Phytopathology® 2022 112:1795-1807 https://doi.org/10.1094/PHYTO-09-21-0370-R
Population Biology
Evidence of Coevolution Between Cronartium harknessii Lineages
and Their Corresponding Hosts, Lodgepole Pine and Jack Pine
Chandra H. McAllister,1 Catherine I. Cullingham,2 Rhiannon M. Peery,1 Michael Mbenoun,1 Eden McPeak,1
Nicolas Feau,3,4 Richard C. Hamelin,3 Tod D. Ramsfield,5 Colin L. Myrholm (retired),5 and Janice E. K. Cooke1,†
1
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada
Department of Biology, Carleton University, Ottawa, Ontario, Canada
3
Department of Forest Science, University of British Columbia, Vancouver, British Columbia, Canada
4
Pacific Forestry Centre, Canadian Forest Service, Natural Resources Canada, Victoria, British Columbia, Canada
5
Northern Forestry Centre, Canadian Forest Service, Natural Resources Canada, Edmonton, Alberta, Canada
Accepted for publication 11 February 2022.
2
ABSTRACT
Variation in rate of infection and susceptibility of Pinus spp. to the fungus
Cronartium harknessii (syn. Endocronartium harknessii), the causative agent
of western gall rust, has been well documented. To test the hypothesis that
there is a coevolutionary relationship between C. harknessii and its hosts, we
examined genetic structure and virulence of C. harknessii associated with
lodgepole pine (P. contorta var. latifolia), jack pine (P. banksiana), and their
hybrids. A secondary objective was to improve assessment and diagnosis of
infection in hosts. Using 18 microsatellites, we assessed genetic structure of
C. harknessii from 90 sites within the ranges of lodgepole pine and jack pine.
We identified two lineages (East and West, FST = 0.677) associated with host
genetic structure (r = 0.81, P = 0.001), with East comprising three sublineages.
In parallel, we conducted a factorial experiment in which lodgepole pine, jack
pine, and hybrid seedlings were inoculated with spores from the two primary
genetic lineages. With this experiment, we refined the phenotypic categories
associated with infection and demonstrated that stem width can be used as a
quantitative measure of host response to infection. Overall, each host
responded differentially to the fungal lineages, with jack pine exhibiting more
resiliency to infection than lodgepole pine and hybrids exhibiting intermediate
resiliency. Taken together, the shared genetic structure between fungus and
host species, and the differential interaction of the fungal species with the
hosts, supports a coevolutionary relationship between host and pathogen.
Cronartium harknessii E. Meinecke (syn. Endocronartium harknessii (J.P. Moore) Y Hiratsuka; syn. Peridermium harknessii J. P.
Moore), the causative agent of western gall rust (WGR), belongs to
the order Peridermium (Basidiomycota), which includes many of the
world’s most economically impactful plant pathogens (Pendleton
et al. 2014; Vogler and Bruns 1998; Wingfield et al. 2004). C. harknessii is closely related to other pine stem and cone rusts in the family Cronartiaceae (Pendleton et al. 2014), including C. quercuum
f. sp. fusiforme causing fusiform rust and C. ribicola (J.C. Fisch. in
Rabh.) causing white pine blister rust (Sniezko et al. 2014). Unlike
other closely related rust species, C. harknessii is considered autoecious and does not require an alternate host to complete its life cycle.
This pine-to-pine infection route makes C. harknessii a “short-cycled
species” (Vogler and Bruns 1998) and can result in rapid spread of
this pathogen across its native range. The range of C. harknessii
spans most of Canada and the United States (Hopkin et al. 1988;
Ramsfield and Vogler 2010), occurring on both natural Pinus host
species, including jack pine (P. banksiana), lodgepole pine (P. contorta var. latifolia), and ponderosa pine (P. ponderosa), as well as
exotic hosts such as Scots pine (P. sylvestris) (Peterson 1960). The
large geographical range of C. harknessii host species, as well as the
diversity in host genetics and resistance (Wu et al. 1996; Wu and
Ying 1998; Yanchuk et al. 1988; Yang et al. 1997, 1999), has created
an environment in which a pathogen could rapidly evolve, supporting a coevolutionary relationship between the different tree species
and the fungus (Reznick 2001). Coevolutionary relationships (for a
review, see Burdon and Thrall 2009 and Occhipinti 2013) have previously been observed for rust fungi (Hart 1988), including studies
of white pine blister rust, where host resistance and diverse landscape
genetics of both the fungus and the host were observed (Kim et al.
2003; Richardson et al. 2008).
Several studies have reported quantitative disease resistance to
C. harknessii in lodgepole pine, jack pine. and their hybrids
(Yanchuk et al. 1988; Yang et al. 1997, 1999), with some studies
reporting more resistance to C. harknessii in jack pine than lodgepole
pine (Wu and Ying 1998; Yang et al. 1997). Wu et al. (1996) also
reported increased resistance in lodgepole × jack pine hybrids in the
hybrid zone of Alberta relative to lodgepole pine. Major gene resistance and quantitative disease resistance in Pinus spp. to close relatives of C. harknessii have also been well documented, for example,
southern pines (P. taeda, P. virginiana, P. elliottii, and P. palustris)
resistance to C. quercuum f. sp. fusiforme (Schmidt et al. 2000;
Sniezko et al. 2014; Wilcox et al. 1996) and P. lambertiana, P. monticola, P. strobiformis, and P. flexilis resistance to C. ribicola (Kinloch
et al. 1970, 1999; Schoettle et al. 2014).
Many studies have examined infection of Pinus species by
C. harknessii and related Cronartium species with the goal of identifying and characterizing host resistance to the pathogen. In contrast,
far fewer studies have investigated the underlying coevolutionary
†
Corresponding author: J. E. K. Cooke; janice.cooke@ualberta.ca
C. McAllister and C. Cullingham contributed equally to this work.
Data Availability: Genetic data for lodgepole pine, jack pine, and C. harknessii
are available on Dataverse: https://dataverse.scholarsportal.info/dataverse/ualberta.
Funding: Funding for this research was provided to J. E. K. Cooke through
grants from Alberta Agriculture and Forestry and Alberta Innovates Bio Solutions
(grant AIBIO 14-009) and the Natural Sciences and Engineering Research Council of Canada Strategic Grants Program (STPGP 52100-18).
*The e-Xtra logo stands for “electronic extra” and indicates there are supplementary materials published online.
The author(s) declare no conflict of interest.
Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Keywords: community genetics, hybrids, Pinus banksiana, Pinus contorta,
population genetics, rust, western gall
Vol. 112, No. 8, 2022 1795
relationship of the Pinus–Cronartium pathosystems, including the
coevolutionary relationship of C. harknessii with lodgepole and jack
pine. However, understanding such relationships is important for the
long-term goals of breeding for durable resistance. In one of the only
studies to examine the relationship of C. harknessii with lodgepole
and jack pine at the population level, Li et al. (2001) used random
amplified polymorphic DNA (RAPD) markers to genetically distinguish C. harknessii collected from lodgepole and jack pine. An early
form of molecular markers, RAPD markers exhibit a number of limitations, including behavior as dominant markers (i.e., heterozygosity
cannot be determined) and challenges with reproducibility (Perez
et al. 1998). The study also used a limited sampling design, analyzing
68 isolates sampled at 13 locations in western Canada. Finally, no
genetic analyses of ancestry were conducted for the host pines to
confirm the species designations. Because two of the sampling locations were located within the lodgepole pine × jack pine hybrid
zone, which is now known to be considerably more complex than
when the Li et al. (2001) study was carried out (Burns et al. 2019;
Cullingham et al. 2012), it is possible that galls could have been
collected from hybrid pines within these locations.
Taking advantage of new molecular markers developed using
genomic data, together with a more comprehensive understanding of
the extent and mosaic nature of the lodgepole pine × jack pine hybrid
zone (Burns et al. 2019; Cullingham et al. 2012), we tested the
hypotheses that C. harknessii comprises two separately evolving lineages in the northern half of its range and that these lineages show
the hallmarks of coevolution with their lodgepole and jack pine hosts.
To address these hypotheses, we performed an analysis of the genetic
structure of C. harknessii across Canada, ranging from western British Columbia to eastern Ontario and north to the Northwest Territories, using microsatellite loci developed from a draft genome
assembly of C. harknessii. The resulting genetic structure of C. harknessii was then compared with the genetic structure of the two host
species (Burns et al. 2019) using a community genetic approach
(James et al. 2011) to determine whether genetic differentiation
covaries with host genetic differences. We then used the fungal
genetic data to select C. harknessii genetic material associated with
either lodgepole pine or jack pine to carry out inoculation trials
comparing relative virulence in lodgepole pine, jack pine, and lodgepole × jack pine hybrid provenance material. As part of this study,
our aim was to improve assessment of infection in juvenile trees to
decrease the time of assessing symptoms and to decrease the subjective nature of the disease scoring. To assess host–pathogen interactions and develop a refined phenotyping schema, we used the tornneedle method developed by Myrholm and Hiratsuka (1993),
because of its increased effectiveness and reliability of spore delivery
over that of other inoculation methods (Blenis and Hiratsuka 1986;
Blenis and Pinnell 1988; Burnes et al. 1988; Myrholm and Hiratsuka
1993). Our premise was that early, visually discernable, and reliably
detected symptoms of disease preceding gall formation will enable
earlier scoring of more virulent pathogen variants and/or more susceptible host genotypes. We measured disease progression using this
refined categorical visual index, adapted from Klein et al. (1991), as
well as using stem as a continuous variable that can be more objectively measured. Evidence of shared genetic structure between the
fungus and host species, and differential interaction of the fungal
species with the hosts, would support the hypothesis of a coevolutionary relationship between host and pathogen.
MATERIALS AND METHODS
Fungal samples. A total of 425 single-gall samples of C. harknessii were included in this study. Samples were collected between
May and July (2014 to 2016) from 90 sites within the distribution
range of lodgepole pine and jack pine across six Canadian provinces
and territories (Table 1). Most of the samples were collected as
mature sporulating galls, from which spore propagules of the rust fungus were extracted following the procedure described by Ramsfield
1796 PHYTOPATHOLOGY ®
and Vogler (2010) and stored at –80 C. To supplement sample numbers from sites where not enough sporulating galls could be found,
immature (nonsporulating) and late sporulating galls were also collected, and slices of the inner wood tissue were harvested from these
and preserved at –80 C. Thus, 138 samples were made up of or
included a substantial amount of inner gall wood tissue (Northwest
Territories = 47, Alberta = 28, Manitoba = 24, British Columbia = 17,
Saskatchewan = 17, Ontario = 5).
Fungal DNA extraction and sample identity. DNA was extracted
using the CTAB (hexadecyl trimethyl ammonium bromide)-based
protocol as described by Roe et al. (2010), using 60 mg of starting material to improve extraction yields. Material for extraction
was ground into a fine powder under liquid nitrogen using a Retsch
MM301 Mixer Mill (Haan, Germany) for 90 s at 25 Hz, and inner
gall tissue was ground by hand in liquid nitrogen using mortar and
pestle. DNA yields and purity were assessed with Infinit M200 PRO
NanoQuant spectrometer (Tecan Trading AG, Zurich, Switzerland),
and working solutions were adjusted to 25 îg/µl for spore samples
and 40 îg/µl for gall tissue samples. The identity of C. harknessii
was confirmed for selected samples from various locations across the
sampling area (Supplementary Table S1) by sequencing the ITS and
IGS-1 regions of the ribosomal RNA gene cluster and comparing the
sequences generated to records of C. harknessii and other related rust
fungi in GenBank (sequencing methods are included in the supplementary material, Supplementary Fig. S1).
Microsatellite development and typing. A customized Python
script (available upon request) was used to scan the 92.0-Mb draft
genome of C. harknessii (GCA_000500795) for bi- to hexa-nucleotide
tandem sequences with at least three repeats. For each of the 3,654
loci detected, a pair of oligonucleotide PCR primers were predicted
within the 150-bp flanking regions using Primer3 web software vers.
4.0.0 (Koressaar and Remm 2007; Untergasser et al. 2012), with the
following parameters: optimum Tm = 60 C and of 18- to 23-bp
primer lengths. A set of 48 markers with di-, tri- or tetra-nucleotide
repeat motifs were initially selected and evaluated for PCR amplification and polymorphism. Each marker was chosen from a unique
assembly scaffold and included a perfect tandem repeat motif, with a
minimum of nine, eight, and six counts for di-, tri- and tetra-nucleotide
motifs, respectively.
The 48 selected markers were screened against a panel of 22 exploratory samples of C. harknessii from British Columbia, Alberta, and
Saskatchewan. A mock control made up of DNA from healthy jack
pine tissue was also included to ensure that markers did not amplify the
host DNA. A cost-effective approach with fluorescent-labeled M13
universal primer (59-TGTAAAACGACGGCCAGT-39) (Schuelke
2000) was used for genotyping. The sequence-specific reverse primer
was also modified by attaching a GTTT “PIG-tail” to its 59 end to
improve genotyping accuracy (Brownstein et al. 1996). All primers
were ordered from Integrated DNA Technologies (Coralville, Iowa)
and four fluorescent labels (FAM, NED, PET, and VIC) were used.
