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Vol. 98, No. 1, 2008 51
Ecology and Epidemiology
Real-Time PCR-Based Monitoring of DNA Pools in the Tri-Trophic
Interaction Between Norway Spruce, the Rust Thekopsora areolata,
and an Opportunistic Ascomycetous Phomopsis sp.
Ari M. Hietala, Halvor Solheim, and Carl Gunnar Fossdal
Norwegian Forest and Landscape Institute, Pb. 115, NO-1431 Ås, Norway.
Accepted for publication 19 June 2007.
ABSTRACT
Hietala, A. M., Solheim, H., and Fossdal, C. G. 2008. Real-time PCR-
based monitoring of DNA pools in the tri-trophic interaction between
Norway spruce, the rust Thekopsora areolata, and an opportunistic
ascomycetous Phomopsis sp. Phytopathology 98:51-58.
The difficulty in subculturing biotrophic fungi complicates etiological
studies related to the associated plant diseases. By employing internal
transcribed spacer rDNA-targeted quantitative real-time polymerase chain
reaction, we now show that the heteroecious rust Thekopsora areolata,
commonly associated in natural conditions to sapling shoots and cones of
Norway spruce and leaves of wild bird cherry, frequently infects nursery-
grown seedlings of the conifer. A spatial sampling scheme was used to
investigate seedlings and saplings of Norway spruce showing phloem
necrosis: the highest concentration of DNA of T. ar eo lat a was recorded in
the area with necrotic phloem. The separate analysis of bark and wood
tissues suggested that the initial spread of the rust to healthy tissues
neighboring the infection site takes place in the bark. A Phomopsis
species found to coexist with T. a re ola ta in several seedlings showed very
high DNA levels in the upper part of the lesion, and even in the visually
healthy proximal tissues above the lesions, which indicates that the
ascomycete, most probably a secondary invader following primary
infection by T. a re ola ta , has a latent stage during early host colonization.
We hypothesize that this hemibiotrophic mode of infection contributes to
the successful coexistence of Phomopsis with a biotrophic rust.
Additional keywords: hemibiotrophy, infection, Picea abies.
Thekopsora areolata (Fr.) P. Magn. (Pucciniastrum areolatum
(Fr.) Otth, Pucciniastrum padi (Schm. & Kunze) Diet.) is a
Eurasian rust fungus recorded from England through the whole of
Europe and from Russia to Kamtschatka and Japan (6,10,21). The
fungus alternates between conifers and broadleaved trees in order
to complete its life cycle with five distinct spore stages. Its main
hosts are Norway spruce (Picea abies (L.) Karst.) and wild bird
cherry (Prunus padus L.) (20).
T. areolata overwinters as telia in the leaves of wild bird cherry
shed on the ground. In spring during rainy weather, the telio-
spores germinate and form basidiospores in synchrony with the
flowering of Norway spruce. Female flowers of spruce, that even-
tually give rise to cones, are infected by basidiospores carried by
air currents. Following the formation of pycnia on the outer sides
of the cone scales and spermatization, dikaryotic hyphae form
aecidia on both sides of the cone scales during the summer (6).
The aecidia mature and open next spring and release aecidio-
spores, which infect cherry leaves. The scales of infected cones
are characteristically spread apart from each other and no seeds
are formed in infected cones, which often remain attached to
shoots longer than the healthy ones. Basidiospores of T. areolata
may also infect actively growing shoots of spruce, the fast-grow-
ing terminal shoots of spruce saplings being especially suscep-
tible. Infected shoots usually become crooked, S-formed, with
some dead tissue in the crooked part, and the shoots often die
above the crook (13,19).
In a project focused on diseases of Norway spruce, we have
been investigating the etiology of bark necrosis in nursery seed-
lings. Seedlings showing typical symptoms of T. areolata infec-
tion were often observed in forest nurseries, but no fruit bodies of
the rust were observed in these seedlings. An ascomycete, a
Phomopsis species, was now frequently codetected with T. a re o-
lata in these diseased shoots of Norway spruce. While necrotro-
phic fungi can trigger a hypersensitive response and programmed
cell death in the host plant to feed subsequently as saprotrophs
(7), rusts rely on haustoria in extracting nutrients from living host
cells and enable their parasitic relationships with plants by long-
term suppression of host defense responses (23). Due to this nutri-
tional mode, biotrophic fungi such as rusts are difficult to culture
on artificial media; however, the development of real-time poly-
merase chain reaction (PCR) technology has provided a powerful
tool for quantitative detection of unculturable microbes in their
natural substrates (9). To study the interaction of T. areol a ta ,
Phomopsis sp., and the hosting Norway spruce, we developed
real-time PCR assays to quantify their DNA pools at the advanc-
ing margins of the lesions in diseased shoots.
