- ข้อความ
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Species level identi¢cation of coni
Species level identi¢cation of conifer associated Ceratocystis sapstain
fungi by PCR-RFLP on a L-tubulin gene fragment
Peter A. Loppnau, Colette Breuil
Department of Wood Science, University of British Columbia, 2424 Main Mall, Vancouver, BC, Canada V6T1Z4
Received 15 November 2002; accepted 27 March 2003
First published online 17 April 2003
Abstract
The genus Ceratocystis includes several morphologically similar species commonly found as agents of sapstain in coniferous trees. In
this paper we describe a simple and reliable polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique
that aids in the identification and differentiation of these fungi. PCR was used to amplify a 1.3-kb fragment of the L-tubulin gene from
C. coerulescens, C. pinicola, C. douglassi, C. resinifera, C. rufipenni, C. polonica and C. adiposa. The PCR amplicon from representative
isolates was sequenced. This information was utilized to select restriction enzymes that generated species-specific RFLP patterns. This
approach was tested on our collection of over 200 Ceratocystis isolates and identified the fungi with a high level of confidence, reducing
the time needed to identify these species by classical methods.
2 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords: L-Tubulin; Polymerase chain reaction-restriction fragment length polymorphism; Ceratocystis; Sapstain; Blue stain
1. Introduction
The genus Ceratocystis sensu stricto Ellis and Halsted is
a small group of saprobes and parasites of many commercially
important crop plants and trees. They are distinguished
from other ophiostomatoid fungi by their £askshaped
ascomata, Chalara anamorphs, and sensitivity to
cycloheximide [1,2]. Several species have been described as
growing on coniferous hosts and are capable of staining
the sapwood blue or gray. The growth and staining of
these fungi in wood is rapid and they cause considerable
value loss to the wood products industry.
The conifer associated Ceratocystis species are morphologically
similar yet appear to occupy di¡erent ecological
niches [3] and are intersterile [4]. They display a range of
behaviors, from saprobic to pathogenic growth. C. coerulescens
has been isolated from Picea and Pinus in both
continental Europe and North America. C. pinicola has
been isolated from Scots pine and Corsican pine, in Great
Britain. C. douglassi is a common saprophyte found on
Douglas ¢r lumber in western North America [5]. These
three species colonize and stain the butts of wind blown
trees and broken roots or freshly cut logs or lumber [6]. C.
resinifera is found on Picea and Pinus species of continental
Europe and North America. In addition to saprobic
growth, this species has also been described as a weak
pathogen, growing rapidly from wound sites in living Norway
spruce. C. coerulescens, C. pinicola, C. douglassi and
C. resinifera are all thought to have non-speci¢c relationships
with bark beetles and other insect vectors, sporulate
readily in culture, and produce a strong aromatic aroma,
possibly to attract their vectors [3,6].
Another three species of Ceratocystis form close symbiotic
relationships with bark beetles. In association with
beetle attack these fungi are pathogenic to healthy trees.
The fungi sporulate scarcely in culture and do not produce
a fruity odor. C. ru¢penni, found in western North America,
is vectored by the North American spruce beetle Dendroctonus
ru¢pennis and is a pathogen of Engelmann and
white spruces [7]. C. polonica is found across Eurasia and
Japan on Picea species and is vectored by Ips typographus.
It is best known in Norway where it is an aggressive
0378-1097 / 03 / $22.00 2 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
doi:10.1016/S0378-1097(03)00256-8
* Corresponding author. Tel. : +1(604) 822-9738;
Fax: +1(604) 922-9104.
E-mail address: breuil@interchange.ubc.ca (C. Breuil).
FEMS Microbiology Letters 222 (2003) 143^147
www.fems-microbiology.org
pathogen of Norway spruce [8]. C. laricicola, also found
across Eurasia and Japan, is restricted to larch and associated
with the bark beetle Ips cembrae.
C. adiposa is distantly related and morphologically distinct
from the above-described species. However, we include
it in this study since it has been isolated from
wood chips and sawn lumber in North America [9].
Species of Ceratocystis can be di⁄cult to di¡erentiate.
For example, C. coerulescens, C. resinifera, and C. ru¢penni
overlap in host and geographical range. Although some
morphological di¡erences exist, such as the diameter of
perithecial bases and ascospore length, there is considerable
overlap in these characters and they can require
months to develop in culture. The polymerase chain reaction-
restriction fragment length polymorphism (PCRRFLP)
method has proved useful for the rapid and sensitive
detection and di¡erentiation of many plant pathogens.
Ribosomal DNA (rDNA) is a common target sequence
and has been used extensively for the di¡erentiation of
sapstain species [10,11]. However, these methods have
been unable to separate closely related Ceratocystis species
due to low sequence heterogeneity [12]. The L-tubulin gene
has also been successfully used for PCR-RFLP based differentiation
of species of Babesia and Theileria [13] and
distinguishing between groups of Leishmania [14]. Tubulin
proteins have a relatively high degree of conservation at
the amino acid and nucleotide level, but a relatively high
degree of variability in the intronic sequences.
In this paper we describe a rapid and reliable PCRRFLP
identi¢cation method to di¡erentiate species of Ceratocystis
using a fragment of the L-tubulin gene sequence,
which had higher sequence variability than rDNA. This
method was used to con¢rm the identity and to di¡erentiate
Ceratocystis sapstain fungi, which our lab routinely
isolates from stained logs and lumber in Canada.