From the 45 markers, we identified 18 microsatellite markers
that were amplified in two multiplex panels of nine markers each
(Supplementary Table S2). Genotyping of population samples was
carried out in 15 ll of PCR reactions containing 1.5 µl of 10×
ThermoPol Buffer (New England BioLabs, Ipswich, MA), 1.5 µl of
dNTPs (2 mM each) (Invitrogen, Waltham, MA), 0.48 µl of forward primer (2.5 µM), 0.48 µl of reverse primer and M13 primer
(10 µM), Taq DNA polymerase (BioLabs), and 1.5 µl of DNA template. PCR cycling conditions were as follows: 5 min of initial
denaturation at 94 C, followed by 30 cycles of 30 s at 94 C, 45 s
at 58 C, and 45 s at 72 C, then eight cycles of 30 s at 94 C, 45 s
at 53 C, and 45 s at 72 C, and 10 min of final extension at 72 C.
Reaction products were pooled according to two multiplex panels
of nine markers each; thereafter, 2.5 µl of each pool was mixed
with 0.25 µl of GeneScan 500 LIZ size standard (Applied Biosystems, Waltham, MA) and 8 µl of HI-DI Formamide and run on an
ABI 3730 DNA Analyzer (Applied Biosystems). Results were analyzed with GeneMapper v4.0 (Applied Biosystems).
TABLE 1. Sampling information for Cronartium harknessii samples collected and genotyped at 18 microsatellite locia
Province/siteb
AB
ABW
CH
ED
FM
HI
HK
HO
MT
SH
SL
WC
WC2
BC
BCC
BCGI
BCN
BCQ
BCS
BCSM
BCSR
MT
VI
MB
MB0
MB1
MB2
SKMB
WB
NT
NT1
NT2
NT3
NT4
ON
CL
GL
ML
ON0
ON2
ON3
ONH
ONP
ONR
ONS
TB
NA
SK
HB
SK1
SKMB
SKN
a
Jack
pine
Lodgepole
pine
28
75
15
20
NA
Total
6
109
15
20
2
14
15
9
2
6
8
7
9
2
97
25
31
2
1
8
8
3
3
16
43
1
5
15
16
6
47
4
10
18
15
87
13
12
10
1
6
4
7
4
10
2
13
5
28
4
9
7
8
2
12
2
15
9
2
6
8
5
2
9
2
97
25
31
2
1
8
8
3
3
16
43
1
5
15
16
6
47
4
10
18
15
87
13
12
10
1
6
4
7
4
10
2
13
5
28
4
9
7
8
Comparative
analysis
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
Host pine species for the collected C. harknessii samples are indicated
based on host ranges. Individuals that were sampled within the lodgepole
pine × jack pine hybrid zone are listed in the not applicable (NA) column.
Sampling sites used for the comparative population structure analysis with
the pine host are indicated with a “y” in the comparative analysis column.
b
AB = Alberta; ABW = Alberta west; BC = British Columbia; BCC = British
Columbia central; BCGI = British Columbia Glacier; BCN = British
Columbia north; BCQ = British Columbia Quesnel; BCS = British Columbia
Castlegar; BCSM = British Columbia Smithers; BCSR = British Columbia
Salmon River; CH = Cypress Hills; CL = Chapleau; ED = Edmonton;
FM = Fort McMurray; GL = Gibson Lake; HB = Hudson Bay; HI = Hinton;
HK = Hotchkiss; HO = Hondo; MB = Manitoba; MB0 = Manitoba 0; MB1 =
Manitoba 1; MB2 = Manitoba 2; ML = Malette; MT = Mountain; NA = not
available; NT = Northwest Territories; NT1 = Northwest Territories 1;
NT2 = Northwest Territories 2; NT3 = Northwest Territories 3; NT4 =
Northwest Territories 4; ON = Ontario; ON0 = Ontario 0; ON2 = Ontario 2;
ON3 = Ontario 3; ONH = Ontario Huung; ONP = Ontario Phillip; ONR =
Ontario Dryden; ONS = Ontario seed; SH = Swan Hills; SK = Saskatchewan;
SK1 = Saskatchewan 1; SKMB = Saskatchewan–Manitoba; SKN =
Saskatchewan north; SL = Smoky Lake; TB = Thunder Bay; VI = Vancouver
Island; WB = Woodbridge; WC = Whitecourt; and WC2 = Whitecourt 2.
Microsatellite and community ecology analyses. Before analysis of genetic data, the microsatellite dataset was clone corrected
in R ver. 4.0.4 (R Core Team 2021) using the poppr package ver.
2.9.1 (Kamvar et al. 2014) to ensure only one representative of
each multilocus genotype was included. We used the genetic distance option for clone correction to make sure individuals with
missing data were not included as unique genotypes. To identify
populations without a priori knowledge, we used STRUCTURE ver.
2.3.4, using the admixture model (Falush et al. 2003; Pritchard et al.
2000), with allele frequencies correlated, and examined likelihoods
for K = 1 to 5, with five iterations each of 100,000 burn in Markov
Chain Monte Carlo (MCMC), and 250,000 data collection MCMC.
We examined the resulting output using STRUCTURE HARVESTER
(Earl and vonHoldt 2012) to identify populations based on both the
mean log likelihood (Ln) probability of the data (Pritchard et al.
2000), and the DK statistic (Evanno et al. 2005). We further examined the clusters identified in STRUCTURE (using the same parameters
as above, except testing K = 1 to 10 for one of the clusters, see
Results) to detect additional hierarchical population structure. We
generated a principal component analysis (PCA) plot and a distancebased network in R to confirm the clustering solution using the vegan
package ver. 2.5-4 (Dixon 2003; Oksanen et al. 2018) and poppr,
respectively. Additionally, we used the find.clusters, Bayesian information criterion (BIC) and percent variation methods, and dapc functions in the R package adegenet ver. 2.1.3 (Jombart et al. 2010). We
estimated genetic diversity using Shannon’s index (Shannon 1948),
Simpson’s index (Simpson 1949), and a measure of evenness (E5;
Ludwig and Reynolds 1988) using rarefaction and all samples in
poppr for each of the identified clusters (see Results). Genetic differentiation among clusters was estimated in Arlequin 3.5 using the FST
like estimator (Excoffier and Lischer 2010). Significant linkage identified via the index of association (IA) (Brown et al. 1980) and r d
(Agapow and Burt 2001) was used to determine whether clonal versus sexual reproduction was more likely within C. harknessii using
999 permutations. To determine the most likely reproductive mode,
we examined multilocus linkage in a hierarchical fashion: the entire
dataset, within each main lineage, and within sublineages. Genetic
differentiation and linkage were estimated using the clonecorrected data.