MATERIALS AND METHODS
Sampling. A total of 100 2-year-old containerized nursery
seedlings of Norway spruce that showed necrotic stem lesions
with a slightly swollen bark area (Fig. 1) were collected from
three different nurseries. To first screen the seedlings for the pres-
ence of candidate pathogens, a 5-mm-long bark sample was taken
from the middle of the lesion from 50 randomly selected seed-
lings and processed for DNA analysis as described below. After
this initial screen, 18 seedlings were sampled by taking 5-mm-
long samples from the edges of the lesion area in order to obtain
spatial colonization profiles. To examine tissue-specific coloniza-
tion, the bark in the lesion area was separated from the wood with
a scalpel in five seedlings, and the samples were processed sepa-
rately for DNA isolation. In addition to diseased nursery seed-
Corresponding author: A. M. Hietala
E-mail address: Ari.Hietala@Skogoglandskap.no
doi:10.1094/ PHYTO-98-1-0051
© 2008 The American Phytopathological Society
52 PHYTO PATH OLOGY
lings, T. areolata-infected cones and shoots of naturally grown
saplings of Norway spruce and the leaves of wild bird cherry
were collected for DNA isolation/colonization profiling and inter-
nal transcribed spacer (ITS) rDNA sequence comparisons. These
samples were collected from various localities in Norway, the
sampling area stretching from Kristiansand in southern Norway to
Stjørdal in central Norway covering a distance of 800 km (Table 1).
DNA isolation, cloning, and sequence analysis. For DNA
isolation, infected bark and wood samples from Norway spruce
were excised, frozen immediately in liquid N2, and ground in
liquid N2-chilled containers for 2 min in an MM 300 mill (Retsch
Gmbh, Haan, Germany). Uredinia samples from T. areolata-
infected leaves of wild bird cherry and aecidia samples from
Norway spruce cones were ground with a pestle in an Eppendorf
tube. To obtain negative references for testing the specificity of
the real-time PCR assays used for quantifying Norway spruce and
T. areolata DNA in diseased seedlings, the following strains of
fungi commonly associated with diseased shoots of Norway
spruce seedlings were cultured on cellophane-coated malt extract
agar (2% malt extract and 1.5% agar) for DNA isolation: Alter-
naria sp. (strain 2006-210), Botrytis cinerea Pers. (strain 1983-
145/1 Øa), Epicoccum nigrum Link (strain 1959-1898/1/1), Fu-
sarium sp. (strain 2006-221), Gremmeniella abietina (Lagerberg)
Morelet (strain 2002-26/2), Herpotrichia juniperi (Duby) Petrak
(strain 1973-53), Nectria fuckeliana Booth (strain 1973-74),
Phoma sp. (strain 2006-229), Phomopsis sp. (strain 2002-62/1),
Rhizosphaera kalkhoffii Bubak (strain 1997-1/6), Sirococcus
conigenus (D.C.) P.F. Cannon & Minter (strain 2000-58/2), and
Tiarasporella parca (Berk and Broome) Whithey (strain 1991-
727/28) (all these isolates were obtained from the culture collec-
tion of the Norwegian Forest and Landscape Institute). In addi-
tion, aecidia samples of Cronartium ribicola (collection Ru29)
and C. flaccidum (collection Ru30) were included as negative
reference material. DNA isolation was performed with the Plant
DNA Mini Isolation Kit (Qiagen, Hilden, Germany) according to
the manufacturer’s instructions.
Amplification was carried out with ITS1-F and ITS4-B primers
(5) in 25-µl reaction volumes using HotStarTaq DNA Polymerase
(Qiagen) according to the manufacturer’s instructions. After gel
electrophoresis, the PCR products were purified with GFX PCR
DNA and Gel Band Purification kit (Amersham Biosciences,
Little Chalfont, England), ligated into a cloning vector (pGEM-T
Easy; Promega, Madison, WI), and transformed into Escherichia
coli (DH5-α). For sequencing, plasmid DNA was isolated and
purified using Wizard Plus Minipreps (Promega). Three distinct
colonies were sequenced in both directions on an ABI PRISM
3100 Genetic Analyzer (Applied Biosystems, Foster City, CA).
The alignments of complementary strands and the similarity
studies within sequenced samples and other known published ITS
regions in the NCBI GenBank sequence database were deter-
mined with Seqman software (Lasergene, DNASTAR Inc., Madi-
son, WI).
TABLE 1. Rust samples sequenced for internal transcribed spacer rDNA region in the study
Code
Rust
Host
Source
Spore stage
Locality
Collectora
Accession
no.