2. Materials and methods
2.1. Fungal isolates
Isolates of Ceratocystis for L-tubulin sequence analysis
were obtained from various collections in North America
and Europe. These included two isolates of C. coerulescens
from Canada and Germany, three isolates of C. pinicola
from the UK, one isolate of C. douglassi from the USA,
six isolates of C. resinifera from Canada, two isolates of
C. ru¢penni from Canada, one isolate of C. polonica from
Norway, and one isolate of C. adiposa from Canada. Isolate
numbers and GenBank accession numbers for the
L-tubulin sequences are shown in Fig. 1. Ceratocystis isolates
for L-tubulin PCR-RFLP analysis were isolated from
logs at sites across Canada during the summer of 2000
following previously described methodology [9]. This collection
included isolates of C. resinifera : 40 from Williams
Lake, British Columbia, eight from Cranbrook, British
Columbia, 42 from Edson, Alberta, 34 from Big River,
Saskatchewan, 17 from Kirkland Lake, Ontario, 19 from
LaSarre, Quebec, 17 from Saint-Emile, Quebec, ¢ve from
Fore“t Montmorency, Quebec, and 18 from Plaster Rock,
New Brunswick. As well isolates of C. coerulescens were
obtained: one from Merrit, British Columbia, one from
Saint-Emile, and 36 from Cranbrook. Isolates of C. ru¢-
penni from previous collections, two from Prince George,
British Columbia and one from Edmonton, Alberta, were
also analyzed. Further details regarding this collection can
be obtained by request from the authors. Isolates were
stored as mycelial plugs of Oxoid malt extract agar in
20% glycerol at 4‡C for short periods or at 380‡C for
extended periods. Taxonomic characterization of isolates
followed morphological descriptions [3,5] as well as analysis
of rDNA sequences [12,15].
Fig. 1. One most parsimonious tree produced from the alignment of DNA sequences of a fragment of the Ceratocystis L-tubulin gene (tree length = 409,
CI =0.919, RI =0.876). Bootstrap values (1000 replicates) greater than 50% are indicated at the top of the branches. The bar indicates tree scale. Gen-
Bank accession numbers of published sequences: CL13-12 (AY140945), C313 (AY140943), 126-1E1 (AY140935), 0901 (AY140932), 206-291
(AY140937), 9320810 (AY140939), C324 (AY140944), BW2-111 (AY140942), D53-5A3 (AY140946), 123-314 (AY140933), Ai12-33 (AY140940), Ai13-14
(AY140941), 125-214 (AY140934), C609 (AY142242), 92.633.254.7 (AY140938), 152-5-1 (AY140936).
144 P.A. Loppnau, C. Breuil / FEMS Microbiology Letters 222 (2003) 143^147
2.2. PCR reactions and DNA sequencing
DNA for PCR ampli¢cations was extracted as described
previously [16]. A phenol chloroform extraction was performed
prior to isopropanol precipitation. PCR was performed
using a Hybaid Touch Down Thermal Cycler. The
50-Wl reactions contained 200 ng of genomic DNA,
40 pmol of each forward T10 (5P-ACG-ATA-GGT-TCACCT-
CCA-GAC-3P) [17] and reverse BT12 (5P-GTT-GTCAAT-
GCA-GAA-GGT-CTC-3P) primers, 100 WM dNTPs,
and 1U Taq polymerase. A hot start PCR was employed
using: initial denaturation at 94‡C for 4 min, ¢ve cycles of
denaturation at 94‡C for 50 s, primer annealing at 47‡C
for 50 s, and primer extension at 72‡C for 1min. This was
followed by an additional 30 cycles of denaturation at
94‡C for 50 s, primer annealing at 55‡C for 50 s, and
primer extension at 72‡C for 1min . Final chain elongation
was 72‡C for 10 min.
Templates for sequencing reactions were ampli¢ed as
described above and puri¢ed from 1% agarose gels using
a QIAquick Gel Extraction Kit (Qiagen). PCR products
were ligated to pCR-II0 TOPO (Invitrogen). Sequencing
of both strands was carried out using an ABI Prism1
BigDye1 Terminator Cycle Sequencing Ready Reaction
kit (PE Applied Biosystems). Sequencing products were
analyzed at the UBC Nucleic Acid and Protein Service
Laboratory (University of British Columbia, Vancouver,
BC, Canada) on an ABI 373 DNA sequencer (PE Applied
Biosystems). Direct sequencing was performed using the
primers T8 (5P-GAC-CGA-AGA-TGA-AGT-TGT-CG-
fungi by PCR-RFLP on a L-tubulin gene fragment
Peter A. Loppnau, Colette Breuil
Department of Wood Science, University of British Columbia, 2424 Main Mall, Vancouver, BC, Canada V6T1Z4
Received 15 November 2002; accepted 27 March 2003
First published online 17 April 2003
Abstract
The genus Ceratocystis includes several morphologically similar species commonly found as agents of sapstain in coniferous trees. In
this paper we describe a simple and reliable polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique
that aids in the identification and differentiation of these fungi. PCR was used to amplify a 1.3-kb fragment of the L-tubulin gene from
C. coerulescens, C. pinicola, C. douglassi, C. resinifera, C. rufipenni, C. polonica and C. adiposa. The PCR amplicon from representative
isolates was sequenced. This information was utilized to select restriction enzymes that generated species-specific RFLP patterns. This
approach was tested on our collection of over 200 Ceratocystis isolates and identified the fungi with a high level of confidence, reducing
the time needed to identify these species by classical methods.
2 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords: L-Tubulin; Polymerase chain reaction-restriction fragment length polymorphism; Ceratocystis; Sapstain; Blue stain
1. Introduction
The genus Ceratocystis sensu stricto Ellis and Halsted is
a small group of saprobes and parasites of many commercially
important crop plants and trees. They are distinguished
from other ophiostomatoid fungi by their £askshaped
ascomata, Chalara anamorphs, and sensitivity to
cycloheximide [1,2]. Several species have been described as
growing on coniferous hosts and are capable of staining
the sapwood blue or gray. The growth and staining of
these fungi in wood is rapid and they cause considerable
value loss to the wood products industry.
The conifer associated Ceratocystis species are morphologically
similar yet appear to occupy di¡erent ecological
niches [3] and are intersterile [4]. They display a range of
behaviors, from saprobic to pathogenic growth. C. coerulescens
has been isolated from Picea and Pinus in both
continental Europe and North America. C. pinicola has
been isolated from Scots pine and Corsican pine, in Great
Britain. C. douglassi is a common saprophyte found on
Douglas ¢r lumber in western North America [5]. These
three species colonize and stain the butts of wind blown
trees and broken roots or freshly cut logs or lumber [6]. C.
resinifera is found on Picea and Pinus species of continental
Europe and North America. In addition to saprobic
growth, this species has also been described as a weak
pathogen, growing rapidly from wound sites in living Norway
spruce. C. coerulescens, C. pinicola, C. douglassi and
C. resinifera are all thought to have non-speci¢c relationships
with bark beetles and other insect vectors, sporulate
readily in culture, and produce a strong aromatic aroma,
possibly to attract their vectors [3,6].