To examine whether the observed genetic structure was spatially
associated with host species lodgepole pine and jack pine, we paired
fungal samples with previously genotyped pine samples. Pine genotypes were generated using 28 species discriminating single nucleotide polymorphisms developed to refine their spatial distribution (for
genotyping details, see Burns et al. 2019). We grouped fungal and
pine samples based on geographic proximity using ArcMap10.5
(ESRI, Redlands, CA), where samples within 100 km were considered a sampling site (and <100 km when within the lodgepole × jack
pine hybrid zone). We examined genetic population structure for
both pine and fungus using PCA of the site-specific allele frequencies
using the package vegan in R. To examine whether the host species’
genetic structure was in concordance with the fungal population
structure, we compared the resulting PCA plots using a Procrustes
transformation (Gower 1975). This transformation calculates a
correlation-type coefficient “r” that indicates whether the plots share
the same structure.
Plant tissue, experimental design, and inoculation. To examine the interaction between different sources of the fungal pathogen
and disease progression in lodgepole, jack pine, and lodgepole ×
jack pine hybrids, we obtained pine seed from the Alberta Tree
Improvement and Seed Centre (ATISC) (Smoky Lake, Alberta,
Canada) and the National Tree Seed Centre (Fredericton, New
Brunswick, Canada). Seeds were obtained for five provenances:
Jacobie Creek (lodgepole pine; British Columbia), Shelter Creek
(lodgepole pine; Alberta), Blue Ridge (lodgepole pine × jack pine
hybrids; Alberta), Stoney Mountain (jack pine; Alberta), and Cyril
Lake (jack pine; Ontario). An overview of the seed origin, provenance, and species can be found in Table 2. We selected Alberta
Vol. 112, No. 8, 2022 1797
provenances based on the hybrid zone delineation by Cullingham
et al. (2012) to ensure accurate species assignment. Species assignments for Alberta provenances were confirmed by ATISC.
Seeds were surface sterilized in 1% vol/vol sodium hypochlorite
(bleach) according to Groome et al. (1991), then placed in sterile
stratification boxes between moistened Versa-Pak germination paper
(Seedburo, Des Plaines, IL) and subjected to moist chilling in the
dark at 4 C for 2 weeks. Moist-chilled seeds were surface sterilized
using the same protocol before sowing in 6- × 12-cell styroblocks
(61 cu. in/cavity, Beaver Plastics, Spruce Grove, Canada) containing
a prewetted 2:1 peat/vermiculite mixture. Two to three seeds were
sown per cavity, then lightly covered with dry vermiculite. The
experiment was carried out in a controlled environment growth
chamber with illumination of 180 lmol, 16 h photoperiod, 22 C
days/16 C nights, and 55% humidity. Styroblocks were enclosed in
tented clear polyethylene bags for the first 2 weeks to create the high
humidity conditions facilitating germination. After removal of the
styroblocks from the bags, seedlings were fertilized weekly with
0.5 g liter−1 per block N-P-K, 20-20-20, for the remainder of the
experiment, with seedlings being watered as needed. Plants were
culled to one seedling/cell 3 weeks after sowing. A fully randomized blocked design was used for the experiment, with n = 165
cells/provenance.
Eight weeks after sowing, plants were inoculated using the tornneedle method described by Myrholm and Hiratsuka (1993) with
one of three treatments: 1) “mock” inoculum, consisting of only
talc powder; 2) “C.harkWEST” inoculum, consisting of C. harknessii spores obtained from multiple galls on lodgepole pine near Hinton, Alberta; or 3) “C.harkEAST” inoculum, consisting of a mix of
genetically identical C. harknessii spores from the E1 lineage (see
Results) obtained from jack pine in northern Ontario. All spores
were mixed together in a 3:1 ratio with talc (spores/talc) before
inoculation. A total of 50 to 55 seedlings per provenance were
inoculated with each of the three treatments. Directly after inoculation, blocks were moistened with water, covered with clear polyethylene bags as described above to increase humidity and
facilitate infection, and incubated at 16 C in the dark for 2 days
(Myrholm and Hiratsuka 1993). After 48 h, bags were removed,
and previously described lighting and temperature conditions were
reinstated for the remainder of the experiment. Directly after inoculation, remaining spores were tested for germination rates by plating on 1.5% water agar at ambient temperature. After 24 h, both
C.harkWEST and C.harkEAST inoculum showed average germination
rates of 23%.
To examine potential differences between host pine species,
including environmental factors, and disease progression we conducted a phenology study covering a west to east transect through
Alberta. Six sites were visited multiple times for 7 weeks, and gall
stage was recorded (Supplementary Table S3).
Phenotyping. Plants were phenotyped weekly for WGR disease
progression from 8 to 26 weeks postinoculation (wpi) using a categorical visual assessment scale based on that of Klein et al. (1991).
This modified scale included subcategories of the categories used
by Klein et al. (1991), giving a total of 10 phenotypes. A brief
description of each phenotypic category can be found in Table 3
and images are shown in Supplementary Figure S2.
To develop a quantitative, more objective means to phenotype disease severity, we measured stem width and dry weight of seedlings
at 26 wpi. Plants were cut at the base of the hypocotyl, just above the
soil, and terminal buds were removed. Stems were carefully stripped
of all needles, lateral branches, and buds before taking stem-width
measurements using a digital caliper (Marathon, No. CO 030200).
Measurements were taken at the base of the hypocotyl as well as at
either the point of inoculation or at the point of greatest stem width
associated with the developing gall. In a small number of cases
where swelling and gall formation extended to the base of the stem,
hypocotyl base measurements were exchanged for measurements
taken below the removed terminal bud. Data were expressed as a
difference in stem width, calculated by subtracting the base hypocotyl measurement from the point of inoculation/point of greatest
stem width. Stems were flash frozen in liquid nitrogen after caliper
measurements.
