19.2 32.3 19.2 28.1 19.2 38.2 19.2 32.4 19.2 33.4 19.2 29.7 19.2 1 25.8 20.0 26.3 20.0 26.3 20.0 27.5 20.0 36.8 20.0 37.3 20.0 35.6 20.0 32.3 20.0 2 26.1 19.8 26.7 19.8 26.4 19.8 27.5 19.8 35.9 19.8 36.7 19.8 35.4 19.8 32.2 19.8 C. r os ea v s F. g ra m in ea ru m 3 26.9 19.9 26.9 19.9 27.0 19.9 27.6 19.9 35.7 19.9 36.8 19.9 35.7 19.9 32.6 19.9 1 36.4 22.0 0.0 22.0 29.5 22.0 28.2 22.0 26.2 22.0 37.4 22.0 N/A 22.0 32.8 22.0 2 37.4 21.5 0.0 21.5 29.2 21.5 34.1 21.5 26.3 21.5 N/A 21.5 N/A 21.5 33.0 21.5 3 36.4 22.5 0.0 22.5 30.6 22.5 31.8 22.5 28.2 22.5 38.1 22.5 38.7 22.5 35.4 22.5 4 37.2 21.9 0.0 21.9 30.4 21.9 33.5 21.9 26.7 21.9 N/A 21.9 N/A 21.9 33.6 21.9 Ze ara len on e 5 36.6 20.7 0.0 20.7 28.7 20.7 32.4 20.7 26.2 20.7 38.8 20.7 N/A 20.7 31.8 20.7 1 36.8 20.4 37.9 20.4 28.9 20.4 32.9 20.4 38.6 20.4 38.4 20.4 N/A 20.4 31.3 20.4 2 35.9 20.1 38.7 20.1 29.5 20.1 28.1 20.1 38.5 20.1 37.7 20.1 N/A 20.1 30.8 20.1 3 36.1 19.8 38.5 19.8 29.4 19.8 32.0 19.8 38.2 19.8 37.4 19.8 38.1 19.8 31.3 19.8 4 36.2 20.2 38.7 20.2 30.1 20.2 32.7 20.2 38.5 20.2 36.8 20.2 37.2 20.2 31.7 20.2 Ct rl z ea ra len on e 5 36.3 20.0 38.6 20.0 29.1 20.0 32.9 20.0 N/A 20.0 38.5 20.0 N/A 20.0 31.2 20.0 1 38.0 22.6 N/A 22.6 28.6 22.6 37.3 22.6 N/A 22.6 38.1 22.6 N/A 22.6 35.2 22.6

Ch

ip

co

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3 38.3 23.8 N/A 23.8 29.5 23.8 38.6 23.8 N/A 23.8 N/A 23.8 N/A 23.8 35.9 23.8 4 38.2 22.2 N/A 22.2 29.0 22.2 36.3 22.2 N/A 22.2 N/A 22.2 N/A 22.2 35.3 22.2 5 38.0 22.8 N/A 22.8 29.4 22.8 36.6 22.8 N/A 22.8 N/A 22.8 N/A 22.8 35.7 22.8 1 36.0 21.5 N/A 21.5 29.0 21.5 38.4 21.5 38.2 21.5 38.6 21.5 N/A 21.5 32.8 21.5 2 38.0 21.8 N/A 21.8 30.4 21.8 38.4 21.8 N/A 21.8 38.8 21.8 N/A 21.8 33.9 21.8 3 36.6 20.8 N/A 20.8 30.3 20.8 38.0 20.8 N/A 20.8 38.8 20.8 N/A 20.8 33.9 20.8 4 36.1 22.0 N/A 22.0 30.6 22.0 36.9 22.0 N/A 22.0 36.3 22.0 N/A 22.0 32.9 22.0

Ca

n

tu

s

5 36.3 20.5 N/A 20.5 30.0 20.5 35.9 20.5 38.4 20.5 37.1 20.5 N/A 20.5 33.2 20.5 1 38.2 24.6 N/A 24.6 26.9 24.6 38.6 24.6 34.4 24.6 N/A 24.6 N/A 24.6 37.3 24.6 2 35.0 23.6 N/A 23.6 25.1 23.6 35.2 23.6 33.3 23.6 N/A 23.6 N/A 23.6 36.3 23.6 3 36.8 24.2 N/A 24.2 26.1 24.2 N/A 24.2 34.4 24.2 38.3 24.2 N/A 24.2 36.6 24.2 4 33.6 22.8 N/A 22.8 24.7 22.8 35.0 22.8 32.6 22.8 38.5 22.8 N/A 22.8 37.5 22.8

Ap

ro

n

5 35.0 23.5 N/A 23.5 25.4 23.5 37.8 23.5 33.2 23.5 38.2 23.5 N/A 23.5 35.8 23.5 1 36.2 23.7 N/A 23.7 30.6 23.7 36.5 23.7 N/A 23.7 N/A 23.7 N/A 23.7 34.3 23.7 2 38.6 23.4 N/A 23.4 30.8 23.4 38.5 23.4 N/A 23.4 N/A 23.4 N/A 23.4 35.2 23.4 3 38.8 24.4 N/A 24.4 31.9 24.4 37.4 24.4 N/A 24.4 N/A 24.4 N/A 24.4 36.4 24.4 4 37.7 24.1 N/A 24.1 31.6 24.1 N/A 24.1 N/A 24.1 N/A 24.1 N/A 24.1 35.4 24.1

Am

is

ta

r

5 N/A 23.8 N/A 23.8 30.7 23.8 38.7 23.8 N/A 23.8 38.6 23.8 N/A 23.8 35.2 23.8 1 36.0 20.3 N/A 20.3 29.4 20.3 30.6 20.3 N/A 20.3 N/A 20.3 N/A 20.3 32.7 20.3 2 36.0 19.9 N/A 19.9 27.9 19.9 35.7 19.9 37.9 19.9 N/A 19.9 N/A 19.9 33.0 19.9 3 37.0 20.8 N/A 20.8 29.6 20.8 35.0 20.8 N/A 20.8 38.4 20.8 N/A 20.8 32.2 20.8 4 37.0 21.1 N/A 21.1 29.7 21.1 35.6 21.1 N/A 21.1 38.1 21.1 N/A 21.1 33.6 21.1

Ct rl fu n gi ci d es

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