Another three species of Ceratocystis form close symbiotic
relationships with bark beetles. In association with
beetle attack these fungi are pathogenic to healthy trees.
The fungi sporulate scarcely in culture and do not produce
a fruity odor. C. ru¢penni, found in western North America,
is vectored by the North American spruce beetle Dendroctonus
ru¢pennis and is a pathogen of Engelmann and
white spruces [7]. C. polonica is found across Eurasia and
Japan on Picea species and is vectored by Ips typographus.
It is best known in Norway where it is an aggressive
0378-1097 / 03 / $22.00 2 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
doi:10.1016/S0378-1097(03)00256-8
* Corresponding author. Tel. : +1(604) 822-9738;
Fax: +1(604) 922-9104.
E-mail address: breuil@interchange.ubc.ca (C. Breuil).
FEMS Microbiology Letters 222 (2003) 143^147
www.fems-microbiology.org
pathogen of Norway spruce [8]. C. laricicola, also found
across Eurasia and Japan, is restricted to larch and associated
with the bark beetle Ips cembrae.
C. adiposa is distantly related and morphologically distinct
from the above-described species. However, we include
it in this study since it has been isolated from
wood chips and sawn lumber in North America [9].
Species of Ceratocystis can be di⁄cult to di¡erentiate.
For example, C. coerulescens, C. resinifera, and C. ru¢penni
overlap in host and geographical range. Although some
morphological di¡erences exist, such as the diameter of
perithecial bases and ascospore length, there is considerable
overlap in these characters and they can require
months to develop in culture. The polymerase chain reaction-
restriction fragment length polymorphism (PCRRFLP)
method has proved useful for the rapid and sensitive
detection and di¡erentiation of many plant pathogens.
Ribosomal DNA (rDNA) is a common target sequence
and has been used extensively for the di¡erentiation of
sapstain species [10,11]. However, these methods have
been unable to separate closely related Ceratocystis species
due to low sequence heterogeneity [12]. The L-tubulin gene
has also been successfully used for PCR-RFLP based differentiation
of species of Babesia and Theileria [13] and
distinguishing between groups of Leishmania [14]. Tubulin
proteins have a relatively high degree of conservation at
the amino acid and nucleotide level, but a relatively high
degree of variability in the intronic sequences.
In this paper we describe a rapid and reliable PCRRFLP
identi¢cation method to di¡erentiate species of Ceratocystis
using a fragment of the L-tubulin gene sequence,
which had higher sequence variability than rDNA. This
method was used to con¢rm the identity and to di¡erentiate
Ceratocystis sapstain fungi, which our lab routinely
isolates from stained logs and lumber in Canada.
2. Materials and methods
2.1. Fungal isolates
Isolates of Ceratocystis for L-tubulin sequence analysis
were obtained from various collections in North America
and Europe. These included two isolates of C. coerulescens
from Canada and Germany, three isolates of C. pinicola
from the UK, one isolate of C. douglassi from the USA,
six isolates of C. resinifera from Canada, two isolates of
C. ru¢penni from Canada, one isolate of C. polonica from
Norway, and one isolate of C. adiposa from Canada. Isolate
numbers and GenBank accession numbers for the
L-tubulin sequences are shown in Fig. 1. Ceratocystis isolates
for L-tubulin PCR-RFLP analysis were isolated from
logs at sites across Canada during the summer of 2000
following previously described methodology [9]. This collection
included isolates of C. resinifera : 40 from Williams
Lake, British Columbia, eight from Cranbrook, British
Columbia, 42 from Edson, Alberta, 34 from Big River,
Saskatchewan, 17 from Kirkland Lake, Ontario, 19 from
LaSarre, Quebec, 17 from Saint-Emile, Quebec, ¢ve from
Fore“t Montmorency, Quebec, and 18 from Plaster Rock,
New Brunswick. As well isolates of C. coerulescens were
obtained: one from Merrit, British Columbia, one from
Saint-Emile, and 36 from Cranbrook. Isolates of C. ru¢-
penni from previous collections, two from Prince George,
British Columbia and one from Edmonton, Alberta, were
also analyzed. Further details regarding this collection can
be obtained by request from the authors. Isolates were
stored as mycelial plugs of Oxoid malt extract agar in
20% glycerol at 4‡C for short periods or at 380‡C for
extended periods. Taxonomic characterization of isolates
followed morphological descriptions [3,5] as well as analysis
of rDNA sequences [12,15].
Fig. 1. One most parsimonious tree produced from the alignment of DNA sequences of a fragment of the Ceratocystis L-tubulin gene (tree length = 409,
CI =0.919, RI =0.876). Bootstrap values (1000 replicates) greater than 50% are indicated at the top of the branches. The bar indicates tree scale. Gen-
Bank accession numbers of published sequences: CL13-12 (AY140945), C313 (AY140943), 126-1E1 (AY140935), 0901 (AY140932), 206-291
(AY140937), 9320810 (AY140939), C324 (AY140944), BW2-111 (AY140942), D53-5A3 (AY140946), 123-314 (AY140933), Ai12-33 (AY140940), Ai13-14
(AY140941), 125-214 (AY140934), C609 (AY142242), 92.633.254.7 (AY140938), 152-5-1 (AY140936).
