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Journal of Microbiological Methods
Direct colony PCR-SSCP for detection of multiple pythiaceous
oomycetes in environmental samples
Ping Kong*, Patricia A. Richardson, Chuanxue Hong
Department of Plant Pathology, Physiology, and Weed Science, Virginia Polytechnic Institute and State University,
Hampton Roads Agricultural Research and Extension Center, 1444 Diamond Springs Road, Virginia Beach, VA 23455, United States
Received 2 July 2004; received in revised form 26 October 2004; accepted 27 October 2004
Available online 8 December 2004
Abstract
Colony PCR was developed for detection of pythiaceous species recovered on selective agar plates without DNA extraction.
A minute amount of mycelia from a single colony was picked up with a pipette tip and added directly to the PCR mix as
template for DNA amplification. Successful amplification was achieved in over 95% of the colonies recovered from plant
tissues, irrigation water and soil with species-specific primers or oomycete ITS-1 primers. PCR was inhibited in the case of
colonies emerging from unwashed pine bark potting mix plates. Direct colony PCR with ITS-1 primers combined with single-
strand conformation polymorphism analysis (SSCP) was used to determine population levels of single and multiple species in
plant and environmental samples. Application of this technique for disease diagnosis and monitoring pathogen sources was
explored, and the potential for studying diversity and population dynamics of other cultivated microbial communities in the
environment is discussed.
D 2004 Elsevier B.V. All rights reserved.
Keywords: Direct colony PCR; ITS-1 SSCP; Phytophthora; Pythium; Plant tissues; Soil; Irrigation water
1. Introduction
Pythiaceae is a large family of algae-like micro-
organisms (oomycetes) that includes Phytophthora
and Pythium with about 300 described species (http://
www.cabi-bioscience.org). Pythiaceous species have
been recovered from areas ranging from undisturbed
0167-7012/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.mimet.2004.10.019
* Corresponding author. Tel.: +1 757 3633941; fax: +1 757
3633950.
E-mail address: pkong@vt.edu (P. Kong).
terrestrial ecosystems to brackish salt-water marshes.
Many of them are active saprophytes on decaying
organic substrates while others are agriculturally and
economically important plant pathogens (Van der
Plaats-Niterink, 1981; Dick, 1990; Erwin and Ribeiro,
1996). Identification and detection of pythiaceous
oomycetes in environments is important not only in
disease diagnosis and management but also in
ecological and evolutionary studies of microbial
communities.
Isolation of pathogens from diseased plants or
infested environments on a selective medium fol-
61 (2005) 25–32
P. Kong et al. / Journal of Microbiological Methods 61 (2005) 25–3226
lowed by morphological examination is a conven-
tional method for detection and identification of
Phytophthora and Pythium species (Waterhouse,
1963; Van der Plaats-Niterink, 1981; Dick, 1990). In
addition to detection of certain species, it can be used
to determine species populations by grouping the
recovered colonies that have similar morphology.
However, biological and/or morphological examina-
tion is tedious and requires considerable expertise.
Species identification often takes weeks and may be
impossible when no diagnostic structures are pro-
duced. As a result, many DNA-based techniques, such
as PCR, RFLP, RAPD, single strand-conformation
polymorphism (SSCP), DNA probing and sequenc-
ing, have been used to assist detection and differ-
entiation of species from culture (Forster and Coffey,
1991; Briard et al., 1995; Wang and White, 1997;
Levesque et al., 1998; Ristaino et al., 1998; Matsu-
moto et al., 1999; Cooke et al., 2000; Martin, 2000;
Bailey et al., 2002; Kong et al., 2003b, 2004). Most of
these methods are PCR-based and allow identifying a
culture without morphological examination. However,
none of these techniques are directly adaptable to field
use. They usually require subculturing and DNA
extraction, which are time-consuming and labor-
intensive when a large number of isolates need to be
identified. Although PCR can be a culture-independ-
ent technique that allows species-specific detection of
Pythiaceae from plants, soil, and irrigation water, it
requires extracted DNA to minimize the inhibitory
effect of contaminants from environmental samples
(Ersek et al., 1994; Tooley et al., 1997; Kageyama et
al., 1997; Kong et al., 2003a,c; Wang et al., 2003).