Quantitative phenotypic data (stem width and stem weight)
were analyzed to examine their relationship to visual disease
phenotype using analysis of variance. Data were examined using
the Shapiro-Wilk test for normality and transformed using BoxCox before these analyses. After significant association between
qualitative phenotype categories and quantitative data (see
Results), we used a Tukey’s posthoc test to further explore this
relationship. We then used linear regression to assess whether
the two quantitative phenotypes were highly correlated. After
linear regression, we used stem-width measures to examine the
effect of treatment and provenance using a generalized linear
mixed effect model (GLMM) implemented in the lme4 package
ver. 1.1-26 (Bates et al. 2015), controlling for experimental
block effects (random effect) using a Gaussian distribution with
a log link. For treatment, effects were measured based on comparison with the mock, whereas for the provenances, they were
measured in comparison with Jacobie Creek. Analyses were
conducted in R 3.3.2, with the exception of the GLMM (conducted in R 4.0.4).
RESULTS
Microsatellite and community ecology analyses. We generated genotypes for 411 C. harknessii samples using 18 microsatellite loci. The percentage of missing data was low (1.2%), and there
were no more than three loci missing for any individual genotype,
that is, 339 individuals had complete genotypes. After clone correction, a total of 124 unique genotypes were represented. Using
these genotypes, we identified two population clusters with STRUCTURE, which we will define as “East” (N = 50) and “West” (N = 74)
for the remainder of the article. This was supported by DK (DK2 =
4,460), likelihood, and Q-value plots (Fig. 1); genetic differentiation was highly significant among clusters (FST = 0.677, P =
0.001). STRUCTURE analysis of the East and West populations separately indicated that the West population was one single cluster,
whereas the East was comprised of additional clusters (Fig. 1).
TABLE 2. Overview of pine seed provenances used in this studya
Provenance
Jacobie Creek
Shelter Creek
Blue Ridge
Stoney Mountain
Cyril Lake
a
Received
from
Accession
or seedlot
Collection site
Species
Sampling coordinates
Elevation
(m)
NTSC
ATISC
ATISC
ATISC
NTSC
7172010.0
2932
2312
2368
9630232.0
British Columbia, Canada
Alberta, Canada
Alberta, Canada
Alberta, Canada
Ontario, Canada
Pinus contorta var. latifolia
P. contorta var. latifolia
P. contorta × P. banksiana
P. banksiana
P. banksiana
52.53333, –121.88330
54.4000, –119.35000
54.10000, –115.53333
56.2667, –111.60000
50.50000, –84.41666
990
1,425
777
610
304
Seed represents bulked seed collections from either the Alberta Tree Improvement and Seed Centre (ATISC) (Smoky Lake, Alberta, Canada) and the National
Tree Seed Centre (NTSC) (Fredericton, New Brunswick, Canada).
1798 PHYTOPATHOLOGY ®
Two and three clusters were supported for the East cluster, which
we examined further using PCA, a genetic distance network, and
discriminant analysis of principal components (DAPC). The PCA
and network clearly demonstrated there were two clusters (Supplementary Fig. S3), whereas the third cluster was weakly defined.
The DAPC find.clusters BIC plot did not present a clear solution
for which K was most supported. Using the interpretation from the
authors of the program, we chose to investigate K = 3 and K = 4
(Supplementary Fig. S4). In both DAPC analyses, we retained 18
PCs (which explained at least 80% of the variation), and we
retained all discriminant functions. DAPC de novo assigned clusters for K = 3 and K = 4 were an exact match to the substructure
found with STRUCTURE and PCA analyses. Differentiation among
clusters was significant (P = 0.001), with differentiation greatest
between the East and West clusters (E1-West = 0.816, E2-West =
0.775, E3-West = 0.815), whereas differentiation within in the East
clusters was also high (E1-E2 = 0.0.756, E1-E3 = 0.730, E2-E3 =
0.763). We present the geographic distribution for three East clusters,
which demonstrates some geographic partitioning; however, individuals from each cluster were often colocated (Fig. 2). The West cluster
comprised 183 individuals and 74 genetic clones, whereas the East
cluster had 228 individuals and only 50 clones. Diversity and evenness were higher in the West cluster than in the East clusters (Table 4).
Measures of linkage disequilibrium were significant using the entire
dataset (IA = 7.073, P = 0.001) and for the East cluster (IA = 3.572,
P = 0.001); however, linkage was not significant at the lowest hierarchical level (West, East 1, East 2, and East 3) (Table 4).
We selected 280 pine samples that had been previously genotyped from over 900 samples (Burns et al. 2019). These individuals
were located less than l100 km from our fungal samples (N = 166),
which resulted in 14 sampling groups (Supplementary Fig. S5).
PCA of the allele frequencies for both datasets were generated and
found to be significantly correlated based on a Procrustes analysis
(r = 0.81, P = 0.001; Fig. 2). The East population of C. harknessii
was aligned with jack pine, whereas the West population was
aligned with lodgepole pine.
Phenotyping. Visual analysis of all seedlings from 8 to 26 wpi
showed that mock inoculated individuals displayed a variety of phenotypes from complete wound healing to overall stem swelling not
associated with the point of inoculation. These phenotypes correlate
with categories 0, 1a, 2a, and 3a in the phenotypic index developed
for this study (Table 3; Supplementary Fig. S2). Phenotypes 0, 1a,
2a, and 3a were observed in both mock and C. harknessii inoculated individuals but did not lead to gall formation, and thus were
not included in further analyses. Gall formation (phenotype 3d) was
reliably preceded by phenotypes 2c (necrotic canker), 3b (swelling
without discoloration), and 3c (swelling with discoloration) and
were relatively straightforward to identify. In contrast, phenotypes
1b (vibrant discoloration) and 2b (discolored, indented, and/or scablike canker), although not occurring in mock individuals and therefore likely associated with infection, were more subjective measures
and did not progress to gall formation in all instances. Progression
of gall maturation along a transect within Alberta encompassing
pure lodgepole and jack pine stands and within the lodgepole ×
jack hybrid zone showed a trend toward earlier maturation in jack
pine-dominated locations, followed by locations within the hybrid
zone, then the lower elevation lodgepole pine stands surveyed in
this study (Fig. 3).
In inoculation experiments, a proportion of individuals developed
galls regardless of provenance or C. harknessii spore origin (C.harkWEST
or C.harkEAST) from 8 to 26 wpi (Fig. 4; Supplementary Fig. S5).
The number of individuals per provenance that developed phenotypes
related to disease, including galls, followed the pattern of jack pine
< hybrid < lodgepole pine for both sources of inocula. Inoculation of
jack pine with C.harkEAST led to a greater proportion of individuals
developing disease-associated phenotypes earlier and a greater proportion of individuals developing galls than inoculation with C.harkWEST.