144 P.A. Loppnau, C. Breuil / FEMS Microbiology Letters 222 (2003) 143^147
2.2. PCR reactions and DNA sequencing
DNA for PCR ampli¢cations was extracted as described
previously [16]. A phenol chloroform extraction was performed
prior to isopropanol precipitation. PCR was performed
using a Hybaid Touch Down Thermal Cycler. The
50-Wl reactions contained 200 ng of genomic DNA,
40 pmol of each forward T10 (5P-ACG-ATA-GGT-TCACCT-
CCA-GAC-3P) [17] and reverse BT12 (5P-GTT-GTCAAT-
GCA-GAA-GGT-CTC-3P) primers, 100 WM dNTPs,
and 1U Taq polymerase. A hot start PCR was employed
using: initial denaturation at 94‡C for 4 min, ¢ve cycles of
denaturation at 94‡C for 50 s, primer annealing at 47‡C
for 50 s, and primer extension at 72‡C for 1min. This was
followed by an additional 30 cycles of denaturation at
94‡C for 50 s, primer annealing at 55‡C for 50 s, and
primer extension at 72‡C for 1min . Final chain elongation
was 72‡C for 10 min.
Templates for sequencing reactions were ampli¢ed as
described above and puri¢ed from 1% agarose gels using
a QIAquick Gel Extraction Kit (Qiagen). PCR products
were ligated to pCR-II0 TOPO (Invitrogen). Sequencing
of both strands was carried out using an ABI Prism1
BigDye1 Terminator Cycle Sequencing Ready Reaction
kit (PE Applied Biosystems). Sequencing products were
analyzed at the UBC Nucleic Acid and Protein Service
Laboratory (University of British Columbia, Vancouver,
BC, Canada) on an ABI 373 DNA sequencer (PE Applied
Biosystems). Direct sequencing was performed using the
primers T8 (5P-GAC-CGA-AGA-TGA-AGT-TGT-CG-
0/5000
Species level identi¢cation of conifer associated Ceratocystis sapstainfungi by PCR-RFLP on a L-tubulin gene fragmentPeter A. Loppnau, Colette Breuil Department of Wood Science, University of British Columbia, 2424 Main Mall, Vancouver, BC, Canada V6T1Z4Received 15 November 2002; accepted 27 March 2003First published online 17 April 2003AbstractThe genus Ceratocystis includes several morphologically similar species commonly found as agents of sapstain in coniferous trees. Inthis paper we describe a simple and reliable polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniquethat aids in the identification and differentiation of these fungi. PCR was used to amplify a 1.3-kb fragment of the L-tubulin gene fromC. coerulescens, C. pinicola, C. douglassi, C. resinifera, C. rufipenni, C. polonica and C. adiposa. The PCR amplicon from representativeisolates was sequenced. This information was utilized to select restriction enzymes that generated species-specific RFLP patterns. Thisapproach was tested on our collection of over 200 Ceratocystis isolates and identified the fungi with a high level of confidence, reducingthe time needed to identify these species by classical methods.2 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.Keywords: L-Tubulin; Polymerase chain reaction-restriction fragment length polymorphism; Ceratocystis; Sapstain; Blue stain1. IntroductionThe genus Ceratocystis sensu stricto Ellis and Halsted isa small group of saprobes and parasites of many commerciallyimportant crop plants and trees. They are distinguishedfrom other ophiostomatoid fungi by their £askshapedascomata, Chalara anamorphs, and sensitivity tocycloheximide [1,2]. Several species have been described asgrowing on coniferous hosts and are capable of stainingthe sapwood blue or gray. The growth and staining ofthese fungi in wood is rapid and they cause considerablevalue loss to the wood products industry.The conifer associated Ceratocystis species are morphologicallysimilar yet appear to occupy di¡erent ecologicalniches [3] and are intersterile [4]. They display a range ofbehaviors, from saprobic to pathogenic growth. C. coerulescenshas been isolated from Picea and Pinus in bothcontinental Europe and North America. C. pinicola hasbeen isolated from Scots pine and Corsican pine, in GreatBritain. C. douglassi is a common saprophyte found onDouglas ¢r lumber in western North America [5]. Thesethree species colonize and stain the butts of wind blowntrees and broken roots or freshly cut logs or lumber [6]. C.resinifera is found on Picea and Pinus species of continentalEurope and North America. In addition to saprobicgrowth, this species has also been described as a weakpathogen, growing rapidly from wound sites in living Norwayspruce. C. coerulescens, C. pinicola, C. douglassi andC. resinifera are all thought to have non-speci¢c relationshipswith bark beetles and other insect vectors, sporulatereadily in culture, and produce a strong aromatic aroma,possibly to attract their vectors [3,6].Another three species of Ceratocystis form close symbioticrelationships with bark beetles. In association withbeetle attack these fungi are pathogenic to healthy trees.The fungi sporulate scarcely in culture and do not producea fruity odor. C. ru¢penni, found in western North America,is vectored by the North American spruce beetle Dendroctonusru¢pennis and is a pathogen of Engelmann andwhite spruces [7]. C. polonica is found across Eurasia andJapan on Picea species and is vectored by Ips typographus.It is best known in Norway where it is an aggressive0378-1097 / 03 / $22.00 2 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.doi:10.1016/S0378-1097(03)00256-8* Corresponding author. Tel. : +1(604) 822-9738;Fax: +1(604) 922-9104.E-mail address: breuil@interchange.ubc.ca (C. Breuil).FEMS Microbiology Letters 222 (2003) 143^147www.fems-microbiology.orgpathogen of Norway spruce [8]. C. laricicola, also foundacross Eurasia and Japan, is restricted to larch and associatedwith the bark beetle Ips cembrae.C. adiposa is distantly related and morphologically distinctfrom the above-described species. However, we includeit in this study since it has been isolated fromwood chips and sawn lumber in North America [9].Species of Ceratocystis can be di⁄cult to di¡erentiate.For example, C. coerulescens, C. resinifera, and C. ru¢pennioverlap in host and geographical range. Although somemorphological di¡erences exist, such as the diameter ofperithecial bases and ascospore length, there is considerableoverlap in these characters and they can requiremonths to develop in culture. The polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP)method has proved useful for the rapid and sensitivedetection and di¡erentiation of many plant pathogens.Ribosomal DNA (rDNA) is a common target sequenceand has been used extensively for the di¡erentiation ofsapstain species [10,11]. However, these methods havebeen unable to separate closely related Ceratocystis speciesdue to low sequence heterogeneity [12]. The L-tubulin genehas also been successfully used for PCR-RFLP based differentiationof species of Babesia and Theileria [13] anddistinguishing between groups of Leishmania [14]. Tubulinproteins have a relatively high degree of conservation atthe amino acid and nucleotide level, but a relatively highdegree of variability in the intronic sequences.In this paper we describe a rapid and reliable PCRRFLPidenti¢cation method to di¡erentiate species of Ceratocystisusing a fragment of the L-tubulin gene sequence,which had higher sequence variability than rDNA. Thismethod was used to con¢rm the identity and to di¡erentiateCeratocystis sapstain fungi, which our lab routinelyisolates from stained logs and lumber in Canada.2. Materials and methods2.1. Fungal isolatesIsolates of Ceratocystis for L-tubulin sequence analysiswere obtained from various collections in North Americaand Europe. These included two isolates of C. coerulescensfrom Canada and Germany, three isolates of C. pinicolafrom the UK, one isolate of C. douglassi from the USA,six isolates of C. resinifera from Canada, two isolates ofC. ru¢penni from Canada, one isolate of C. polonica fromNorway, and one isolate of C. adiposa from Canada. Isolatenumbers and GenBank accession numbers for theL-tubulin sequences are shown in Fig. 1. Ceratocystis isolatesfor L-tubulin PCR-RFLP analysis were isolated fromlogs at sites across Canada during the summer of 2000following previously described methodology [9]. This collectionincluded isolates of C. resinifera : 40 from WilliamsLake, British Columbia, eight from Cranbrook, BritishColumbia, 42 from Edson, Alberta, 34 from Big River,Saskatchewan, 17 from Kirkland Lake, Ontario, 19 fromLaSarre, Quebec, 17 from Saint-Emile, Quebec, ¢ve fromFore“t Montmorency, Quebec, and 18 from Plaster Rock,New Brunswick. As well isolates of C. coerulescens wereobtained: one from Merrit, British Columbia, one fromSaint-Emile, and 36 from Cranbrook. Isolates of C. ru¢-penni from previous collections, two from Prince George,
British Columbia and one from Edmonton, Alberta, were
also analyzed. Further details regarding this collection can
be obtained by request from the authors. Isolates were
stored as mycelial plugs of Oxoid malt extract agar in
20% glycerol at 4‡C for short periods or at 380‡C for
extended periods. Taxonomic characterization of isolates
followed morphological descriptions [3,5] as well as analysis
of rDNA sequences [12,15].
Fig. 1. One most parsimonious tree produced from the alignment of DNA sequences of a fragment of the Ceratocystis L-tubulin gene (tree length = 409,
CI =0.919, RI =0.876). Bootstrap values (1000 replicates) greater than 50% are indicated at the top of the branches. The bar indicates tree scale. Gen-
Bank accession numbers of published sequences: CL13-12 (AY140945), C313 (AY140943), 126-1E1 (AY140935), 0901 (AY140932), 206-291
(AY140937), 9320810 (AY140939), C324 (AY140944), BW2-111 (AY140942), D53-5A3 (AY140946), 123-314 (AY140933), Ai12-33 (AY140940), Ai13-14
(AY140941), 125-214 (AY140934), C609 (AY142242), 92.633.254.7 (AY140938), 152-5-1 (AY140936).
144 P.A. Loppnau, C. Breuil / FEMS Microbiology Letters 222 (2003) 143^147
2.2. PCR reactions and DNA sequencing
DNA for PCR ampli¢cations was extracted as described
previously [16]. A phenol chloroform extraction was performed
prior to isopropanol precipitation. PCR was performed
using a Hybaid Touch Down Thermal Cycler. The
50-Wl reactions contained 200 ng of genomic DNA,
40 pmol of each forward T10 (5P-ACG-ATA-GGT-TCACCT-
CCA-GAC-3P) [17] and reverse BT12 (5P-GTT-GTCAAT-
GCA-GAA-GGT-CTC-3P) primers, 100 WM dNTPs,
and 1U Taq polymerase. A hot start PCR was employed
using: initial denaturation at 94‡C for 4 min, ¢ve cycles of
denaturation at 94‡C for 50 s, primer annealing at 47‡C
for 50 s, and primer extension at 72‡C for 1min. This was
followed by an additional 30 cycles of denaturation at
94‡C for 50 s, primer annealing at 55‡C for 50 s, and
primer extension at 72‡C for 1min . Final chain elongation
was 72‡C for 10 min.
Templates for sequencing reactions were ampli¢ed as
described above and puri¢ed from 1% agarose gels using
a QIAquick Gel Extraction Kit (Qiagen). PCR products
were ligated to pCR-II0 TOPO (Invitrogen). Sequencing
of both strands was carried out using an ABI Prism1
BigDye1 Terminator Cycle Sequencing Ready Reaction
kit (PE Applied Biosystems). Sequencing products were
analyzed at the UBC Nucleic Acid and Protein Service
Laboratory (University of British Columbia, Vancouver,
BC, Canada) on an ABI 373 DNA sequencer (PE Applied
Biosystems). Direct sequencing was performed using the
primers T8 (5P-GAC-CGA-AGA-TGA-AGT-TGT-CG-
British Columbia and one from Edmonton, Alberta, were
also analyzed. Further details regarding this collection can
be obtained by request from the authors. Isolates were
stored as mycelial plugs of Oxoid malt extract agar in
20% glycerol at 4‡C for short periods or at 380‡C for
extended periods. Taxonomic characterization of isolates
followed morphological descriptions [3,5] as well as analysis
of rDNA sequences [12,15].
Fig. 1. One most parsimonious tree produced from the alignment of DNA sequences of a fragment of the Ceratocystis L-tubulin gene (tree length = 409,
CI =0.919, RI =0.876). Bootstrap values (1000 replicates) greater than 50% are indicated at the top of the branches. The bar indicates tree scale. Gen-
Bank accession numbers of published sequences: CL13-12 (AY140945), C313 (AY140943), 126-1E1 (AY140935), 0901 (AY140932), 206-291
(AY140937), 9320810 (AY140939), C324 (AY140944), BW2-111 (AY140942), D53-5A3 (AY140946), 123-314 (AY140933), Ai12-33 (AY140940), Ai13-14
(AY140941), 125-214 (AY140934), C609 (AY142242), 92.633.254.7 (AY140938), 152-5-1 (AY140936).