Bu4 2 Thekopsora areolata Picea abies Seedling None detected Buskerud, Øvre Eiker, Hokksund 1 EF363336
Bu5 10 Thekopsora areolata Picea abies Seedling None detected Buskerud, Øvre Eiker, Hokksund 1 DQ445891
LR 4B Thekopsora areolata Picea abies Seedling None detected Telemark, Sauherad, Gvarv 1 DQ445892
LR 14BA Thekopsora areolata Picea abies Seedling None detected Telemark, Sauherad, Gvarv 1 DQ445894
Sk1 10 Thekopsora areolata Picea abies Seedling None detected Sør-Trøndelag, Melhus, Skjerdingstad 1 DQ445901
Spl 1 5 Thekopsora areolata Picea abies Seedling None detected Sør-Trøndelag, Melhus, Skjerdingstad 1 DQ087229
Spl3 5 Thekopsora areolata Picea abies Seedling None detected Sør-Trøndelag, Melhus, Skjerdingstad 1 DQ087229
Thek 1 Thekopsora areolata Picea abies Cone Aecidia Østfold, Eidsberg, Ramstad 1 DQ445895
Thek 2 Thekopsora areolata Picea abies Cone Aecidia Østfold, Eidsberg, Ramstad 1 DQ087230
Thek 3 Thekopsora areolata Picea abies Cone Aecidia Østfold, Eidsberg, Ramstad 1 DQ445896
Thek 4 Thekopsora areolata Picea abies Cone Aecidia Hedmark, Trysil, Bekkvang 1 DQ445897
Thek 5 Thekopsora areolata Picea abies Cone Aecidia Hedmark, Trysil, Bekkvang 1 DQ445898
Thek 7 Thekopsora areolata Picea abies Cone Aecidia Nord-Trøndelag, Stjørdal, Hegra 2 DQ445899
Sta1 Thekopsora areolata Picea abies Sapling Aecidia Hedmark, Stange, Tangen 1 DQ445902
Sta2 Thekopsora areolata Picea abies Sapling None detected Hedmark, Stange, Tangen 1 DQ445903
2A Thekopsora areolata Prunus padus Leaf Uredinia Nord-Trøndelag, Stjørdal, Lånke 2 DQ445904
Ru4 Thekopsora areolata Prunus padus Leaf Uredinia Aust-Agder, Birkenes, Birkeland 1 DQ087231
Ru5 Thekopsora areolata Prunus padus Leaf Uredinia Vest-Agder, Kristiansand, Kroken 1 DQ445900
Hal1 Thekopsora areolata Prunus padus Leaf Uredinia Østfold, Halden, Lundene 1 DQ445905
Ru6 Pucciniastrum ebilobii Epilobium angustifolium Leaf Uredinia Vest-Agder, Kristiansand, Foss 1 DQ445906
Ru11 Pucciniastrum ebilobii Epilobium watsonii Leaf Uredinia Vest-Agder, Kristiansand, Foss 1 DQ445907
Ru29 Cronartium ribicola Pinus peuce Stem Aecidia Buskerud, Øvre Eiker, Hokksund 1 DQ445908
a 1, H. Solheim; and 2, A. Hietala.
Fig. 1. The 2-year-old Norway spruce seedling Bu4 showing typical symp-
toms of Thekopsora areolata infection: crooked stem with dark brown,
slightly swollen bark area with resin flow (bar = 1 cm).
Vol. 98, No. 1, 2008 53
Oligonucleotide primers used for quantification of the host
and pathogen. The real-time PCR primers used for monitoring
T. areolata colonization in infected seedlings were designed with
the Primer Express software 1.5a provided with Applied Bio-
systems real-time quantitative PCR systems (Applied Biosystems)
by employing a conserved and species-specific sequence area in
the ITS rDNA gene cluster. The amount of Norway spruce DNA
in analyzed samples from infected nursery seedlings was esti-
mated by using the polyubiquitin primer/probe set previously de-
scribed (9). In addition, we monitored the presence of G. abietina
and Phomopsis sp., pathogenic fungi commonly associated with
necrotic stem lesions in Norway spruce seedlings, with primer/
probe sets described by Børja et al. (3). The primer/probe sets
used are shown in Table 2.
PCR conditions and specificity of primers. The real-time
PCR detection of T. areo lat a DNA was performed in SYBR
Green PCR Mastermix (P/N 4309155; Applied Biosystems),
while amplification of Norway spruce, G. abietina, and Phomop-
sis DNA was performed with TaqMan Universal PCR Master Mix
(P/N 4304437; Applied Biosystems). A primer concentration of
50 nM was chosen for the T. a re ola t a primers, while the primer
and probe concentrations of 150 and 333 nM (9), respectively,
were used for detecting the DNA of Norway spruce. For G. abie-
tina and Phomopsis sp., a primer concentration of 300 nM and a
probe concentration of 400 nM were used (3). All PCR analyses
were performed in singleplex conditions.
To construct standard curves for quantification of Norway
spruce and pathogen DNA, DNA isolated from a visually healthy
control seedling where DNA of T. areolata or Phomopsis sp.
could not be detected by real-time PCR, from aecidia of T. areo-
lata formed on a naturally infected cone of the sample Thek2
(Table 1), and from a pure culture of Phomopsis sp. (strain 2002-
62/1) was quantified by a Versafluor fluorometer (Bio-Rad,
Hercules, CA) and a PicoGreen DNA quantification kit (Molecu-
lar Probes, Eugene, Oregon) and then pooled together in known
concentrations. The host DNA standard curve samples (100, 10,
1, and 0.1 ng of Norway spruce DNA) were all spiked with 1 ng
of T. areolata and 1 ng of Phomopsis sp. DNA. The T. a re ol ata
DNA standard curve samples (100, 10, 1, 0.1, 0.01, and 0.001 pg
of T. areolata DNA) were all spiked with 1 ng of Norway spruce
and 1 ng of Phomopsis sp. DNA. The Phomopsis DNA standard
curve samples (1,000, 100, 10, 1, and 0.1 pg of Phomopsis sp.