Individuals from the Cyril Lake provenance inoculated with
C.harkWEST showed virtually no gall formation, whereas those inoculated with C.harkEAST led to approximately 40% of seedlings exhibiting
galls by 26 wpi. Inoculation of hybrids (Blue Ridge provenance)
resulted in 50% of seedlings exhibiting galls by 26 wpi with both
inocula. Lodgepole pine from both Shelter Creek and Jacobie Creek
started to exhibit gall formation at 15 wpi for the C.harkWEST inocula
and over 75% exhibited galls by 26 wpi. The C.harkEAST was not as virulent in lodgepole pine, with galls starting to show at week 20, with
<60% of seedlings exhibiting galls in both provenances at 26 wpi.
Differences in stem width (N = 589, df = 8, F = 92.48, P < 0.001)
and stem dry weight (N = 172, df = 8, F = 19.20, P < 0.001) measured at 26 wpi (Fig. 5A and B; Supplementary Fig. S6) were statistically significant across qualitative disease phenotypes (Fig. 5A
and B) following normalization using Box-Cox power transformation (Supplementary Fig. S7). Tukey’s posthoc tests indicated that
gall formation (phenotype 3d) had both significantly higher stemwidth difference and dry weight relative to all other phenotypes.
Smaller but still significant increases in both quantitative parameters
were also observed in relation to phenotype 3c (swelling with necrosis or discoloration), compared with the other phenotype categories.
TABLE 3. Enhanced phenotypic scale of western gall rust disease progression in young pine seedlings inoculated via the torn-needle method described by
Myrholm and Hiratsuka (1993)
Phenotypic
category
General
phenotype
0
1a
1b
2a
2b
2c
Healed
Discoloration
Discoloration
Canker
Canker
Canker
3a
Swelling
3b
Swelling
3c
Swelling
3d
Gall
Leads to gall
formation
Phenotypic description
Fully healed wound
Yellow to pink, with no visible canker formation; discoloration is not vibrant
Vibrant purple to red, with no visible canker formation
Unhealed wound: no discoloration, a scab or wound that did not heal fully
Generally purple or red discolored, indented, and/or scab-like
Necrotic tissues developed; these may be directly associated with point of
inoculation or with an area near the inoculation point
General swelling of the stem: swelling is not restricted to area around
inoculation point, may be associated with general variation in stem width or
swelling caused by bending of the stem; not associated with discoloration
(1b) or canker formation (2b, 2c)
Enlargement of stem specifically associated around point of inoculation;
swelling is in a ball or gall shape; no discoloration or canker formation
associated with swelling
Enlargement of stem specifically associated around point of inoculation;
swelling is in a ball or gall shape; also associated with discoloration (1b) or
canker (2b, 2c)
Fully formed gall; well-defined start and end points of gall along stem length
(clear constrictions)
No
No
Yes
No
Yes
Yes
No
Yes
Yes
Yes
Vol. 112, No. 8, 2022 1799
Fig. 1. Cronartium harknessii STRUCTURE results for all samples (top panel), the East cluster (middle panel), and the West cluster (bottom panel). Each individual is
represented by a bar along the x-axis. Included are the admixture plots, which include ancestry coefficients (y-axis) for individuals (x-axis) and the DK/mean probability plots for each analysis (number of K tested: 1 to 5, 1 to 10 for the West). The number of populations supported for all the data were K = 2, K = 2 and 3 for
the East and K = 1 for the West.
Fig. 2. Distribution and population structure for all Cronartium harknessii samples, with sample sizes at each site represented by the size of the circle (N = 7
to 31). Included in the inset is the Procrustes plot, which shows the amount of error (indicated by the arrows) between the principal component analysis positioning of the C. harknessii sample sites versus lodgepole pine and jack pine sample sites. Colors of the points correspond to the C. harknessii lineages
(blue = East, yellow = West). WGR, western gall rust, Cronartium harknessii.
1800 PHYTOPATHOLOGY ®
Linear regression analysis comparing the difference in stem-width
and dry-weight data showed a positive correlation R2adj = 0.41
(Fig. 5C, N = 172). Therefore, we focused on using stem width difference as a quantitative rather than categorical measure of C. harknessii infection.
To further evaluate the use of difference in stem width as a quantitative measure of C. harknessii infection, a generalized linear mixed
model was used to analyze effects on difference in stem width by
provenance, treatment (mock, C.harkWEST, or C.harkEAST), and provenance by treatment interaction, while controlling for block. The
TABLE 4. Genetic diversity and multilocus linkage estimates for Cronartium harknessii across the entire dataset (All), the highest hierarchical clusters (East,
West), and the clusters identified in the East (E1, E2, and E3)a
Cluster
All
East
E1
E2
E3
West
a
Shannon’s
index
3.75
(3.775, 4.065)
3.01
(2.88, 3.132)
1.10
(0.851, 1.487)
2.31
(1.764, 2.184)
1.89
(3.602, 3.925)
3.54
(3.602, 3.925)
Simpson’s
index
0.949
(0.939,
0.879
(0.862,
0.431
(0.306,
0.840
(0.778,
0.800
(0.765,
0.955
(0.949,
0.556)
0.894)
0.556)
0.945)
0.854)
0.971)
Evenness
0.447
(0.330,
0.376
(0.347,
0.379
(0.300,
0.607
(0.345,
0.718
(0.605,
0.638
(0.493,
IA
r d
7.073
0.418
3.572
0.251
–0.556
–0.081
–0.328
–0.043
–0.179
–0.031
–0.009
–0.001
0.452)
0.408)
0.383)
0.668)
0.767)
0.648)
The linkage measures (IA and r d ) in bold are significant (P = 0.001) based on permutation testing, and confidence intervals for the diversity measures are
included in parentheses. The diversity measures were estimated using the entire data set (N = 411; E1 = 96, E2 = 45, E3 = 87, West = 183), whereas the
linkage estimates were estimated using the clone-corrected data.
Fig. 3. Progression of gall maturation monitored along a transect of pine depicted from west to east. The westernmost locations represent lodgepole pine,
[William A. Switzer Provincial Park (WASPP), W. McLeod River Provincial Recreation Area (W MRPRA), and E. McLeod River (E MRPRA)]; the intermediate locations represent lodgepole × jack pine hybrids [Windfall and Bridge Lakes Natural Area (BLNA)], and the easternmost location represents jack pine
(Conklin Rd). Sampling sites are ordered from west at the top of the plot to east at the bottom of the plot. Host species assignments are based on the analyses
of Burns et al. (2019). Gall stage was recorded during Spring 2021 at these six sites. Week count was initiated at the first observed occurrence of aecia starting to develop at a site and continued until aeciospores were either present or had been released.