144 P.A. Loppnau, C. Breuil / FEMS Microbiology Letters 222 (2003) 143^147
2.2. PCR reactions and DNA sequencing
DNA for PCR ampli¢cations was extracted as described
previously [16]. A phenol chloroform extraction was performed
prior to isopropanol precipitation. PCR was performed
using a Hybaid Touch Down Thermal Cycler. The
50-Wl reactions contained 200 ng of genomic DNA,
40 pmol of each forward T10 (5P-ACG-ATA-GGT-TCACCT-
CCA-GAC-3P) [17] and reverse BT12 (5P-GTT-GTCAAT-
GCA-GAA-GGT-CTC-3P) primers, 100 WM dNTPs,
and 1U Taq polymerase. A hot start PCR was employed
using: initial denaturation at 94‡C for 4 min, ¢ve cycles of
denaturation at 94‡C for 50 s, primer annealing at 47‡C
for 50 s, and primer extension at 72‡C for 1min. This was
followed by an additional 30 cycles of denaturation at
94‡C for 50 s, primer annealing at 55‡C for 50 s, and
primer extension at 72‡C for 1min . Final chain elongation
was 72‡C for 10 min.
Templates for sequencing reactions were ampli¢ed as
described above and puri¢ed from 1% agarose gels using
a QIAquick Gel Extraction Kit (Qiagen). PCR products
were ligated to pCR-II0 TOPO (Invitrogen). Sequencing
of both strands was carried out using an ABI Prism1
BigDye1 Terminator Cycle Sequencing Ready Reaction
kit (PE Applied Biosystems). Sequencing products were
analyzed at the UBC Nucleic Acid and Protein Service
Laboratory (University of British Columbia, Vancouver,
BC, Canada) on an ABI 373 DNA sequencer (PE Applied
Biosystems). Direct sequencing was performed using the
primers T8 (5P-GAC-CGA-AGA-TGA-AGT-TGT-CG-
การแปล กรุณารอสักครู่..
Species level identi¢cation of conifer associated Ceratocystis sapstain
fungi by PCR-RFLP on a L-tubulin gene fragment
Peter A. Loppnau, Colette Breuil
Department of Wood Science, University of British Columbia, 2424 Main Mall, Vancouver, BC, Canada V6T1Z4
Received 15 November 2002; accepted 27 March 2003
First published online 17 April 2003
Abstract
The genus Ceratocystis includes several morphologically similar species commonly found as agents of sapstain in coniferous trees. In
this paper we describe a simple and reliable polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique
that aids in the identification and differentiation of these fungi. PCR was used to amplify a 1.3-kb fragment of the L-tubulin gene from
C. coerulescens, C. pinicola, C. douglassi, C. resinifera, C. rufipenni, C. polonica and C. adiposa. The PCR amplicon from representative
isolates was sequenced. This information was utilized to select restriction enzymes that generated species-specific RFLP patterns. This
approach was tested on our collection of over 200 Ceratocystis isolates and identified the fungi with a high level of confidence, reducing
the time needed to identify these species by classical methods.
2 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords: L-Tubulin; Polymerase chain reaction-restriction fragment length polymorphism; Ceratocystis; Sapstain; Blue stain
1. Introduction
The genus Ceratocystis sensu stricto Ellis and Halsted is
a small group of saprobes and parasites of many commercially
important crop plants and trees. They are distinguished
from other ophiostomatoid fungi by their £askshaped
ascomata, Chalara anamorphs, and sensitivity to
cycloheximide [1,2]. Several species have been described as
growing on coniferous hosts and are capable of staining
the sapwood blue or gray. The growth and staining of
these fungi in wood is rapid and they cause considerable
value loss to the wood products industry.
The conifer associated Ceratocystis species are morphologically
similar yet appear to occupy di¡erent ecological
niches [3] and are intersterile [4]. They display a range of
behaviors, from saprobic to pathogenic growth. C. coerulescens
has been isolated from Picea and Pinus in both
continental Europe and North America. C. pinicola has
been isolated from Scots pine and Corsican pine, in Great
Britain. C. douglassi is a common saprophyte found on
Douglas ¢r lumber in western North America [5]. These
three species colonize and stain the butts of wind blown
trees and broken roots or freshly cut logs or lumber [6]. C.
resinifera is found on Picea and Pinus species of continental
Europe and North America. In addition to saprobic
growth, this species has also been described as a weak
pathogen, growing rapidly from wound sites in living Norway
spruce. C. coerulescens, C. pinicola, C. douglassi and
C. resinifera are all thought to have non-speci¢c relationships
with bark beetles and other insect vectors, sporulate
readily in culture, and produce a strong aromatic aroma,
possibly to attract their vectors [3,6].
Another three species of Ceratocystis form close symbiotic
relationships with bark beetles. In association with
beetle attack these fungi are pathogenic to healthy trees.
The fungi sporulate scarcely in culture and do not produce
a fruity odor. C. ru¢penni, found in western North America,
is vectored by the North American spruce beetle Dendroctonus
ru¢pennis and is a pathogen of Engelmann and
white spruces [7]. C. polonica is found across Eurasia and
Japan on Picea species and is vectored by Ips typographus.
It is best known in Norway where it is an aggressive
0378-1097 / 03 / $22.00 2 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
doi:10.1016/S0378-1097(03)00256-8
* Corresponding author. Tel. : +1(604) 822-9738;
Fax: +1(604) 922-9104.
E-mail address: breuil@interchange.ubc.ca (C. Breuil).
FEMS Microbiology Letters 222 (2003) 143^147
www.fems-microbiology.org
pathogen of Norway spruce [8]. C. laricicola, also found
across Eurasia and Japan, is restricted to larch and associated
with the bark beetle Ips cembrae.
