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Previous Article | Next Article 
Applied and Environmental Microbiology, November 2000, p. 4758-4763, Vol. 66, No. 11
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Identification of a Universally Primed-PCR-Derived
Sequence-Characterized Amplified Region Marker for an Antagonistic
Strain of Clonostachys rosea and Development of a
Strain-Specific PCR Detection Assay
Sergey A.
Bulat,1
Mette
Lübeck,2,*
Irina A.
Alekhina,1
Dan Funck
Jensen,2
Inge M. B.
Knudsen,2 and
Peter
Stephensen
Lübeck2,
Laboratory of Eukaryote Genetics, Petersburg
Nuclear Physics Institute, Gatchina 188350, Russia,1 and Plant Pathology
Section, Department of Plant Biology, The Royal Veterinary and
Agricultural University, DK-1871 Frederiksberg C,
Denmark2
Received 17 March 2000/Accepted 11 August 2000
 |
ABSTRACT |
We developed a PCR detection method that selectively recognizes a
single biological control agent and demonstrated that universally primed PCR (UP-PCR) can identify strain-specific markers. Antagonistic strains of Clonostachys rosea (syn. Gliocladium
roseum) were screened by UP-PCR, and a strain-specific marker
was identified for strain GR5. No significant sequence
homology was found between this marker and any other
sequences in the databases. Southern blot analysis of the PCR
product revealed that the marker represented a single-copy sequence
specific for strain GR5. The marker was converted into a
sequence-characterized amplified region (SCAR), and a specific PCR
primer pair was designed. Eighty-two strains, isolated primarily from
Danish soils, and 31 soil samples, originating from different localities, were tested, and this specificity was confirmed. Two strains responded to the SCAR primers under suboptimal PCR conditions, and the amplified sequences from these strains were similar, but not identical, to the GR5 marker. Soil assays in which total DNA was
extracted from GR5-infested and noninoculated field soils showed that
the SCAR primers could detect GR5 in a pool of mixed DNA and that no
other soil microorganisms present contained sequences amplified by the
primers. The assay developed will be useful for monitoring
biological control agents released into natural field soil.
| |
INTRODUCTION |
Clonostachys rosea
Schroers, Samuels, Seifert & Gams (syn. Gliocladium roseum
Bain.) is a common saprophyte in soil worldwide (10, 22,
25). It is antagonistic to other fungi, including important plant
pathogens, in soil and plant material (11, 12, 24, 25, 29,
31). The mode of action responsible for this antagonism is not
well understood, but mycoparasitism, substrate competition, antibiosis,
and induced resistance all may play a role (25). Several
strains of C. rosea, including GR5, with antagonistic
activities against seed-borne diseases of cereals (12) were
analyzed by universally primed PCR (UP-PCR) and found to be genetically
very similar but not clonal (6). The release of biocontrol
agents into the environment has created a demand for methods for
monitoring and distinguishing them from indigenous strains. Monitoring
a selected Clonostachys strain in soil using dilution plates
has the disadvantage that it is not possible to distinguish the
released strain from indigenous strains of the same species
(13). Therefore, methods with high degrees of specificity and sensitivity are needed for detection of individual strains.
PCR has become an attractive tool for detection of specific
microorganisms in microbial ecology, and much effort has been devoted
to the development of primers that recognize specific species (8,
16, 19, 30). Genetically modified microorganisms can be
recognized by PCR because the foreign sequence they carry differs from
the background, thus allowing the design of primers that selectively
recognize the target gene (3, 27, 28). PCR also can be used
to recognize individual wild-type strains if unique sequences can be
identified (1, 8, 19). Thus, PCR techniques also may be used
to analyze environmental samples without culturing the microorganisms.
The UP-PCR technique (7) is similar to the randomly
amplified polymorphic DNA (RAPD) technique (32), but longer
primers (approximately 16 to 21 nucleotides) with unique designs are
used (6, 8). The reactions are carried out at relatively
high annealing temperatures and result in highly reproducible
amplification products (fingerprints) from single organisms.
