Rutgers Turf Symposium January 14, 2011

1. Using Genomics to Unravel the Mysteries of
Sclerotinia homoeocarpa
Lane Tredway, Ignazio Carbone, Bangya Ma, Alex Putman, and
Troy Taylor
Department of Plant Pathology
North Carolina State University

2. Much of North Carolina is in the Transition Zone
Piedmont
Blue Ridge
Coastal Plain
Transition Zone
both warm- and cool-season grasses marginally
adapted

3. fairy ring spring dead spot
bacteria?
sting nematodes
diagnostics
online decision aids
fungicide evaluation

4. The Most Important
Turf Disease
• infects all grasses grown for
turf
• develops under a broad range
of environmental conditions
• ~40% of fungicides applied to
turf in US are for control of
dollar spot
• develops resistance to
fungicides very quickly

5. Historical Perspective
• dollar spot first observed in
1920’s
• S. homoeocarpa described by
F.T. Bennet in 1937
• Jackson determined that
Sclerotinia was an improper
designation in 1973
• In 1979, Kohn proposed Lanzia
or Moellerodiscus as likely
classifications

6. Holst-Jensen et al., 1997
Mycologia 89:885
• used rDNA sequences to study
phylogenetic relationships in
the Sclerotineaceae and
Rutstroemiaceae
• confirmed that S. homoeocarpa
belongs to Family
Rutstroemiaceae and is most
closely related to the genera
Poculum and Rutstroemia

7. Rutstroemiaceae
• widespread in temperate
climates
• produce substratal stroma,
not sclerotia
• includes the genera
Lambertella, Lanzia,
Poculum, Rutstroemia
• saprophytic fungi that
grown on dead wood and
bark

8. Banner Maxx (1 fl oz)
EC50 = 0.011
Historical Perspective:
Fungicide Resistance
• resistance to anilazine and
cadmium fungicides first
reported in 1960’s
• benzimidazole resistance Banner Maxx (1 fl oz)
EC50 = 0.054
reported in 1970’s
• DMI and dicarboxamide
resistance reported in
1980’s
• carboxamide resistance.....
Plots treated with Banner Maxx
1 fl oz/1000 ft2
14 day interval

9. Project Objectives
• develop a global sample of S.
homoeocarpa populations
• determine the genetic
structure of populations
• investigate the molecular
mechanisms governing
fungicide sensitivity
• understand the population
dynamics leading to fungicide
resistance

11. Developing a global sample of S. homoeocarpa populations

12. Multilocus Sequence Analysis of S. homoeocarpa Populations

13. Multilocus Sequence Analysis of S. homoeocarpa Populations

14. Multilocus Sequence Analysis of S. homoeocarpa Populations

15. Multilocus Sequence Analysis of S. homoeocarpa Populations

16. Sclerotinia homoeocarpa Genome Project
• collaboration among NC State (Tredway and Carbone) and Ohio State
(Boehm and Mitchell)
• sequencing funded by Center for Turfgrass Science at NC State and
performed by J. Craig Venter Institute
• selected Ohio State lab strain (MB01) for analysis
• combination of Sanger and Illumina sequencing techniques employed
to yield 70x coverage
• isolate from seashore paspalum sequenced independently by
Harmon

19. Comparison of S. homoecarpa genome to species in Family Sclerotineaceae

20. Characterization of Mating Type Loci
• isolate MB01 contains the MAT1-1 idiomorph
• Florida isolate contains MAT1-2 idiomorph
• PCR assay developed to determine mating type distribution in
populations
• current data suggests that both mating types are present in
populations from cool- and warm-season turfgrasses

21. Characterization of Mating Type Loci
• isolate MB01 contains the MAT1-1 idiomorph
• Florida isolate contains MAT1-2 idiomorph
• PCR assay developed to determine mating type distribution in
populations
• current data suggests that both mating types are present in
populations from cool- and warm-season turfgrasses
PCR assay for detection of

22. Development of
Microsatellite Markers
• 8090 microsatellites loci annealing temperature
detected in genome
scaffolds
• 791 candidate loci selected
for further analysis
• 22 tested and discarded
• 1 informative locus identified
• 36 more in various stages of
testing
testing assay in diverse sample population

23. Sensitivity to DMI fungicides in Fungal Pathogens
• DMIs inhibit the production of ergosterol, a key component of fungal
cell membranes
• bind to cytochrome P450 lanosterol 14a-demethylase gene (CYP51)
• known resistance mechanisms
• point mutations in CYP51 leading to reduced binding affinity
• increased constituitive expression of CYP51
• energy-dependent drug efflux mechanisms (ATP-binding
cassette transporter proteins)

24. Characterization of ShCYP51
• initial 547-bp fragment amplified using degenerate primers developed
based on CYP51 sequence in Monolinia fructicola and Sclerotinia
sclerotiorum
• remainder of coding sequence and upstream promoters sequenced
by genomic walking
!"#$%!&'()'*+,-./! !"#$%(0-1-2("$34!&'!
introns at 247-299 bp and 498-555 bp

