Early detection of P. ramorum lineages in
Oregon forests using genetic markers
Sydney E. Everhart1, Meredith M. Larsen2, Alan Kanaskie3, and Niklaus J. Grünwald1,2
of Botany and Plant Pathology, Oregon State University, 2Horticulture Crops Research Unit, USDA-ARS, Corvallis, OR
3Oregon Department of Forestry, Salem, OR
2-plex
8-plex
Table 1. Primers used to reduce the number of reactions from 11 to three: an 8-plex and 2-plex reaction
Aerial surveys
Red trees are tanoaks dead
for 1 year, grey ones have
been dead 2 or more years.
Dark green is Doug-fir.
Photo by Ebba Peterson.
Ground inspections
Field scouting is used to
identify affected plants,
looking for typical
symptoms, bleeding
cankers and leaf blight.
SSR Locus
ILVOPrMS145
PrMS39
PrMS9C3
ILVOPrMS79
Ivors18
Ivors64
PrMS45
PrMS6
Assay
8-plex
8-plex
8-plex
8-plex
8-plex
8-plex
8-plex
8-plex
ILVOPrMS131
KI82
2-plex
2-plex
PrMS43
single 6-FAM 114-500
VIC 150-414
NED 92-260
Forward primer
TGGCAGTGTTCTTCAACAGC
GCACGGCCAGAGATTGATAG
TCACACGAAGCAGCAACTCT
AGGCGGAAAACGTCAGAAC
TGCCATCACAACACAAATCC
GCGCTAAGAAAGACACTCCG
CGTGCTGCATCTGGTGTAGT
AATCGATCTCTCGGCTTTGA
Reverse primer
GTTTATTCCCGTGAACAGCGTATC
GTTTATCTGCCGACGTGAAGAAGT
GTTTAGCGGCACTACGGAATACAT
GTTTCTCGAGAGGCTGGAAGTACG
GTTTGTGCTATCTTTCCTGAACGG
GTTTCAACATGTAGCCATTGCAGG
GAAAGTCCGGATTTGCGTTA
TATAGCCCCAGCTGCAACA
Conc.
(µM)
0.04
0.07
0.10
0.15
0.80
0.35
0.15
0.15
CGGCCGTTTTTGTAAGTTTG GTTTCAGATCAAACCAAAATCTGCTC
CCACGTCATTGGGTGACTTC GTTTCGTACAAGTCACGACTCCCC
0.3
0.30
AAATATGCAAAAAGGCAGGA
0.3
GTTTCCGCGTAACCTAGTCTGCTC
To allow for faster genotyping, we developed an 8-plex multiplex assay using
the Qiagen Type-it Microsatellite PCR Kit. This multiplex method typically uses
only 5ng of DNA and has been successful on DNA samples with concentrations
below detectable levels.
To facilitate the updating and harmonizing of information in forest and
nursery databases we have included four Prospero et al. markers (PrMS39,
PrMS45, PrMS6, PrMS9c3) that historically have been used at both Oregon
State University and ARS. In addition, we added 3 markers (KI18, KI64, KI82)
typically used at the ARS lab and published by Ivors et al.
8-plex P. ramorum Microsatellite Marker Allelic Size Range (bp)
dye
In addition to the 8-plex SSR method, we are using a second 2-plex SSR assay
(Table 1), which includes additional markers from Ivors et al. (KI82) and from
Vercauteren et al. (ILVOPrMS131). These makers were not suitable for inclusion
in the 8-plex assay, due to each having a large overlapping allelic range.
Multiplexing 10 loci in only 2 reactions can be used on DNA
below detectable levels for rapid multilocus
genotype identification
|
165
6-FAM
VIC
NED
PET
We have successfully used this assay to genotype isolates that were previously
unable to be amplified. Lesser quality DNA samples (such as those derived
from baited water) can now be tested.