Vol. 112, No. 8, 2022 1801
mixed model would not converge with the provenance by treatment
interaction; therefore, we examined this interaction using a generalized
linear model. There were two significant interactions: C.harkWEST
with Cyril Lake (t = –3.184, P < 0.01) and with Stoney Mountain
(t = –2.927, P < 0.01). The mixed linear model examining provenance and treatment as individual fixed effects with block as a random effect indicates the two lineages had significant effects on
stem width compared with the mock (P < 0.001), and the provenances had significantly different stem widths, with jack pine < hybrid
pine < lodgepole pine (Table 5; Fig. 6A). To summarize, inoculation with either C.harkWEST or C.harkEAST significantly increased
stem-width difference relative to the mock inoculated individuals in
both lodgepole pine provenances (Shelter Creek and Jacobie Creek)
(Fig. 6A). In contrast, inoculation with C.harkEAST but not
Fig. 4. Percentage of lodgepole, hybrid (lodgepole × jack), and jack pine seedlings from five provenances showing disease progression every 2 weeks from 8 to
26 weeks postinoculation (wpi). Spores for inoculation were harvested from sporulating galls near Hinton, Alberta, Canada (C.harkWEST inoculum) or northern
Ontario, Canada (C.harkEAST inoculum). Seedlings were phenotyped using the new phenotypic scale designed in this study (Table 2; Supplementary Fig. S2). Mock
inoculated seedlings were not included because they did not display these phenotypic categories. N = 34 to 44 for each provenance per treatment over time.
1802 PHYTOPATHOLOGY ®
C.harkWEST resulted in significant increases in stem-width difference
in both jack pine provenances (Stoney Mountain and Cyril Lake),
whereas inoculation with C.harkWEST but not C.harkEAST
led to significant increases in stem-width difference for the hybrid
provenance (Blue Ridge). These results for stem-width differences mirror the trends observed for the percentage of galls formed across provenances for the two inocula (Fig. 6B). No significant differences were
observed between mock inoculated individuals across provenances.
Fig. 5. Assessment of western gall rust infection using novel quantitative measures, stem-width difference (mm), and dry weight (mg), relative to traditional
qualitative phenotypic indexing using one-way analysis of variance and mixed effect model analyses. Tukey’s posthoc tests were used to assess significant differences among groups A and B, a = 0.05. A, Stem-width differences (mm) at 26 weeks postinoculation (wpi) (y-axis) versus qualitative disease phenotype
(x-axis). B, Dry weight from a subset of samples collected at 26 wpi (y-axis) versus qualitative disease phenotype (x-axis). C, Linear regression analysis of
the normalized quantitative data, difference in stem width (mm) versus dry weight (mg).
Vol. 112, No. 8, 2022 1803
DISCUSSION
In Canada, lodgepole pine and jack pine are economically and
environmentally important pine species. C. harknessii does not often
cause mortality in mature pines, but it can result in timber volume
reduction as a result of defects and weak points leading to stem
breakage and can have mortality impacts on juvenile trees (Woods
et al. 2000). Given that much of the lodgepole pine distribution in
North America has been impacted by mountain pine beetle (Audley
et al. 2020; Hodge et al. 2017), the age structure has shifted to juvenile trees, which are more susceptible to major stem damage (Woods
et al. 2010). Combining this with impacts of climate change and the
increase in replanting of harvested natural forest areas, C. harknessii
has the potential to become an even more significant pathogen in
Canada and the United States (Kliejunas 2011). Management of
C. harknessii in this changing landscape is dependent upon understanding the diversity of C. harknessii in natural populations, determining whether coevolution of pathogen and host species is
occurring and how this relates to virulence of the pathogen because
deployment of resistant stock has been identified as a key management strategy for this pathogen (Weng et al. 2015).
Our microsatellite data support previous RAPD data identifying two
genetic lineages of C. harknessii (Li et al. 2001). Although these two
lineages are significantly differentiated and strong linkage suggests
there is no sexual reproduction between the East and West, ITS and
IGS-1 sequencing does not support the recognition of these lineages as
distinct species (Supplementary Fig. S1). In agreement with Yang et al.
(1999), we found that the geographic range of each lineage is highly
correlated with the geographic ranges of their host species. Our results
conflict with Li et al. (2001) because we found the West lineage to be
more genetically diverse than the East lineage. However, the East lineage showed more genetic structure, including additional lineages. The
lineages in the east did not show strong geographic structuring, but we
do note that the E1 lineage was only found in the east, whereas E2 and
E3 lineages were found both in the central and eastern portions of the
C. harknessii range (Fig. 2). We also did not find evidence of sexual
reproduction among the East lineages despite finding individuals from
different lineages in similar locations, which warrants additional investigation into potential differences in virulence.
Using a community genetics approach, we were able to investigate
the relationship of C. harknessii lineages and host species within the
mosaic hybrid zone, where lodgepole pine transitions to jack pine.
Yang et al. (1999) hypothesized that there are differing levels of
gene exchange and recombination between fungal lineages in the
hybrid zone causing differences in levels of infection in areas of close
proximity. In this study, the C. harknessii western genetic lineage
was never found on jack pine, nor was the C. harknessii eastern
genetic lineage found on lodgepole pine. Indeed, the two lineages
were never found together at a single sampling location. Also,
TABLE 5. Results from generalized linear mixed model of a full factorial
growth chamber experiment examining the impact of five pine provenances
and three inoculation treatments on difference in stem width in seedlings 26
weeks postinoculationa
Factor
(Intercept)
C.harkEAST
C.harkWEST
Shelter Creek (lodge)
Blue Ridge (hybrid)
Cyril Lake (jack)
Stoney Mountain
(jack)
a
Estimate
Standard
error
t value
P value
0.704
0.655
0.846
0.109
–0.242
–0.521
–0.416
0.084
0.078
0.075
0.055
0.068
0.083
0.075
8.373
8.392
11.224
1.979
–3.532
–6.240
–5.547
<0.001
<0.001
<0.001
0.048
<0.001
<0.001
<0.001
Seedlings were inoculated via torn-needle method 8 weeks after sowing.
Difference in stem width was determined by measuring the difference
between the point of inoculation or the point of greatest swelling near the
point of inoculation and the base of the hypocotyl.