DNA) were all spiked with 1 ng of Norway spruce and 1 ng of
T. areolata DNA. All the standard curve samples were replicated
three times within a single PCR run. To ensure that the cycle
threshold (Ct) values (Ct determines the PCR cycle at which the
reporter fluorescence exceeds that of the background) from the
experimental samples fell within the standard curves and to in-
vestigate the presence of compounds inhibitory to PCR, 3- or
4-log dilution series were prepared for all the experimental
samples. Each experimental sample had undiluted DNA as the
most concentrated, and all log dilutions of a sample were used as
templates in real-time PCR. For both of the series, the experi-
mental and standard curve samples, 3 µl of the DNA solution was
used as the template for each 25-µl PCR. For experimental
samples, each reaction was repeated twice in a separate PCR run.
PCR cycling parameters were 95°C for 10 min followed by 40
cycles of 95°C for 15 s and 60°C for 1 min. For T. areolata, the
PCR cycling was followed by a melting curve analysis: after a
denaturation step at 95°C for 15 s, the sample was cooled to 60°C
for 1 min followed by a linear temperature increase to 95°C,
while continuously monitoring the fluorescent signal. Fluores-
cence emissions were detected with an ABI Prism 7700 (Applied
Biosystems). Data acquisition and analysis were performed with
the Sequence Detection System software package (1.7a; Applied
Biosystems). Genomic DNA from healthy Norway spruce and
from the included reference fungi were used as negative controls
for the T. areolata and Phomopsis markers, while fungal DNA
served as a negative control for the host marker. To further verify
the specificity of the real-time PCR data from experimental
samples, selected DNA samples from seedlings were PCR ampli-
fied both by the basidiomycete-specific primer pair ITS1-F and
ITS4-B and by the universal fungal-specific primer pair ITS1-F
and ITS4 (5), followed by sequencing. For this purpose, the PCR
products purified with MinElute PCR Purification Kit (Qiagen)
were directly sequenced in both directions on an ABI PRISM
3100 Genetic Analyzer (Applied Biosystems) by using the ITS
primers as sequencing primers.
Calculation of fungal colonization levels. Standard curves for
Norway spruce, T. areolata, and Phomopsis sp. were constructed
based on the relationship of Ct values and known host and patho-
gen DNA concentrations: the Ct values were plotted against log-
transformed DNA amounts, and linear regression equations were
calculated for the quantification of DNA pools in unknown
samples.
RESULTS
Alignment of Thekopsora sequences from different sources.
All the T. areolata samples studied yielded an 812-bp amplifi-
cation product with the ITS1-F and ITS4-B primers employed.
The corresponding ITS rDNA sequences comprised a 50-bp 3′
end for the 18S rDNA gene, 210 bp in the ITS1 region, 162 bp for
the 5.8S rDNA gene, 219 bp in the ITS2 region, and a 171-bp 5′
end for the 28S rDNA gene. The ITS1-5.8S-ITS2 sequences from
T. a re o la t a samples from different hosts/tissue types showed little
variation; the similarities among T. areolata sequences obtained
from seedlings, saplings, and cones of Norway spruce and from
TABLE 2. Primers/probes used for monitoring pathogen colonization
Species detected
Primer/probe seta
Product size (bp)
Fluorescence
label
Amplicon
melt point
Thekopsora areolata Forward: 5′-TTTGTGCATTGATGGACTTTAAATAAT-3′ 72 SYBR Green 69.6
Reverse: 5′-CCAAGAGATCCATTGTTAAAAGTTATATGAT-3′
Gremmeniella abietina Forward: 5′-CACCCGTGCCTATATTACTCTGTTG-3′ 90 NAb
Reverse: 5′-TATAGTACTCAGACGGTAAATAATTCATAGGTT-3′
Probe: 5′-TTCCCGGGCCTCAACCCCC-3′ FAM
Phomopsis sp. Forward: 5′-GCACCCAGAAACCCTTTGTG-3′ 116 NA
Reverse: 5′-AAGAGTTGACTTGGCCGCC-3′
Probe: 5′-CGGTAACGAGGAGCAGCCCGC-3′ FAM
Picea abies Forward: 5′-TGGTCGTACTCTGGCCGATTATA-3′ 96 NA
Reverse: 5′-ACACCTAGCGGCACACAGTTAA-3′
Probe: 5′-TGCTCCGTCTCCGTGGTGGCT-3′ VIC
a The primer set for detection of Thekopsora areolata DNA was designed in the present study, while the primer/probe sets for detection of the other two pathogens
and host DNA were designed by Børja et al. (3) and by Hietala et al. (9), respectively.
b Not applicable.
54 PHYTO PATH OLOGY
leaves of wild bird cherry were 99 to 100, 98 to 99, 98 to 100, and
98 to 99%, respectively, while the overall sequence similarity
within this material was 98 to 100%. The conserved sequence was
compared with sequences from the included reference rusts and
with sequences available at the NCBI GenBank sequence data-
base. Among all the sequences available, the rust fungi Puccinia
kuehnii (W. Krüger) E.J. Butler, C. ribicola J.C. Fischer, C. flac-
cidum Alb. & Schwein, and Peridermium harknessii J.P. Moore
showed the greatest similarity to T. a re ola ta, but in addition to
numerous point mutations in the ITS1 and ITS2 regions, these
reference species had a number of inserts in these regions (Fig. 2).