Our objective in this study was to use UP-PCR to develop a PCR
detection method for a single C. rosea strain (GR5) from
natural field soil. GR5 has promise as a biocontrol agent of F. culmorum in barley, based on in planta bioassays (unpublished
results). By converting one of the UP-PCR-derived markers into a
sequence-characterized amplification region (SCAR), we developed a
simple PCR procedure for direct detection of GR5 released into field soil.
| |
MATERIALS AND METHODS |
Fungal strains, growth conditions, and DNA extraction.
Strains of C. rosea and Gliocladium spp. (Table
1) were stored at 
80°C in 10%
glycerol and grown on potato dextrose agar (Difco Laboratories,
Detroit, Mich.). Fresh fungal mycelium (4 to 7 days old) of each
isolate was scraped off petri dishes and added to one-third of the
conical part of a 1.5-ml microcentrifuge tube. Total DNA was extracted
basically as described by Bulat et al. (6), by adding 600 µl of buffer (50 mM Tris-HCl [pH 7.8]), 50 mM EDTA, 150 mM NaCl,
2.5% N-lauroyl sarcosine, 500 mM 2-mercaptoethanol, 600 µg of proteinase K [Sigma Chemical Co., St. Louis, Mo.] per ml) at
65°C for 4 h with frequent mixing. The NaCl concentration of the
solution was then adjusted to 1 M, and two extractions to denature the
proteins were carried out as follows. First, an equal amount of
phenol-chloroform mixture (1:1) was added, left for 15 min at room
temperature (20 to 25°C), and centrifuged at 12,000 × g for 2 min, and the aqueous phase was transferred to a new tube.
An equal volume of chloroform-octanol mixture (24:1) was then added and
left for 15 min at room temperature. After centrifugation at
12,000 × g for 2 min, the aqueous phase again was
transferred, and the DNA was precipitated with isopropyl alcohol (0.6 volume) for a few minutes at 21°C. The precipitate was rinsed once
with 70% ethanol, vacuum dried, and dissolved in TE buffer (1 mM
Tris-HCl [pH 7.8], 0.1 mM EDTA). The final DNA concentration was 50 to 100 mg/ml. The DNA quality and optimal concentration for PCR for
each strain were adjusted by testing dilution series in UP-PCR using a
single UP primer.
UP-PCR amplification.
The UP primers (Table
2) were used individually and in pairwise
combinations (a total of 21 combinations). UP-PCR was performed as
described by Bulat et al. (6) and Lübeck et al.
(18) in a 20-µl volume containing 20 mM Tris-HCl (pH 8.8),
10 mM (NH4)2SO4, 0.2 mM
deoxynucleoside triphosphates (dNTPs), 2.8 mM MgCl2, 40 ng
of primers, between 10 and 100 ng of total DNA, and 2 to 3 U of
Tsp (Thermus sp.) DNA polymerase (courtesy of
O. K. Kaboev, Petersburg Nuclear Physics Institute, St.
Petersburg, Russia). The temperature optimum for Tsp
polymerase is 69°C.
Alternatively, UP-PCR was performed in a 20-µl volume containing 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 0.1% Triton X-100, 0.4 mM dNTPs, 3.5 mM MgCl2, 40 ng of primers, between 10 and 100 ng of total
DNA, and 1 U of DynaZyme version 2.0 polymerase (Finnzymes OY, Espoo,
Finland). The temperature optimum for DynaZyme is 72°C.
We used a high-ramping TC-1000M thermal cycler (Petersburg Nuclear
Physics Institute) with 0.5-ml narrow tubes (Lenmedpolimer, St.
Petersburg, Russia). PCR conditions were as follows (the times include
temperature-ramping periods): 30 cycles with DNA denaturation at 92°C
for 50 s (first denaturation step at 94°C for 2.5 min), primer
annealing at 52 to 55°C for 90 s, and primer extension at 69°C
for 60 s. The rate of ramping between the different temperatures was about 4°C/s. A final extension was performed at 69°C for 3 min.