Sometimes even after elaborate purification steps, it is
still necessary to dilute the DNA or use inhibitor
blocking agents in the subsequent assay preparations
to eliminate inhibition (Ersek et al., 1994; Wilson,
1997; Kageyama et al., 1997; Kong et al., 2003c). For
environmental bacterium detection, direct PCR with-
out DNA extraction has been reported, but serial
dilution or pretreatment of the samples was still
required (Herrick et al., 1993; Fode-Vaughan et al.,
2001; Schaad et al., 2002). PCR with diluted DNA
may result in a false detection due to low concen-
tration of target DNA, especially with low copy
number target sequences (Judelson and Tooley, 2000).
When multiple species are present in a sample, mixed
DNA can result in PCR bias and other technique-
related problems and affect accuracy of detection
(Ishii and Fukui, 2001; Hongoh et al., 2003; Ercolini,
2004).
An alternative approach that may avoid these
problems is direct PCR on isolation plates of environ-
mental samples. In this way, mixed individuals can be
separated and enriched. Direct colony PCR requires
isolation but does not require subculturing, DNA
extraction or sample pretreatment. Therefore, it can be
a rapid, accurate and cost effective approach for
pathogen detection in environmental samples. Direct
colony PCR has been successfully used with intact
cultures to screen genetically engineered bacterial
cells (Gussow and Clackson, 1989; Joshi et al., 1991;
Hiraishi, 1992) and fungi (Aufuavre-Brown et al.,
1993; van Zeijl et al., 1997). However, direct colony
PCR on environmental samples has had only limited
uses. In addition, the applications of colony PCR
focus on bacterial samples (Herrick et al., 1993; Fode-
Vaughan et al., 2001; Schaad et al., 2002), and a
pretreatment, such as addition of chemicals into the
PCR reaction mix, or nested PCR is required
(Lichtensteiger et al., 1996; Sheu et al., 2000).
In our previous studies, we had success with PCR
amplification of Phytophthora and Pythium DNA
using a small amount of boiled mycelium preparation
from pure cultures (Kong et al., 2003a,b,c, 2004). We
also had success with direct PCR using zoospore
suspensions and individual chlamydospores of Phy-
tophthora nicotianae and Phytophthora cinnamomi
without DNA extraction and sample pretreatment
(Kong et al., 2003a,c). Similar methods have also
been reported in other oomycete studies (Wangsom-
boondee and Ristaino, 2002; Bailey et al., 2002), but
PCR on pythiaceous colonies recovered directly from
environmental samples has not been reported. We also
developed a PCR-SSCP assay to differentiate multiple
species within the genera of Phytophthora (Kong et
al., 2003b) and Pythium (Kong et al., 2004) using a
single pair of oomycete primers and DNA from pure
cultures. The objective of this study was to fill a
technical gap by assessing the potential of direct
colony PCR on field samples in an attempt to create
an effective tool for disease diagnosis and for studies
of pythiaceous community structure and dynamics.
The efficacy of direct colony PCR was tested using
species-specific primers (Kong et al., 2003a,c) and
oomycete primers (Cooke et al., 2000) and templates
P. Kong et al. / Journal of Microbiological Methods 61 (2005) 25–32 27
from pure cultures and colonies directly from isolation
plates of plant tissue, soil, and irrigation water.
2. Materials and methods
2.1. Isolates, culture, and DNA preparation
Four isolates of P. nicotianae (1B11, 1E2, 23B8,
and 23B9) and four of P. cinnamomi (1A10, 1E7,
28D5, and 28D9) (Kong et al., 2003a,c) were used in
this study. Cultures were grown on clarified V8 juice
agar, and PARP-V8 and PARPH-V8 selective agar
(Ferguson and Jeffers, 1999) at room temperature in
the dark for 2 days or 2 weeks. Two-day-old cultures
were used to test DNA amplification of intact mycelia.