Specificity of the primer/probe sets used and the standard
curves constructed. The primer pair now employed for
quantifying T. areo lat a colonization in diseased Norway spruce
seedlings amplified an 81-bp area between the 3′ end of the ITS1
region and the 5′ end of the 5.8S rDNA gene (Fig. 2). Puccinia
Fig. 2. CLUSTAL W (1.82) multiple sequence alignment of a conserved internal transcribed spacer (ITS) rDNA sequence of Thekopsora areolata (identical
sequences obtained from infected seedlings [sample Spl 3 5, accession no. DQ087229] and cones [sample Thek 2, DQ087230] of Norway spruce and from leaves
of wild bird cherry [sample Ru4, DQ087231]) and its closest matches at the NCBI GenBank Sequence, Puccinia kuehnii (AJ406049), Cronartium ribicola
(L76499), Peridermium harknessii (L76510), and Cronartium flaccidum (AY566270). The 3′ end of 18S gene and the 5′ end of 28S gene are not shown in
alignment. The line symbol (–) indicates a gap in the alignment, a dot (.) denotes identical nucleotides between the sequences at the given positions. The real-time
PCR primers used for quantification of the rust colonization levels in infected seedlings and saplings of Norway spruce are underlined and in bold.
Vol. 98, No. 1, 2008 55
kuehnii, C. ribicola, C. flaccidum, and Peridermium harknessii
(Fig. 2), the included reference rusts and all the other fungal se-
quences available in the NCBI GenBank sequence database (data
not shown), differed from T. areolata in several nucleotides in the
binding sites of both primers. The primer set developed for moni-
toring T. areolata did not detect the DNA of Norway spruce, and
the primer/probe set used for detecting DNA of Norway spruce
did not detect the DNA of T. areo lat a (data not shown). In addi-
tion, these two sets did not detect the DNA of the included refer-
ence fungi when 1 ng of DNA from these was used as the tem-
plate. In the standard curve samples constructed for Norway
spruce, there was a linear relationship between the Ct values and
the log-transformed amount of DNA in the range of 100 to 0.1 ng
of host DNA (Fig. 3A). In the standard curves for T. areolata and
Phomopsis sp., there was a linear relationship between the Ct
values and the log-transformed amount of pathogen DNA in the
range of 100 to 0.001 pg and 1,000 to 0.1 pg, respectively (Fig.
3B and C). The DNA amount standard curves for Norway spruce,
T. areolata, and Phomopsis sp., based on the relationship of the
log amount of template (x) generated from known host and patho-
gen DNA concentrations and their corresponding Ct values (y),
were y = 29.858 – 3.591x, y = 23.804 – 3.4951x, and y = 30.337 –
3.381x, respectively. For the Norway spruce, T. areolata, and
Phomopsis sp. standard curves, the PCR amplification efficiencies
(E), calculated with the equation E = (10(–1/slope) – 1) × 100, were
89.9, 93.2, and 97.6%, respectively. The melting curve analysis
showed a single peak with a melt point of 69.6°C in the standard
curve samples of T. areolata.
Symptoms of the disease and colonization profiles of T.
areolata and other fungi monitored. The diseased seedlings and
saplings of Norway spruce showed a few centimeter long, dark
brown, slightly swollen necrotic bark area with resin flow, and
many plants were crooked in the infected area (Fig. 1). On some
occasions, the lesions were not distinct and the necrotic tissue was
longer (not shown). In the middle of the lesions, both the outer
bark and the phloem were dark and necrotic, while at proximal
areas above and below this region, the phloem was light brown,
eventually transitioning to a green color when examining more
distal areas. The change in the phloem color from dark brown to
light brown was abrupt, while the transition from light brown to
green phloem was often gradual. Fruit bodies (aecidia, pycnia)
were not observed in the examined seedlings. Similar symptoms
as those observed in the nursery seedlings were also noted in the
5- to 10-m-long saplings included as reference material. Aecidia
were observed in one of the leader shoots of these saplings.
T. areolata was detected by real-time PCR in all the seedlings
and saplings showing the symptoms described above. As with the
standard curve samples, a single peak with a melt point of 69.6°C
was observed for experimental samples amplified with the T. areo -
lata primer pair. To test for the presence of substances inhibitory
to PCR, log-dilution series were prepared for all experimental
samples. The differences in the corresponding Ct values between
the undiluted and 10-fold diluted DNA samples were, particularly
for T. areolata, often well below the expected difference of
approximately 3.3 cycles (for example, the Thekopsora Ct values
of 17.1, 18.3, and 21.7 were obtained for an undiluted, 10-fold,
and 100-fold diluted sample from seedling Sk4, but only the latter
two values are reliable as they gave the expected difference in the
Ct value), which indicates the presence of substances inhibitory to
PCR in the undiluted samples and/or excess levels of the tem-
plate. In contrast, the differences in the corresponding Ct values
between the 10- and 100-fold diluted DNA samples were in the
range also observed between the log dilutions of the correspond-
ing standard curves. This compatibility indicates that substances
inhibitory to PCR amplification were not present in the diluted
samples, and the ten- and hundred-fold diluted DNA samples
were subsequently used for calculating the amounts of host and
pathogen DNA present in the experimental samples. Within the
area colonized by T. areo lat a, the host DNA yields were in
general the lowest in the middle of the lesion area with dark
brown phloem, while the highest host DNA yields were invariably
obtained in the lower lesion area with light brown phloem. When
normalized against host DNA yield and expressed as fungal/host
DNA amount (%), the highest levels of T. areolata DNA were
recorded in the area with dark brown phloem having a relatively
low yield of host DNA, whereas in the lesion margins, the DNA
levels of the rust declined steeply (Fig. 4).