We also performed UP-PCRs in a model PTC-150 MiniCycler (MJ Research,
Watertown, Mass.) with standard 0.5-ml PCR tubes (Biozym Diagnostik
GmBH, Oldendorf, Germany). PCR conditions were as follows (the times
exclude temperature-ramping periods): 30 cycles with DNA denaturation
at 92°C for 50 s (first denaturation step at 94°C for 3 min),
primer annealing at 53 to 56°C for 70 s, and primer extension at
72°C for 60 s. The ramping rate was about 2.4°C/s. A final
extension was performed at 72°C for 3 min.
Amplification products were initially resolved electrophoretically in
1.7% agarose gels at 300 V for 30 to 40 min. Products from some of the
amplifications also were resolved electrophoretically in 20-cm-long
5.5% polyacrylamide gels at 170 V for 10 h with cold
Tris-borate-EDTA buffer.
Elution of PCR products, Southern blot hybridization, and
sequencing.
The DNA band of interest was cut from an acrylamide
gel and placed in a 1.5-ml microcentrifuge tube. The gel slice was
crushed with a pestle directly in the tube, and the tube was placed in boiling water for 7 min. The resulting DNA solution was reamplified using the UP primers AA2M2 and AS15inv (Table 2) but with 13 instead of
30 cycles. The PCR products were extracted with chloroform-octanol (24:1) and precipitated with 0.7 volume of isopropanol for 10 to 30 min
at room temperature. The pellet was washed with 70% ethanol, dried,
and redissolved in TE buffer. The resulting DNA was then ready
to be sequenced.
For Southern blot hybridization, genomic DNA was digested with
HindIII (Fermentas AB, Vilnius, Lithuania) and
electrophoresed in a 0.7% agarose gel. The DNA was blotted onto a
nylon filter (Hybond N, Amersham Pharmacia Biotech Ltd.,
Buckinghamshire, United Kingdom), and labeling and hybridization were
carried out as described by Bulat et al. (6).
The PCR product was sequenced in both directions using the two primers
AA2M2 and AS15inv and the ABI Prism Big Dye Sequencing System
(Perkin-Elmer Applied Biosystems, Foster City, Calif.). The sequence
for the marker had no homology with known DNA sequences as determined
by using FASTA (21) and BLAST (2) searches to
screen EMBL/GenBank. The sequences for GR5 and two closely related
strains (GR47 and GJ 98-34) were deposited in GenBank.
Design of SCAR primers and PCR amplification conditions.
From the sequence data, a pair of SCAR primers (GR5f and GR5r) with the
expected size of the diagnostic product of 121 bp was designed (Table
2). Genomic DNA was amplified in PCR mixtures containing 0.6 pmol of
GR5f, 1.4 pmol of GR5r, 20 mM Tris-HCl (pH 8.8), 2.8 mM
MgCl2, 10 mM KCl, 10 mM (NH4)SO4,
0.1% Triton X-100, 0.2 mM dNTPs, and 0.2 U of DynaZyme polymerase in a
volume of 20 µl. The amplification conditions were as follows: 30 cycles with DNA denaturation at 92°C for 50 s (first
denaturation step at 94°C for 3 min), primer annealing at 58°C for
80 s, and primer extension at 72°C for 40 s. The final
extension step was performed at 72°C for 3 min. DNA extracted from
soil samples was tested in dilution series using the SCAR primer pair,
and 1/10 of the amplification products was checked on 1.7% agarose gels.
Extraction and purification of DNA from soil microorganisms.
DNA was extracted from 0.5 g of soil by mixing with a lysing
buffer (50 mM Tris-HCl [pH 7.8], 150 mM NaCl, 2.5%
N-lauroyl sarcosine, 0.5 M 2-mercaptoethanol, 200 µg of
proteinase K [Sigma] ml
1) and incubated for 2 to 4 h at 65°C. The soil particles were precipitated by centrifugation for
1 min at 10,000 × g. This procedure was repeated twice
by adding new lysing buffer to the pelleted soil particles. The
supernatants from the extractions were combined, the NaCl concentration
of the solution was adjusted to 1 M, and the solution was then
extracted twice, first with a phenol-chloroform (1:1) mixture and then
with chloroform-octanol (24:1). The DNA was precipitated with 0.6 volume of isopropyl alcohol. The pellet was rinsed with 70% ethanol,
dried, and dissolved in 300 µl of Tris buffer (10 mM Tris-HCl, pH
8.0) at 50°C for 15 min. The crude DNA solution was purified twice
with the Wizard DNA Clean-Up System (Promega Co., Madison, Wis.)
according to the manufacturer's instructions and eluted with 50 µl
of Tris buffer (10 mM Tris-HCl, pH 8.0).