C. adiposa is distantly related and morphologically distinct
from the above-described species. However, we include
it in this study since it has been isolated from
wood chips and sawn lumber in North America [9].
Species of Ceratocystis can be di⁄cult to di¡erentiate.
For example, C. coerulescens, C. resinifera, and C. ru¢penni
overlap in host and geographical range. Although some
morphological di¡erences exist, such as the diameter of
perithecial bases and ascospore length, there is considerable
overlap in these characters and they can require
months to develop in culture. The polymerase chain reaction-
restriction fragment length polymorphism (PCRRFLP)
method has proved useful for the rapid and sensitive
detection and di¡erentiation of many plant pathogens.
Ribosomal DNA (rDNA) is a common target sequence
and has been used extensively for the di¡erentiation of
sapstain species [10,11]. However, these methods have
been unable to separate closely related Ceratocystis species
due to low sequence heterogeneity [12]. The L-tubulin gene
has also been successfully used for PCR-RFLP based differentiation
of species of Babesia and Theileria [13] and
distinguishing between groups of Leishmania [14]. Tubulin
proteins have a relatively high degree of conservation at
the amino acid and nucleotide level, but a relatively high
degree of variability in the intronic sequences.
In this paper we describe a rapid and reliable PCRRFLP
identi¢cation method to di¡erentiate species of Ceratocystis
using a fragment of the L-tubulin gene sequence,
which had higher sequence variability than rDNA. This
method was used to con¢rm the identity and to di¡erentiate
Ceratocystis sapstain fungi, which our lab routinely
isolates from stained logs and lumber in Canada.
2. Materials and methods
2.1. Fungal isolates
Isolates of Ceratocystis for L-tubulin sequence analysis
were obtained from various collections in North America
and Europe. These included two isolates of C. coerulescens
from Canada and Germany, three isolates of C. pinicola
from the UK, one isolate of C. douglassi from the USA,
six isolates of C. resinifera from Canada, two isolates of
C. ru¢penni from Canada, one isolate of C. polonica from
Norway, and one isolate of C. adiposa from Canada. Isolate
numbers and GenBank accession numbers for the
L-tubulin sequences are shown in Fig. 1. Ceratocystis isolates
for L-tubulin PCR-RFLP analysis were isolated from
logs at sites across Canada during the summer of 2000
following previously described methodology [9]. This collection
included isolates of C. resinifera : 40 from Williams
Lake, British Columbia, eight from Cranbrook, British
Columbia, 42 from Edson, Alberta, 34 from Big River,
Saskatchewan, 17 from Kirkland Lake, Ontario, 19 from
LaSarre, Quebec, 17 from Saint-Emile, Quebec, ¢ve from
Fore“t Montmorency, Quebec, and 18 from Plaster Rock,
New Brunswick. As well isolates of C. coerulescens were
obtained: one from Merrit, British Columbia, one from
Saint-Emile, and 36 from Cranbrook. Isolates of C. ru¢-
penni from previous collections, two from Prince George,
British Columbia and one from Edmonton, Alberta, were
also analyzed. Further details regarding this collection can
be obtained by request from the authors. Isolates were
stored as mycelial plugs of Oxoid malt extract agar in
20% glycerol at 4‡C for short periods or at 380‡C for
extended periods. Taxonomic characterization of isolates
followed morphological descriptions [3,5] as well as analysis
of rDNA sequences [12,15].
Fig. 1. One most parsimonious tree produced from the alignment of DNA sequences of a fragment of the Ceratocystis L-tubulin gene (tree length = 409,
CI =0.919, RI =0.876). Bootstrap values (1000 replicates) greater than 50% are indicated at the top of the branches. The bar indicates tree scale. Gen-
Bank accession numbers of published sequences: CL13-12 (AY140945), C313 (AY140943), 126-1E1 (AY140935), 0901 (AY140932), 206-291
(AY140937), 9320810 (AY140939), C324 (AY140944), BW2-111 (AY140942), D53-5A3 (AY140946), 123-314 (AY140933), Ai12-33 (AY140940), Ai13-14
(AY140941), 125-214 (AY140934), C609 (AY142242), 92.633.254.7 (AY140938), 152-5-1 (AY140936).
144 P.A. Loppnau, C. Breuil / FEMS Microbiology Letters 222 (2003) 143^147
2.2. PCR reactions and DNA sequencing
DNA for PCR ampli¢cations was extracted as described
previously [16]. A phenol chloroform extraction was performed
prior to isopropanol precipitation. PCR was performed
using a Hybaid Touch Down Thermal Cycler. The
50-Wl reactions contained 200 ng of genomic DNA,
40 pmol of each forward T10 (5P-ACG-ATA-GGT-TCACCT-
CCA-GAC-3P) [17] and reverse BT12 (5P-GTT-GTCAAT-
GCA-GAA-GGT-CTC-3P) primers, 100 WM dNTPs,
and 1U Taq polymerase. A hot start PCR was employed
using: initial denaturation at 94‡C for 4 min, ¢ve cycles of
denaturation at 94‡C for 50 s, primer annealing at 47‡C
for 50 s, and primer extension at 72‡C for 1min. This was
followed by an additional 30 cycles of denaturation at
94‡C for 50 s, primer annealing at 55‡C for 50 s, and
primer extension at 72‡C for 1min . Final chain elongation
was 72‡C for 10 min.