Out of the 18 seedlings examined along the lesion area with a
spatial sampling scheme, Phomopsis sp. was codetected with
T. a re o lat a in five seedlings. Regarding the saplings analyzed as a
reference material, Phomopsis sp. was codetected with T. areolata
Fig. 3. Standard curves for quantifying the DNA amount of Norway spruce,
Thekopsora areolata, and Phomopsis sp; log-transformed DNA amounts were
plotted against cycle threshold (Ct) values, and the linear regression equations
obtained were used for calculating the amounts of the three DNA pools in
experimental samples. The host DNA standard curve (A) samples (100, 10, 1,
and 0.1 ng of Norway spruce DNA) were all spiked with 1 ng of T. areolata
and 1 ng of Phomopsis sp. DNA, and analyzed by the primer/probe se
t
targeting DNA of Norway spruce. The T. a re ol ata DNA standard curve (B)
samples (100, 10, 1, 0.1, 0.01, and 0.001 pg of DNA) were all spiked with 1
ng of Norway spruce and 1 ng of Phomopsis sp. DNA, and analyzed by the
primer set targeting DNA of the rust fungus. The Phomopsis DNA standard
curve (C) samples (1,000, 100, 10, 1 and 0.1 pg of Phomopsis DNA) were all
spiked with 1 ng of Norway spruce and 1 ng of T. areolata DNA, and
analyzed by the primer/probe set targeting DNA of Phomopsis. Three PCR
replicates were run on each sample.
56 PHYTO PATH OLOGY
Fig. 4. The total host DNA yields (columns) and the percentage of Thekopsora areolata (line with filled squares) and Phomopsis (line with open triangles) DN
A
amount in relation to host DNA amount within stem lesions of Norway spruce. The values are the means + standard deviations of duplicated polymerase chain
reactions (PCR) from 10- and 100-fold diluted DNA samples. The x axis labels “U” and “L” indicate the orientation of the lesion (U, upper margin; L, lower
margin), while the color of phloem in each sampled section is indicated by letters (g, green; d, dark brown; l, light brown). To confirm the presence of T. ar eo la t a
and Phomopsis sp. in the studied seedlings, sequence analysis of internal transcribed spacer (ITS) rDNA PCR products was performed on selected samples; filled
squares and open triangles under the x axis indicate positions where the presence of T. areolata or Phomopsis sp., respectively, was verified also by sequence
analysis. A, Host DNA yields and pathogen colonization profiles in the seedling Bu2 and the sapling Sta2: in the seedling the shoot above the lesion was dead and
the lesion area was sampled spatially by taking 5-mm-long stem sections. In the sapling, the shoot above the lesion was alive and the lesion margins were sampled
spatially by taking 5 × 2 mm (length × width) bark sections, while the middle of the lesion (8-cm-long area with dark or light brown phloem) with missing data was
not analyzed. B, Host DNA yields and pathogen colonization profiles in the seedling Sk4. The shoot above the lesion was alive, and the lesion margins were sampled
spatially by taking 5-mm-long stem sections, while the middle of the lesion (4-cm-long area with dark brown phloem) with missing data was not analyzed. C, Hos
t
DNA yields and pathogen colonization profiles in bark and wood of seedling Sk1. The shoot above the lesion was alive. The lesion margins were sampled spatially
by taking 5-mm-long stem sections, while the middle of the lesion (5.5-cm-long area with dark brown phloem) with missing data was not analyzed. From each 5-
mm-long section, the bark and wood were processed separately; the samples shown at identical positions in the panels originate from the same stem sections.
Vol. 98, No. 1, 2008 57
in one of the three saplings analyzed, but its presence was re-
stricted to a single sampling position (Fig. 4A). In contrast to
T. a re o la t a, high levels of Phomopsis sp. DNA were observed
particularly in the upper part of the lesion with light brown phloem
and even in healthy-appearing bark with green phloem, and often
there was no steep decline in its DNA levels even at the lesion
margins (Fig. 4B, upper lesion). The third monitored pathogen,
G. abietina, was not detected in any of the examined Norway
spruce material. The ITS rDNA PCR and sequencing performed
for verification of the real-time PCR data confirmed the presence
of T. a re ola ta and Phomopsis sp. in the seedlings analyzed (Fig.