As a positive control, each DNA sample was amplified in different
concentrations with primers that recognize the ITS1 region of fungal
nuclear ribosomal DNA. This control was performed to ensure the quality
of the extracted DNA, to test for presence of fungal DNA, and to
optimize the DNA concentration to minimize the risk of obtaining a
false-negative result. The amplification conditions for ITS1
amplification using the primer pair X and Y were as described by Bulat
et al. (6). DNA extraction for some of the samples had to be
repeated until they fulfilled this criterion (out of 33 soil samples,
31 were used) (Table 3). Subsequently, DNA from each soil sample was tested with the SCAR primer pair for
presence of the GR5-specific sequence.
Inoculation of C. rosea strain GR5 into field
soil.
The soil used for this experiment was field soil from two
Danish barley fields (B23DK and B24DK) (Table 3). The B24DK soil sample
was a composite sample from nine collections in the field. To avoid
large soil aggregates and to obtain homogeneous material, the soil was
sieved through a 4-mm sieve prior to inoculation with GR5 germinated
conidia. A conidial suspension of GR5 (108 cells/ml) was
added to 20 ml of potato dextrose broth (Difco) in an 100-ml Erlenmeyer
flask and incubated overnight at 25°C on an orbital shaker
at 180 rpm. The germinated conidia and young hyphae were harvested on a
polyester filter membrane (42-µm mesh) and washed with sterile water.
Two different amounts of the germinated conidia and hyphae (1 and
0.1 ml) were used to inoculate 10 g of soil in 50-ml capped
plastic tubes. DNA from the inoculated soil was extracted and tested as
described above.
Dilution plating for test of indigenous C. rosea.
We
tested for the presence of indigenous C. rosea in two of the
noninoculated soil samples (B23DK and B24DK) by dilution plating of
three replicate subsamples of soil (equal to 10 g [dry weight]). Each sample was homogenized with 90 ml of sterile water, and dilutions were plated on V8 agar (20% Campbell V8 juice, 2% Bacto agar
[Difco], and 0.21% Triton X-100). The medium was adjusted to pH 7 and after autoclaving was amended with antibiotics (chloramphenicol
[0.5 g/liter] and tetracycline [0.25 g/liter]). Isolates were
identified in a stereomicroscope. C. rosea isolates were
recultivated on potato dextrose agar amended with antibiotics and
stored at 80°C in 10% glycerol until use.
Nucleotide sequence accession numbers.
The GenBank accession
numbers for the sequences from strains GR5, GR47, and GJ 98-34 are
AF141913, AF141914, and AF141915, respectively.
| |
RESULTS |
Identification of strain-specific UP-PCR markers.
Seven UP
primers, individually and in pairwise combinations (21 primer
combinations), were tested for the ability to distinguish 14 C. rosea strains (Table 1). The primer
combination AS15inv-AA2M2 amplified a unique 250-bp UP-PCR fragment
from GR5 (Fig. 1). When used as a probe
in Southern blots, this fragment was found as a single copy that was
limited to GR5 (Fig. 2).
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FIG. 1.
UP-PCR banding profiles for C. rosea strains
generated with the AS15inv-AA2M2 UP primer combination. Lanes 1 to 14, strains GR3, IK726, GR6, GR10, GR12, GR11, GR9, GR8, GR4, GR5, GR1,
GR13, GR7, and GR2, respectively. Lanes M, molecular size markers ( phage DNA digested with PstI). Arrows shows the marker of
interest for strain GR5.
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FIG. 2.
Southern blot hybridization analysis of the GR5
AS15inv-AA2M2 marker. Lanes 1 to 14, strains IK726, GR12, GR10, GR13,
GR5, GR3, GR2, GR4, GR7, GR6, GR11, GR9, GR8, and GR1, respectively.