For standard PCR, crude DNA extracts were prepared
from 2-week-old cultures as described previously
(Kong et al., 2003a).
2.2. Isolation of Phytophthora and Pythium species
from plant tissues, soil, and irrigation water
Plant parts used in this study included fine roots of
azalea (Rhododendron sp), daphne (Daphne sp.) and
tomato (Lycopersicon esculentum), stems of tobacco
(Nicotiana tobacum), and leaves of annual vinca
(Catharanthus roseus) (Table 1). Diseased leaves,
stems, and roots were surface-sterilized with 10%
bleach then rinsed thoroughly with sterile distilled
water. These plant tissues were cut into small pieces
and plated equally among three PARP-V8 agar plates.
After diseased plants were removed, soil samples
were taken from the containers and plated on PARP-
V8 agar using a dilution plating method (Johnson and
Curl, 1972). Six replicated plates were used for each
Table 1
Direct colony PCR with species-specific primers for disease diagnosis
Plant Tissue/pieces plated Nu
To
Azalea (Rhododendron sp.) Fine roots/30 47
Tomato (Lycopersicon esculentum) Fine roots/30 27
Daphne (Daphne sp.) Fine roots/18 14
Tobacco (Nicotiana tobacum) Stems/9 8
Annual vinca (Catharanthus roseus) Foliage/30 23
a Total colonies were counted from three PARP agar isolation plates
nicotianae-specific and P. cinnamomi-specific primer pairs.
soil sample. Three of these isolation plates were
washed with running distilled water to remove soil
particles and air-dried in a laminar flow hood before
mycelium was taken to assess the effect of PCR
inhibitors from the soil particles. Three replicated
water samples were collected from an irrigation runoff
ditch and a sprinkler at a nursery in eastern Virginia,
USA. These samples were processed using a filtration
method (Hong et al., 2002) and plated on PARP-V8
and PARPH-V8 selective agar. All isolation plates
were incubated at room temperature in the dark until
colonies were visible (about 2 mm in diameter).
2.3. PCR and colony PCR
Both standard PCR and colony PCR mixtures
contain 10 mM Tris–HCl, 5 mM KCl, 1.5 mMMgCl2,
0.5 AM of each primer (Table 2), 0.2 mM dNTP, and
0.5 units of TaqDNA polymerase (TaKaRa Mirus
Biocorporation, Madison WI, USA). For standard
PCR, aliquots of 23 Al of the PCR reaction mixture
were prepared and 2 Al of DNA from a culture was
added as template. For colony PCR, a minute amount
of mycelia was added to 25 Al aliquots by scratching
2–3 mm across the surface of a colony using a
disposable tip on a 20-Al pipettor, then titurating twice
while gently swirling the tip in the mixture. The
thermocycler was programmed as described previ-
ously according to the primer pair used (Kong et al.,
2003a,b,c) with the exception of an initial denaturing
time of 5 min. Both standard PCR and direct colony
PCR were repeated at least three times for each
sample. Amplicons were visualized with an UV
transilluminator after resolving 5 Al of the products
in 1% agarose gels and staining with ethidium
bromide.
mber of coloniesa
tal Phytophthora nicotianae Phytophthora cinnamomi
0 15
3 0
14 0
8 0
22 0
from a diseased plant and subjected to direct colony PCR with P.
Table 2
Primer pairs used in this study for colony PCR tests
Name Sense Sequence (5V–3V) Size of amplicon (bp) Reference
LPV3a Forward GTGCAGACTGTCGATGTG 450 Kong et al., 2003c
Reverse GAACCACAACAGGCACGT
PNb Forward CCACCACGCAGCAAACTGCGGC 230 Kong et al., 2003a
Reverse TTGAGTACCAGGCCGCTCGTA
ITS-1 Forward GAAGGTGAAGTCGTAACAAGG ca. 300 Cooke et al., 2000
Reverse AGCGTTCTTCATCGATGTGC
a LPV3: P. cinnamomi-specific primer pair.b PN: P. nicotianae-specific primer pair.