4). While these T. areolata sequences obtained by direct sequenc-
ing of the PCR products showed 98 to 100% similarity to those
obtained by cloning from seedlings, saplings, and cones of Norway
spruce and from leaves of wild bird cherry the Phomopsis sp.
sequences obtained here were 99 to 100% similar to those of the
Phomopsis sp. recently characterized by Børja et al. (3).
In order to investigate fungal spread within different tissue
types, five seedlings were sampled in such a way that the bark
was separated from the wood, and these tissues were processed
separately. For both T. areo lat a and Phomopsis sp., the coloni-
zation profiles, when using host DNA yield as the normalizer,
were similar between the tissue types (Fig. 4C), but in the lesion
margins, bark contained regularly higher normalized DNA levels
of the rust than wood.
DISCUSSION
By amplifying the ITS1-5.8S-ITS2 region of T. a re ola t a from
DNA of diseased nursery-grown Norway spruce seedlings and
comparing it to T. a re ola t a sequences obtained from cones and
saplings of Norway spruce and from leaves of wild bird cherry,
we showed that this wide-spread pathogenic rust is commonly
associated with stem lesions in container-grown 2-year-old seed-
lings of the conifer. The symptoms observed in these seedlings
are similar to those observed in saplings infected frequently by
the rust in forest conditions. In laboratory experiments published
in 1900, Klebahn (11) was able to artificially inoculate shoots of
Norway spruce seedlings with basidiospores of the pathogen; no
fruit bodies were formed in those experiments, but the author
noted the strong smell of sugary liquid exuded by pycnia. Based
on fruit body observations, Roll-Hansen (19) in the 1940s showed
the presence of T. areolata on 3- to 4-year-old nursery seedlings
of Norway spruce. Otherwise there are no reports of young spruce
seedlings hosting this rust. This is most likely due to the fact that
the rust is difficult to culture in artificial media, and that the
conspicuous aecidia allowing conventional identification of the
fungus are not formed in the currently produced 2-year-old
Norway spruce seedlings. In Norway spruce saplings, aecidia of
T. areolata develop inside the bark and the poorly developed bark
of young seedlings compared with that of saplings may explain
the absence of fruit bodies in nursery seedlings. An alternative
explanation is that mating, an event preceding aecidia formation,
does not commonly take place in nursery conditions.
Based on inoculation experiments (11,19,22), T. areolata is
regarded to alternate between the conifer spruce (Picea) and
broadleaved trees in the genus Prunus in order to complete its life
cycle. Our comparison of ITS rDNA sequences of T. are ola ta
from the different hosts/tissue types is in agreement with this con-
cept, as ITS rDNA sequence variants specific to the hosts/tissue
types were not detected. According to our preliminary observa-
tions and depending on annual weather conditions and locality of
the nursery, up to four Norway spruce seedlings per a 96-seedling
container tray can show symptoms characteristic of T. areolata
infection. The affected seedlings are typically not located as a
group but scattered within the nursery field and also within the
container trays, which would be in accordance with an airborne
inoculum, i.e., air-current spread of basidiospores formed on
fallen leaves of wild bird cherry. The incidence of T. areolata on
sapling shoots of Norway spruce in forest conditions has been
shown to depend on their distance to wild bird cherry (13). Thus,
eradication of this alternate host from the immediate vicinity of
affected Norway spruce nurseries should be recommended.
Based on a phylogenetic analysis of the 28S rDNA sequence,
Maier et al. (14) concluded that the genus Thekopsora is poly-
phyletic and that T. a re ola ta is closely related to Cronartium spp.
This is compatible with our ITS sequence data as, among the de-
posits in the NCBI GenBank sequence database, the pine stem
rust fungi C. ribicola and C. flaccidum showed high sequence
similarity in the ITS regions to T. areolata. Puccinia kuehnii (a
leaf rust of sugarcane) and Peridermium harknessii (a stem rust of
pine), the other reference species with high sequence similarity to
T. areolata in the ITS regions, were unfortunately not included in
the study of Maier et al. (14).
The recently introduced real-time PCR procedure provides a
robust tool for detecting and quantifying unculturable pathogens,
such as rust fungi, in their natural substrates (2). For designing the
real-time PCR assay, we employed an ITS1-5.8S-rDNA region
that is species-specific to T. areolata to ensure specificity. Due to
the high copy number of ITS rDNA gene cluster in the genome,
the ITS-based detection is about 100 times more sensitive than an
assay detecting a single copy gene (18). The lowest point in the
standard curve for T. a re ola ta was 0.001 pg of DNA, which
equals 0.965 Mbp DNA (1 pg DNA = 965 Mbp [1]). The genome
sizes of T. areolata and other Thekopsora species are not known,
but as reviewed by Leonard and Szabo (12), in the rust genus
Puccinia, the genome sizes range between 67 and 154 Mb. We
thus conclude that our real-time PCR assay should be able to
quantitatively detect even single nuclei of T. areolata.
There was a strong gradient in the amount of T. ar eo l ata DNA
along the lesion length, with the highest levels being recorded in
the area with necrotic phloem. Similar gradients in the amount of
pathogen DNA have been observed in the Norway spruce–Hetero-
basidion annosum pathosystem for example, where pathogen
DNA showed the highest levels next to the inoculation site in the
bark and declined along with the distance away from this site (9).