Lanes M, molecular size markers.  -32P-labeled
AS15inv-AA2M2 PCR product from strain GR5 was used as the probe.
|
|
Screening of C. rosea strains for the GR5-specific
marker.
The SCAR primer pair was tested for specificity against
the 14 strains plus 68 additional strains of C. rosea and
Gliocladium spp. (Table 1; Fig.
3). Only DNA from the GR5 strain could be amplified using the SCAR primers to produce the diagnostic product. We
therefore hypothesize that this sequence is unique for GR5. Under
suboptimal reaction conditions, DNAs from two other strains (GJS 89-34 and GR47 [Table 1]) could be amplified with the SCAR primers, but
both strains could be differentiated from GR5 by UP-PCR (Fig.
4). The fragments, from these strains
were sequenced, and they differed slightly from each other and from the
GR5 sequence (96 to 99% similarity).
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FIG. 3.
Testing of C. rosea strains for the
AS15inv-AA2M2 marker using the SCAR primer set. Lanes 1 to 14, strains
IK726, GR12, GR10, GR13, GR5, GR3, GR2, GR4, GR7, GR6, GR11, GR9, GR8,
and GR1, respectively. Lanes M, molecular size markers ( phage DNA
digested with PstI).
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FIG. 4.
Differentiation of GR5 from GR47 and GJS 89-34 by
UP-PCR. Lanes 1 to 3, profiles generated with UP primer AS15inv. Lanes
4 to 6, profiles generated with UP primer combination AS15inv-AA2M2.
Lanes 7 to 9, profiles generated with UP primer AA2M2. Lanes 1, 4, and
7, strain GR5. Lanes 2, 5, and 8, strain GJS 89-34. Lanes 3, 6, and 9, strain GR47. Lanes M, molecular size markers ( phage DNA digested
with PstI).
|
|
Detection of GR5 in soil.
DNA was extracted from
GR5-inoculated and control field soils from 31 different fields, mostly
Danish (Table 3), and tested in dilution series in PCR using primers
that amplify the ITS1 region of fungal nuclear ribosomal DNA (positive
control for amplification), followed by testing with the SCAR primer
pair. GR5 was isolated in 1991 from a field neighboring soil B24DK. In
some DNA extractions from noninoculated B24DK, a weak product of
similar size was produced using the SCAR primers. This might indicate
that GR5 or strains carrying the GR5-specific SCAR marker were
naturally present in that soil at a low level. In all other field soils
tested, no background level of the GR5 marker was detected. For the two
GR5-inoculated soils we could detect GR5 using the SCAR primer pair
(Fig. 5).
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FIG. 5.
GR5 detection in the B23DK soil using the SCAR primer
set. Lane 1, GR5 (positive control); lanes 2 to 5, soil sample with
large amount of GR5 (0.1, 0.01, 0.001, and 0.0001 µl of eluted DNA,
respectively); lanes 6 to 8, soil sample with a smaller amount of GR5
(0.1, 0.01, and 0.001 µl of eluted DNA, respectively); lanes 9 and
10, noninfested soil sample (negative control) (0.1 and 0.01 µl of
eluted DNA, respectively). Lanes M, molecular size markers.
|
|
We tested two of the noninoculated soils for the presence of indigenous
C. rosea strains by plate diluting on semiselective media.
We found that soil B23DK (Table 3) contained C. rosea at a
level of approximately 4 × 103 CFU/g (dry weight) of
soil and that soil B24DK contained C. rosea at a level of
approximately 103 CFU/g of soil. Representative isolates
from the plate dilutions were screened for the GR5-specific marker
using the SCAR primers, and all were negative.
| |
DISCUSSION |
We have developed a strain-specific PCR-based detection tool for
an antagonistic C. rosea strain, GR5. This strain can
control disease caused by F. culmorum efficiently in an in
planta bioassay that has been shown to correlate well with field
performance (12, 14, 26). The detection assay will be used
to detect and monitor GR5 deliberately released into field soil. We
used UP-PCR to identify a fragment from which SCAR primers were
developed and used for PCR detection of the strain. Markers such as the
one we have developed may be important for detection of other
biocontrol agents or specific pathogenic microorganisms or for
protection of commercial (patent) strains. In general, UP-PCR-derived
markers have little, if any, similarity to sequences in known
databases, and this 250-bp marker also lacked any significant sequence similarity.