P. Kong et al. / Journal of Microbiological Methods 61 (2005) 25–3228
2.4. SSCP analysis of colony PCR products amplified
with ITS-1 primers
Colony PCR products of samples amplified using
the ITS-1 primers were analyzed as previously
described (Kong et al., 2003b, 2004). Briefly, 1 Alof PCR products were mixed with 9 Al of denaturingbuffer containing 95% formamide, 20 mM EDTA and
0.05% bromophenol blue, heated at 96 8C for 10 min
and chilled on ice for 1 min. Five microliters of this
mixture was electrophoresed on an 8% non-denatur-
ing polyacrylamide minigel in pre-chilled 1� TBE
buffer at 200 V at room temperature for 2 h followed
by silver staining. The SSCP banding patterns were
determined using a single-stranded DNA ladder and
comparing with those of known species.
Fig. 1. Agarose gel (1%) electrophoresis of products of direct
colony PCR using intact mycelia from individual colonies and
standard PCR using DNA extract from the same culture. The PCR
were repeated three times. Top left and right: Amplicons of each of
four isolates of P. nicotianae by the species-specific primer pair PN
and oomycete ITS-1 primers, respectively. Bottom left and right:
Amplicons of P. cinnamomi by the species-specific primer pair
LPV3 and oomycete ITS-1 primers, respectively. M is a 100-bp
DNA ladder.
3. Results and discussion
3.1. Direct colony PCR from pure cultures on two
medium types
Colony PCR of pure culture without DNA extrac-
tion was successful and produced bands with the same
intensity as standard PCR using crude DNA for all
eight isolates (Fig. 1). The results were independent of
the type of primers and the copy number of target
DNA. Both high copy number targets from the elicitin
gene of P. nicotianae and the ITS-1 region of rDNA,
and the low copy number LPV, a storage protein gene
repeat of P. cinnamomi, were well amplified. This
indicates that colony PCR can be advantageous over
standard PCR for detection of individual pathogen
DNA targets, especially those with low copy number
in a mixed DNA sample at low concentration. Similar
intensive bands were obtained from isolates cultured
on both non-selective V8 agar and selective PARP-V8
and PARPH-V8 agar media commonly used for
isolation of pythiaceous species in field samples (data
not shown). The efficient amplification of direct
colony PCR on these media implicates a practical
application of this method for assaying environmental
samples. When handling a large number of plates/
colonies, 1–3% colony PCR failure may occur. This
can be improved by carefully harvesting mycelia or
avoiding excess agar in the reaction mixture.
Fig. 2. PCR-SSCP analysis of 47 colonies recovered from aza-
lea roots. The colonies were subjected to direct colony PCR with
ITS-1 primers, and the PCR products were denatured and electro-
phoresed in an 8% native acrylamide gel. Colonies showing the same
banding patterns were grouped and identified with known species.
The identity of each species pattern is listed on the top of each lane;
P. stands for Phytophthora and Py. stands for Pythium; L is a ssDNA
ladder. The population (%) of representative species within the total
ITS-1-amplified colonies is illustrated on top of the gel.
P. Kong et al. / Journal of Microbiological Methods 61 (2005) 25–32 29
3.2. Direct colony PCR for detection of Pythiaceae in
planta using species-specific primers
Colony PCR can be a quantitative tool for
pathogen detection that may provide more informa-
tion than standard PCR detection protocols. Direct
colony PCR was applied to detect the population level
of P. nicotianae and P. cinnamomi in container-grown
plants using species-specific primers (Table 2).