It seems obvious that the necrotic bark with high DNA levels of
the rust represents initial infection sites from which T. areo lat a is
spreading both upwards and downwards to the neighboring
healthy tissues. The steep decline in DNA levels of T. areolata in
the margin areas of the lesion coincided with the change of the
phloem color from dark brown to light brown, indicating that the
rust infection results in host response, leading eventually in host
cell death and degradation of target DNA. The analysis of bark
and wood tissues separately indicated that the rust is able to also
colonize wood in the area with dark brown phloem, but its initial
spread to healthy tissues neighboring the infection site pre-
sumably takes place within the bark.
In 5 out of the 18 seedlings examined in detail with a spatial
sampling scheme, T. areolata was found to coexist with a
Phomopsis sp. showing 99 to 100% ITS rDNA sequence similar-
ity to the Phomopsis species recently characterized from Norway
spruce seedlings (3). Compatible with our data, Cech and Perny
(4), based on fruit body observations and fungal isolations, showed
that Phomopsis spp. are commonly present in T. areol at a-infected
shoots of Norway spruce saplings in forest conditions. Based on
pathogenicity tests, Børja et al. (3) concluded that this Phomopsis
species is a weak pathogen depending on specific host-predisposing
conditions such as abiotic stress or infection by other pathogens.
Hahn (8) also describes Phomopsis occulta as a weak pathogen in
conifers following injuries caused by frost, transplanting, drought,
and parasitic fungi such as the white pine blister rust (Cronartium
ribicola). We consider it highly likely that the Phomopsis sp. now
codetected with T. areolata is a secondary invader benefiting from
the primary infection by the rust. The fact that considerably higher
maximal DNA levels were observed for Phomopsis sp. than
58 PHYTO PATH OLOGY
T. areolata can probably be explained by their differential
karyotic condition; while the vegetative stage of T. areol ata on
Norway spruce is either uni- or binucleate depending on whether
spermatization has taken place, the Phomopsis sp. has multi-
nucleate cells in the vegetative stage (A. M. Hietala, H. Solheim,
and C. G. Fossdal, unpublished observation).
Suppression of host defense responses, such as the hyper-
sensitive response involving death of colonized cells, is essential
for rust fungi, which rely on the success of haustoria, specialized
hyphae for nutrient absorption, in extracting nutrients from living
host cells (16). The fact that the Phomopsis sp. coexisting with
T. areolata in the diseased seedlings showed very high DNA
levels even in the healthy tissues above the lesions indicates that
the fungus has a quiescent stage in the infection process. In
several fruit diseases, Phomopsis species circumvent host defense
responses by having a latent colonization stage during fruit devel-
opment and by triggering the expression of pathogenicity factors
after harvest (24). This transient type of biotrophy in which the
fungus shows a biotrophic phase during early host colonization
and then switches to a necrotrophic phase is referred to as hemi-
biotrophic (15). The initial penetration and invasion processes of a
host by a fungal pathogen in the absence of a significant plant
resistant response ensure successful colonization of host tissue
and subsequent necrotrophic activities. Like hemibiotrophic fungi
during their biotrophic stage, rusts have limited secretory activity,
especially of hydrolytic enzymes, in order to minimize damages
to host cells (15). It remains to be examined whether the detected
Phomopsis species triggers the expression of cell-wall-degrading
enzymes for example after the growing season. In any case, the
mode of infection of the Phomopsis sp. resembles that of a
biotroph as the fungus is apparently able to colonize spruce bark
without triggering host cell death. This colonization mode un-
doubtedly contributes to the successful invasion of living host
tissue by Phomopsis sp. and it may even facilitate its coexistence
with a biotrophic rust.
Real-time PCR is currently the most sensitive quantification
method for nucleic acids. Regarding quantification of infection in
plants, the tool has so far been utilized for monitoring infection
by singular pathogens. The multiplexing option provided by
different fluorescent labels of the probe would allow simultaneous
monitoring of several DNA pools in a single tube (9). Due to the
high throughput nature of real-time PCR, we anticipate that the
tool will become widely used also in ecological studies when
monitoring events such as colonization of a common niche by
several microorganisms. Besides monitoring colonization, real-
time PCR can also be used for monitoring transcriptomes of the
interacting host and fungi during early stages of infection (17), a
situation still poorly understood in rust–host interaction.
ACKNOWLEDGMENTS
This project was financed by the Research Council of Norway (Project
no. 156881/I10) and Skogforsk. The nurseries Buskerud Skogselskaps
planteskole, Skogplanter Midt-Norge AS avd. Skjerdingstad, Sønsterud
planteskole AS and Telemark Skogplanteskule AS provided samples,
mostly via the nursery consultants M. Andersen and A. Strømberg. C.
Kierulf, Skogforsk, brought some samples from Skjerdingstad. Parts of
the laboratory work were performed by our technicians: O. Olsen did
fungal isolations, while I. Heldal and L. Ljevo were responsible for
cloning and sequencing.
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