In previous studies, specific primers have been developed for fungal
subspecies, varieties, and even strains using ribosomal DNA variable
regions (4, 5, 22). Strain differentiation problems often
occur as the sample size increases, however, since only a few strains
have been used for the primer design in many of the reported examples
(see, e.g., references 4 and 15). Similar work has been done with the RAPD technique. Zimand et al.
(33) used a set of nine RAPD primers to differentiate
Trichoderma harzianum strain T-39 from other
Trichoderma strains, and Abbasi et al. (1) used
three RAPD markers to differentiate Trichoderma hamatum
strain 382 from 45 other T. hamatum strains. Abbasi et al.
(1) converted the RAPD markers into SCAR markers, none of
which was unique for the strain. However, their approach was not tested
with noninoculated compost. Abbasi et al. (1) used 180 RAPD
primers to screen their T. hamatum strains in a strategy similar to ours, but we only used 7 different UP primers in this work.
The specificity of the SCAR primers was tested on 77 C. rosea strains and 5 strains from two closely related species, but only GR5 carried this sequence. Of the two strains that responded to
the SCAR primers under suboptimal reaction conditions, one was from
Guyana and the other was from Denmark. The fragments amplified from
these strains were quite similar (96 to 99% similarity) to the GR5
sequence. Although the marker is unique for GR5 among the strains
tested, DNA with a similar but slightly varying sequence composition
does appear to occur in other C. rosea strains.
We also tested the specificity of the primers on DNA in field soil
samples obtained from 31 different locations. One of the samples came
from the same locality from which GR5 originated (B24DK). Very weak
amplification sometimes occurred, indicating the presence of a
background level of GR5 or strains with the GR5 marker in this soil.
Thus, in order to use the marker as a tool for monitoring GR5 in
different soils, it is essential to define the background level of the
marker. In the other 30 soils no background was detected, even though
at least some of these soils contained indigenous C. rosea.
The isolates recovered by a plate dilution technique from these soils
did not respond to the primers. Detection of a small number of target
organisms in an environment requires a high level of sensitivity
(19, 23). The assay we have developed could be used to
detect GR5 deliberately released into field soil. However, the assay is
qualitative, and future work will be devoted to developing a sensitive
quantitative assay for facilitating strain counts. This assay will
enable us to take a background level of the marker into account in
comparative studies and give us the opportunity to identify optimal
levels of GR5 in relation to biocontrol efficacy and to study long-term survival after field release. Because mixtures of different strains for
biocontrol may enhance the performance of a commercial product, we also
are developing similar detection schemes for other antagonistic C. rosea strains.
| |
ACKNOWLEDGMENTS |
We thank Ulf Thrane, Department of Biotechnology, Danish
Technical University, Lyngby, Denmark; Gary Samuels, U.S. Department of
Agriculture, Agricultural Research Service, Beltsville, Md.; and Alison
Stewart, Department of Plant Science, Lincoln University, Canterbury,
New Zealand, for providing us with C. rosea strains. We also
thank three anonymous reviewers for comments on the manuscript and
Karin Olesen for technical assistance.
This investigation was supported by the Danish Ministry of Education, a
program from the Danish Ministry of Environmental Affairs (SMP2), and
the Russian State Program Frontiers in Genetics (in part). Grants from
the Nordic Academy for Advanced Study (NorFA) and the Danish Rectors
Conference financed visits for Sergey Bulat and Irina Alekhina.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Plant Pathology
Section, Department of Plant Biology, The Royal Veterinary and
Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C,
Denmark. Phone: (45) 3528 3304. Fax: (45) 3528 3310. E-mail:
met{at}kvl.dk.
Present address: The Danish Veterinary Laboratory, DK-1790
Copenhagen, Denmark.
| |
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Applied and Environmental Microbiology, November 2000, p. 4758-4763, Vol. 66, No. 11
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