125
|
205
|
245
|
285
|
325
|
ILVOPrMS145
PrMS45
365
|
405
ILVOPrMS79
PrMS9C3
PrMS39 (represented in black in the chromatogram)
PrMS6
Ivors64
Ivors18
NA2 Genotype
Genotyping is key to
identification of P. ramorum
species and clonal lineages
Amplicon
Dye
size (bp)
6-FAM 165-262
NED 126-270
VIC 212-234
6-FAM 330-400
PET 212-290
NED 338-410
VIC 160-196
PET 163-174
We added ILVOPrMS145 from Vercauteren et al designed to elucidate genetic
diversity specifically of EU-1 isolates. Additionally we included ILVOPrMS79
and ILVOPrMS131 also from the Vercauteren et al. which target genetic
diversity of NA-1 and NA-2 isolates.
EU1 Genotype
Only the NA1 clonal lineage
is documented in Oregon
forests. In 2012,
genotyping revealed a
nursery near Cape
Sebastian that had a P.
ramorum isolate that
belonged to the European
clonal lineage EU1. Rapid
identification of clonal
lineage was imperative for
successful eradication.
1. add latest primer set to panel
2. develop faster and more robust
multiplex method for genotyping
3. integrate database of nursery
isolates with forest isolates
• Genotyping needs to be robust to differentiate the four clonal lineages
• Must be faster so that results of detection are reported earlier
• Need to be able to gain more information from existing genotype
information from both nursery and forest isolates
EU2 Genotype
Phytophthora ramorum is a quarantine species that causes Sudden Oak
Death of Tanoak and Coast Live Oak and foliar blight of key ornamental
nursery plants, including Camellia, Viburnum, and Rhododendron.
NA1 Genotype
1Department
Acknowledgements: Funding for this work
Confirm as Phytophthora
Immunostrip test kits are
used to identify whether or
not a Phytophthora is
present, but cannot identify
to species. Tissue samples
taken for pathogen isolation.
Genotyping for identification
Microsatellites identify whether
the isolate is P. ramorum and also
identify the clonal lineage of each
isolate (NA1, NA2, EU1, or EU2)
• Routine genotyping now screens for
all four clonal lineages
• Multiplex method is faster, needing
only 2 reactions for all 10 markers
• Higher sensitivity means low quality
samples can now be tested
Currently underway
• Integration of forest and nursery
genotype databases
• Genotyping of all 2013 samples
(projected completion in mid-March)
• Development of new genotyping
approach using genotyping-by
sequencing (GBS)
40,968 SNPs in order of occurrence on the P. ramorum genome
came from USDA APHIS, USDA ARS, US Forest
Service PSW, and the USDA Forest Service Forest
Health Monitoring Program. Additional financial
support provided by the USDA AFRI-NIFA
Fellowship Grant to S. Everhart. We thank all
colleagues who generously provided isolates.
Citations:
Sample 1
Ivors, K., Garbelotto, M., Vries, I. D., Ruyter-Spira,
C., Te Hekkert, B., Rosenzweig, N., and
Bonants, P. 2006. Microsatellite markers
identify three lineages of Phytophthora
ramorum in US nurseries, yet single lineages
in US forest and European nursery
populations. Mol. Ecol. 15:1493-1505.
Sample 2
Prospero, S., Hansen, E. M., Grünwald, N. J., and
Winton, L. M. 2007. Population dynamics of
the sudden oak death pathogen
Phytophthora ramorum in Oregon from
2001 to 2004. Mol. Ecol. 16:2958-2973.
Sample 3
Heterozygous SNPs are purple (most), homozygous for the alternate allele
are red, and homozygous for the reference allele are blue (fewest)
GBS yields more than 40,000 SNPs for a preliminary set of 3 NA1 strains (above). These SNPs can be
used to track epidemic spread in Oregon forests over time and space.
Vercauteren, A., De Debbelaere, I., Grünwald, N.
J., Bonants, P., Van Bockstaele, E., Maes, M.,
and Heungens, K. 2010. Clonal expansion of
the Belgian Phytophthora ramorum
populations based on new microsatellite
markers. Mol. Ecol. 19:92-107.
Vercauteren, A., Larsen, M., Goss, E.M.,
Grünwald, N.J., Maes, M., and Heungens, K.
2011. Identification of new polymorphic
microsatellite markers in the NA1 and NA2
lineages of Phytophthora
ramorum. Mycologia. 103: 1245–1249.