Colony PCR identified more than 95% of the isolates
recovered from leaf, stem, or roots of diseased vinca,
tobacco, and daphne as P. nicotianae. It also identified
32% and 11% of the isolates recovered from azalea
and tomato roots as P. cinnamomi and P. nicotianae,
respectively (Table 1). The results from vinca,
tobacco, and daphne plants were consistent with those
of the standard PCR assay (data not shown). However,
the low incidence results of the targeted pathogen
from azalea and tomato roots suggested involvement
of other pathogens, while the standard PCR assay
only detected P. cinnamomi (Kong et al., 2003c) or P.
nicotianae (data not shown). Use of standard PCR to
detect such samples may over- or underestimate the
contribution of the targeted pathogen and overlook
involvement of other pathogens.
Direct colony PCR for detection of Pythiaceae in
planta is relatively rapid compared with conventional
isolation and morphological examination or other
culture-dependent PCR techniques requiring subcul-
turing and DNA extraction. Generally, colony PCR
can be performed within 24 h of isolation, and a
quantitative result for disease detection and diagnosis
can be obtained in 2 days.
3.3. Direct colony PCR-SSCP for detection of
Pythiaceae in planta using ITS-1 primers
Although colony PCR with species-specific pri-
mers can be used to detect a suspected target, the
identity of other non-targeted species cannot be
determined. Yet, identification of other species
involved is important to determine the real cause of a
disease. In this study, colony PCR with oomycete-
specific ITS-1 primers (Table 2) was assembled with
SSCP analysis for identification of pythiaceous species
(Kong et al., 2003b, 2004). Colony PCR-SSCP was
used to determine the composition and population size
of organisms recovered from azalea roots where only
32% of the colonies isolated were positive for P.
cinnamomi. Colony PCR detected 36 of 47 recovered
colonies from azalea roots, indicating that 77% of
these isolates are oomycetes. The SSCP grouped these
ITS-1-positive colonies by their respective banding
patterns that were then identified into species by
comparing with known patterns (Fig. 2, bottom). Eight
groups/species including P. cinnamomi, Phytophthora
megasperma, Phytophthora citricola, Phytophthora
cryptogea, Pythium macrosporum, Pythium deliense,
and two unknown oomycetes were recovered. The
population of P. cinnamomi assessed with colony
PCR-SSCP accounted for 41.7% of ITS-1-positive
Fig. 3. Efficacy of direct colony PCR from a pine bark soil mix.
PCR products with ITS-1 primers of two representative colonies
from each of three washed and unwashed isolation plates, and the
reamplified PCR products of the colonies from unwashed plates
were electrophoresed in a 1% agarose gel.
Fig. 4. Direct colony PCR-SSCP analysis of pythiaceous species in
irrigation water. Colonies recovered from recycled irrigation pond
and run-off water in three plates on two selective media (PARP-V8
and PARPH-V8) were subjected to direct PCR amplification with
primers ITS 6/7. The PCR products were then denatured and
electrophoresed in an 8% native acrylamide gel. Colonies showing
the same banding patterns were grouped and identified with known
species. The identity of each species pattern is listed on the top of
each lane; P. stands for Phytophthora and Py. stands for Pythium; L
is a ssDNA ladder. The population (%) of each group within the tota
ITS-1-amplified colonies is illustrated on top of the gel.
P. Kong et al. / Journal of Microbiological Methods 61 (2005) 25–3230
colonies and 31.9% of the total recovered colonies,
consistent with the results obtained using species-
specific colony PCR. P. megasperma was the second
most abundant population (17.6%); Py. macrosporum
and Py. deliense, and P. citricola were the third and
fourth, respectively (Fig. 2, top). These results indicate
that there were at least four species involved in
infection of the azalea plants.
Colony PCR-SSCP with universal ITS-1 primers
can theoretically detect all oomycete species recov-
ered from environmental samples. Since the result is
based on isolated colonies, and each colony contains
only one type of PCR template, colony PCR-SSCP is
not plagued with false detection problems of standard
PCR due to bias in the presence of multiple templates
or different copy numbers of various species in a
sample. This feature makes colony PCR-SSCP supe-
rior to other methods that target multiple species.