1804 PHYTOPATHOLOGY ®
several of our gall collection locations were within or near the
approximated hybrid zone, and we did not detect any evidence of
sexual reproduction between the lineages. These data suggest the two
lineages are distinct to each host species. However, additional intensive sampling in the hybrid zone would be required to determine
whether fungal recombinants occur between the two lineages when
they are in close proximity.
The mechanism leading to differentiation into distinct genetic lineages likely includes intrinsic and extrinsic factors. For intrinsic factors, none of the lineages show evidence of sexual reproduction
between them. This will limit opportunities for admixture between
the lineages. For extrinsic factors, environmental gradients, including
daily temperatures during the early growing season, and elevation
can influence phenology in this pathosystem (Yang et al. 1997),
which can lead to asynchronous sporulation of C. harknessii across
the zone of lodgepole × jack pine introgression. These environmental
gradients also influence the host species, which can further promote
asynchronous sporulation. Elevation, moisture, and temperature
shape the zone of lodgepole and jack pine introgression (Burns et al.
2019; Cullingham et al. 2012), and they influence growth within species (O’Reilly and Owens 1989; Wang et al. 2003). C. harknessii
sporulation needs to be timed to the new growth and extension of
leaders (Moltzan et al. 2001). Based on monitoring throughout the
2021 season, gall maturation in sites from lodgepole pine-dominated
western Alberta was later than jack pine-dominated sites in eastern
Alberta. From this, we hypothesize that asynchrony in sporulation
has contributed to the observed genetic distinction between the two
primary lineages of C. harknessii. There is limited information on
the sexual reproduction of this species; therefore, additional investigation will be required to understand the factors that limit sexual
reproduction among different lineages.
The genetic differentiation between the eastern and western lineages of C. harknessii was accompanied by differences in virulence,
which in turn differed according to host species. Each of the two C.
harknessii lineages was most virulent on their geographically associated host species: the West lineage (C.harkWEST) caused more disease on lodgepole pine than jack pine, whereas the East lineage
(C.harkEAST) caused more disease on jack pine than lodgepole pine.
When virulence on the nongeographically associated host species
was considered, the East lineage (C.harkEAST) was more virulent on
lodgepole pine than the West lineage (C.harkEAST) was on jack
pine. Jack pine showed the greatest resistance to infection regardless of the pathogen source.
Differences between the pine species response to the fungal strains
suggest that there is a host genetic component to fungal resistance.
Our findings further indicate that genetic resistance to C. harknessii
in both lodgepole pine and jack pine is quantitative, in agreement
with previous studies (Klein et al. 1991; Weng et al. 2015; Wu et al.
1996; Yang et al. 1997). Our observation that lodgepole × jack pine
hybrid seedlings exhibited a proportion of gall development for both
C. harknessii lineages intermediate between that of lodgepole and
jack pine suggests genetic loci controlling resistance in pine trees are
genes with traditional Mendelian inheritance (Li and Yeh 2002).
Finally, the gradient in resistance to C. harknessii observed in lodgepole pine could be associated with introgression of alleles underlying
resiliency from jack pine into lodgepole pine (Cullingham et al.
2013; Fraser et al. 2016).
With strong evidence of a genetic component to resistance, there
is an opportunity for identifying genetic resistance markers for
breeding programs. Our refined disease index customized for the
torn-needle method enables more rapid identification of quantitatively resistant families in screening trials, which can be carried
out on seedlings that have only just commenced epicotyl growth.
We additionally have validated differential stem width as a quantitative measure of disease development after C. harknessii inoculation of young seedlings. Phenotypes that can be assessed using a
continuous, quantitative variable such as differential stem width
often lead to better model performance in quantitative genomic
Fig. 6. Effect of spore source and pine seed provenance on both difference in stem width (mm) and proportion of galls when lodgepole, hybrid (lodgepole × jack),
and jack pine seedlings are mock inoculated (M) or treated with C.harkWEST (W) or C.harkEAST (E). A, Difference in stem width (mm) (stem width at inoculation
point or point of greatest swelling minus the stem width at base of hypocotyl) was measured at 26 weeks postinoculation (wpi), and a Tukey’s posthoc test was
used to determine significance among groups (N = 589, a = 0.05), with uppercase letters comparing treatments within a provenance and lowercase letters comparing
a single treatment across provenances. B, Proportion of total seedlings showing striated galls at 26 wpi for each of the 15 treatments.
analyses than do categorical variables such as disease indices
(Kizilkaya et al. 2014), increasing the ability of breeders to ascertain meaningful breeding values. Development of this detailed qualitative categorical assessment of disease progression, which can be
employed in tandem with our new quantitative measure of disease
progression, provides the necessary tools for in-depth, quantitative
genetic analyses of C. harknessii resistance in lodgepole and jack
pine on young seedlings. These tools will provide valuable input to
breeding programs as consistent, easily discernable, early indicators
of susceptibility to C. harknessii and for deploying resilient seedlings after harvest and large-scale disturbances, including fire and
forest insects.
Conclusion. Coupling genetic data from field-collected samples
with growth chamber experiments, we have provided additional
evidence supporting the previously defined relationship of C. harknessii genetic lineages with pine host species, lodgepole pine, and
jack pine. Growth chamber experiments allowed us to control for
environmental variation that the hosts experience on the landscape,
which can affect fungal pathogenicity (Heineman et al. 2010); by
controlling this, we demonstrated that the two C. harknessii lineages have different levels of virulence, and the hosts have varied
levels of resistance. Our findings provide the clearest evidence to
date of these two C. harknessii lineages exhibiting a coevolutionary
history with lodgepole and jack pine, with a genetic component
contributing to both pathogen virulence and host resistance in this
complex pathosystem.
ACKNOWLEDGMENTS
We thank Lindsay Robb of the Alberta Tree Improvement and Seed
Centre (Alberta Ministry of Agriculture, Forestry and Rural Economic
Development) and Dale Simpson of the National Tree Seed Centre
(Natural Resources Canada, Canadian Forest Service) for their assistance in providing seed used in this study; Roger Brett, Brad Tomm,
and Taylor Scarr of Natural Resources Canada, Canadian Forest Service, Alex Woods of the British Columbia Ministry of Forests, and
Rory McIntosh of Saskatchewan Ministry of Environment who provided galls; Marion Mayerhofer, Samson Osadolor, and Laura Manerus
of the University of Alberta, who collected phenology data; Corey
Davis and the Molecular Biology Service Unit for guidance on microsatellite genotyping; and Andy Benowicz and Deogratias Rweyongeza
of the Alberta Ministry of Agriculture, Forestry and Rural Economic
Development for support, expertise, and project advice.
Vol. 112, No. 8, 2022 1805
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