25. ShCYP51 Sequence Similarities
AA position in ShCYP51 82 125
S. homoeocarpa K Y G D V F T F I -- -- L T T P V F G Q D V V Y D C P N
Monilinia fructicola K Y G D V F T F I -- -- L T T P V F G K D V V Y D C P N
Rat K Y G P V F S F T -- -- L T T P V F G K G V A Y D V P N
Human K Y G P V F S F T -- -- L T T P V F G K G V A Y D V P N
Candida albicans K Y G D V F S F M -- -- L T T P V F G K G V I Y D C P N
Monolinia fructicola * * * * * * * * * * * * * * * * * *
CR1 CR2
AA position in ShCYP51 219 284
S. homoeocarpa A A L Y H D L D M G F S -- -- T Y K D G T P V P D K E I
M. fructicola A S L Y H D L D M G F S -- -- S Y K D G T P V P D K E I
Botryotinia fuckeliana Rat A Q L Y A D L D G G F S -- -- T Y K D G R P L T D D E I
Human A Q L Y A D L D G G F S -- -- T Y K D G R P L T D D E V
C. albicans A Q L Y S D L D K G F T -- -- T Y K D G V K M T D Q E I
* * * * * * * * * * * * *
CR3 CR4
Blumeria graminis AA position in ShCYP51 362
S. homoeocarpa A H M M I A L L M A G Q H S S S S S S -- -- L K E T L R L
M. fructicola A N M M I A L L M A G Q H S S S S S I -- -- L K E T L R L
Rat A G M L I G L L L A G Q H T S S T T S -- -- I K E T L R L
Human A G M L I G L L L A G Q H T S S T T S -- -- I K E T L R L
Sclerotinia sclerotiorum C. albicans A N L L I
* *
G I L M G G Q H T S A S T S -- -- I
* * * * *
K E T L R M
* * * * *
CR4 (continued) CR5
0 25 50 75 100 AA position in ShCYP51 457
S. homoeocarpa Y L P F G A G R H R C I G E Q F A T V N
% Similarity M. fructicola Y L P F G A G S H R C I G E Q F A N V Q
Rat Y V P F G A G R H R C I G E N F A Y V Q
Human Y V P F G A G R H R C I G E N F A Y V Q
C. albicans Y L P F G G G R H R C I G E Q F A Y V Q
* * * * * * * * * * * * *
CR6

26. propiconazole sensitivity (EC50 ppm)
0.08!
0.04!
0.00!
0.02!
0.06!
0.10!
0.12!
LWC27!
VGC5!
PST4!
LWC5!
CHCC10!
761ShME-42!
LWC38!
PST48!
LWC10!
LWC33!
PhPG22!
RE18G8!
RE18G45!
263ShCT-3!
PhPG4!
363ShCT18!
RE18G4!
PhPG9!
451ShCT76!
RCC18G2!
537ShCT137!
522ShNY-9!
RE18G35!
RE18G38!
RCC18G15!
RCCPG18!
557ShCT173!
F42!
G72!
677ShRI-9!
G62!
658ShMA-80!
S088!
H127!
G1!
500ShCT123!
RE18G16!
A22!
G5!
W532L mutation!
V102G mutation!
725ShME19!
Upstream mutation!
G79!
D3!
A4!
Three mutations observed in ShCYP51 and promoter

27. Constituitive Expression of CYP51 in S. homoeocarpa
2.00
Relative Expression (CYP51/b-tub)
1.50
1.4 1.4 1.4
1.3 1.3
1.2 1.2
1.00 1.1
0.9
0.7
0.50
0
sensitive isolates insensitive isolates

28. Relative Expression (CYP51/b-tub)
0!
5!
10!
15!
20!
25!
30!
35!
40!
0!
5!
10!
15!
20!
25!
30!
35!
40!
PS PS
C C
H T4! H T4!
C C
C C
1 1
VG 0! VG 0!
R R
E1 C5 E1 C5
8G ! 8G !
LW 45 LW 45
! !
C C
1 1
LW 0! LW 0!
C C
Sensitive isolate!
5! 5!
Insensitive isolate!
A A
4! 4!
A A
22 22
! !
G G
5 5
H ! H !
12 12
7 7
72 S0 ! 72 S0 !
5S 88 5S 88
H ! H !
M M
E!
0h
E!
24 h
0!
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10!
15!
20!
25!
30!
35!
40!
0!
5!
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25!
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35!
40!
PS PS
C C
H T4!
H T4!
C C
C C
1 1
VG 0! VG 0!
R R
E1 C5 E1 C5
8G ! 8G !
LW 45 LW 45
! !
C C
1 1
LW 0! LW 0!
C C
5! 5!
A A
4! 4!
A A
22 22
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G G
5 5
H ! H !
12 12
7 7
72 S0 ! 72 S0 !
5S 88 5S 88
H ! H !
M M
1h
E! E!
48 h
ShCYP51 expression as influenced by propiconazole exposure

29. 18! Sensitive group! A!
Insensitive group!
Relative expression of ShCYP51!
12!
6!
0!
0 h! 1 h! 24 h! 48 h!
18!
Sensitive group! B!
Insensitive group!
12!
6!
0!
0 h! 1 h! 24 h! 48 h!
ShCYP51 expression as influenced by propiconazole exposure

30. Constituitive and Induced Expression of ABCs
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31. Summary and Conclusions
• S. homoeocarpa populations are subdivided among cool- and warm-
season turfgrass hosts
• availability of genome sequence data has accelerated our research in
several directions
• both mating types are present in S. homoeocarpa populations
• while the genetic basis of DMI resistance remains unknown, induced
expression of CYP51 and increased constituitive expression of ABC
genes appear to be involved

32. Acknowledgements
NC State Turf Pathology Collaborators
Lee Butler Ignazio Carbone
Mike Soika Mike Boehm
Bangya Ma John Kaminski
Alex Putman Bruce Clarke
Joseph Roberts Micah Woods
Troy Taylor Ruth Mann
Jordan Teisher Phil Harmon
Jo Anne Crouch
Funding Ned Tisserat
Center for Turfgrass Science at Maria Tomaso-Peterson
NC State University Frank Wong
Alejandro Canegallo