3.4. Direct colony PCR-SSCP for detection of
pythiaceae in soil
Direct colony PCR with ITS-1 primers detected
more than 95% of the colonies recovered from a peat
moss-based mix (Metro Mix) where a diseased
daphne plant was removed, but did not detect colonies
from pine bark mix from a diseased azalea. However,
the PCR was successful when 1 Al of the initial colonyPCR products were reamplified (Fig. 3), indicating
PCR inhibition from the pine bark mix in the first
reaction. When the isolation plates were washed free
of soil particles before direct colony PCR, successful
amplification occurred, resulting in more intensive
bands than the reamplified products (Fig. 3). This
study indicates that efficacy of direct colony PCR on
soil samples is dependent on the inhibitory effect of
different soil types on the PCR reaction. These
problems can be eliminated by removal of the soil
particles from isolation plates.
SSCP analysis of ITS-1 PCR products of the
colonies from the peat moss-based mix from daphne
and the pine bark mix from azalea facilitated
investigation of pathogen survival in these mixes.
Direct colony PCR detected only a single species (P.
nicotianae and P. megasperma, respectively) com-
prising 90% of the isolates from both soil samples.
The population level of the pathogen detected from
the peat moss-based mix coincided with the level
from the removed daphne root tissue (Table 1),
indicating that P. nicotianae has colonized both the
soil and infected plant residues. In contrast, the P.
cinnamomi pathogen detected from the contained
l
P. Kong et al. / Journal of Microbiological Methods 61 (2005) 25–32 31
azalea root tissue (Table 1) was not detected in the
pine bark mix, indicating that this pathogen might
have not colonized the potting media. Similar results
were reported in a previous study, in which P.
cinnamomi was detected in root tissue but not in the
soil (Kong et al., 2003c).
3.5. Direct colony PCR-SSCP for detection of
Pythiaceae populations in recycled irrigation water
Colony PCR-SSCP can be an efficient and econom-
ical method to monitor pythiaceous species in recycled
irrigation water. Unlike potting soil samples, inhibition
was not observed for colony PCR on irrigation run-off
and recycled water samples plated. ITS-1 was success-
fully detected for 39 of 40 colonies recovered. SSCP
analysis readily identified 10 species by pattern type
and 4 unknown (Fig. 4). Three species were found in
both the pond and run-off water samples. Phytophthora
tropicalis was the most common, followed by Phy-
tophthora drechsleri and Pythium dissotocum. Four
species were found only in the pond water, and
Phytophthora citrophthora was the most common.
Seven species were found only in the run-off water and
P. citricola was the most abundant.
This study demonstrated that colony PCR and
colony PCR-SSCP are useful tools for the identifica-
tion and quantitative detection of pythiaceous species
in plant, soil, and water samples. Colony PCR-SSCP
also can be an alternative to multiplex PCR and PCR-
DGGE for analyzing the diversity and dynamics of
populations of other culturable microoganisms in the
environment. Using individual colonies, direct colony
PCR-SSCP can avoid PCR bias due to the presence of
multitemplates, varying copy numbers of target
sequences, or PCR inhibitors in DNA extracts from
environmental samples. Direct colony PCR-SSCP can
detect any number of species in a targeted group and
their population size. Multiplex PCR and PCR-DGGE
require purified DNA from a sample and can detect
only a limited number of species in a targeted group,
and determination of the population size of each
species is difficult. The number of species detected
with multiplex PCR depends on the number and
specificity of primer pairs. PCR-DGGE uses a single
pair of primers, but the number of species detected is
dependent on the separation of individual species of
the sample in a single lane of a gel. Incorrect
interpretation of results can occur due to co-migration
of DNA fragments of some species that have identical
melting behavior, the misdistribution of the bands
because of formation of chimeric or heteroduplex
molecules, or microheterogeneity in sequences
(Muyzer, 1999; Ercolini, 2004). Moreover, direct
colony PCR-SSCP identifies isolates to species with
readily visible banding patterns while PCR-DGGE
requires sequencing of each species-specific band,
which could be costly for species monitoring or
screening purposes.
Acknowledgements
We thank Professor Gary W. Moorman for
critically reading this manuscript.
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