Templates for sequencing reactions were ampli¢ed as
described above and puri¢ed from 1% agarose gels using
a QIAquick Gel Extraction Kit (Qiagen). PCR products
were ligated to pCR-II0 TOPO (Invitrogen). Sequencing
of both strands was carried out using an ABI Prism1
BigDye1 Terminator Cycle Sequencing Ready Reaction
kit (PE Applied Biosystems). Sequencing products were
analyzed at the UBC Nucleic Acid and Protein Service
Laboratory (University of British Columbia, Vancouver,
BC, Canada) on an ABI 373 DNA sequencer (PE Applied
Biosystems). Direct sequencing was performed using the
primers T8 (5P-GAC-CGA-AGA-TGA-AGT-TGT-CG-
fungi by PCR-RFLP on a L-tubulin gene fragment
Peter A. Loppnau, Colette Breuil
Department of Wood Science, University of British Columbia, 2424 Main Mall, Vancouver, BC, Canada V6T1Z4
Received 15 November 2002; accepted 27 March 2003
First published online 17 April 2003
Abstract
The genus Ceratocystis includes several morphologically similar species commonly found as agents of sapstain in coniferous trees. In
this paper we describe a simple and reliable polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique
that aids in the identification and differentiation of these fungi. PCR was used to amplify a 1.3-kb fragment of the L-tubulin gene from
C. coerulescens, C. pinicola, C. douglassi, C. resinifera, C. rufipenni, C. polonica and C. adiposa. The PCR amplicon from representative
isolates was sequenced. This information was utilized to select restriction enzymes that generated species-specific RFLP patterns. This
approach was tested on our collection of over 200 Ceratocystis isolates and identified the fungi with a high level of confidence, reducing
the time needed to identify these species by classical methods.
2 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords: L-Tubulin; Polymerase chain reaction-restriction fragment length polymorphism; Ceratocystis; Sapstain; Blue stain
1. Introduction
The genus Ceratocystis sensu stricto Ellis and Halsted is
a small group of saprobes and parasites of many commercially
important crop plants and trees. They are distinguished
from other ophiostomatoid fungi by their £askshaped
ascomata, Chalara anamorphs, and sensitivity to
cycloheximide [1,2]. Several species have been described as
growing on coniferous hosts and are capable of staining
the sapwood blue or gray. The growth and staining of
these fungi in wood is rapid and they cause considerable
value loss to the wood products industry.
The conifer associated Ceratocystis species are morphologically
similar yet appear to occupy di¡erent ecological
niches [3] and are intersterile [4]. They display a range of
behaviors, from saprobic to pathogenic growth. C. coerulescens
has been isolated from Picea and Pinus in both
continental Europe and North America. C. pinicola has
been isolated from Scots pine and Corsican pine, in Great
Britain. C. douglassi is a common saprophyte found on
Douglas ¢r lumber in western North America [5]. These
three species colonize and stain the butts of wind blown
trees and broken roots or freshly cut logs or lumber [6]. C.
resinifera is found on Picea and Pinus species of continental
Europe and North America. In addition to saprobic
growth, this species has also been described as a weak
pathogen, growing rapidly from wound sites in living Norway
spruce. C. coerulescens, C. pinicola, C. douglassi and
C. resinifera are all thought to have non-speci¢c relationships
with bark beetles and other insect vectors, sporulate
readily in culture, and produce a strong aromatic aroma,
possibly to attract their vectors [3,6].
Another three species of Ceratocystis form close symbiotic
relationships with bark beetles. In association with
beetle attack these fungi are pathogenic to healthy trees.
The fungi sporulate scarcely in culture and do not produce
a fruity odor. C. ru¢penni, found in western North America,
is vectored by the North American spruce beetle Dendroctonus
ru¢pennis and is a pathogen of Engelmann and
white spruces [7]. C. polonica is found across Eurasia and
Japan on Picea species and is vectored by Ips typographus.
It is best known in Norway where it is an aggressive
0378-1097 / 03 / $22.00 2 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
doi:10.1016/S0378-1097(03)00256-8
* Corresponding author. Tel. : +1(604) 822-9738;
Fax: +1(604) 922-9104.
E-mail address: breuil@interchange.ubc.ca (C. Breuil).
FEMS Microbiology Letters 222 (2003) 143^147
www.fems-microbiology.org
pathogen of Norway spruce [8]. C. laricicola, also found
across Eurasia and Japan, is restricted to larch and associated
with the bark beetle Ips cembrae.
C. adiposa is distantly related and morphologically distinct
from the above-described species. However, we include
it in this study since it has been isolated from
wood chips and sawn lumber in North America [9].
Species of Ceratocystis can be di⁄cult to di¡erentiate.
For example, C. coerulescens, C. resinifera, and C. ru¢penni
overlap in host and geographical range. Although some
morphological di¡erences exist, such as the diameter of
perithecial bases and ascospore length, there is considerable
overlap in these characters and they can require
months to develop in culture. The polymerase chain reaction-
restriction fragment length polymorphism (PCRRFLP)
method has proved useful for the rapid and sensitive
detection and di¡erentiation of many plant pathogens.
Ribosomal DNA (rDNA) is a common target sequence
and has been used extensively for the di¡erentiation of
sapstain species [10,11]. However, these methods have
been unable to separate closely related Ceratocystis species
due to low sequence heterogeneity [12]. The L-tubulin gene
has also been successfully used for PCR-RFLP based differentiation
of species of Babesia and Theileria [13] and
distinguishing between groups of Leishmania [14]. Tubulin
proteins have a relatively high degree of conservation at
the amino acid and nucleotide level, but a relatively high
degree of variability in the intronic sequences.
In this paper we describe a rapid and reliable PCRRFLP
identi¢cation method to di¡erentiate species of Ceratocystis
using a fragment of the L-tubulin gene sequence,
which had higher sequence variability than rDNA. This
method was used to con¢rm the identity and to di¡erentiate
Ceratocystis sapstain fungi, which our lab routinely
isolates from stained logs and lumber in Canada.
2. Materials and methods
2.1. Fungal isolates
Isolates of Ceratocystis for L-tubulin sequence analysis
were obtained from various collections in North America
and Europe. These included two isolates of C. coerulescens
from Canada and Germany, three isolates of C. pinicola
from the UK, one isolate of C. douglassi from the USA,
six isolates of C. resinifera from Canada, two isolates of
C. ru¢penni from Canada, one isolate of C. polonica from
Norway, and one isolate of C. adiposa from Canada. Isolate
numbers and GenBank accession numbers for the
L-tubulin sequences are shown in Fig. 1. Ceratocystis isolates
for L-tubulin PCR-RFLP analysis were isolated from
logs at sites across Canada during the summer of 2000
following previously described methodology [9]. This collection
included isolates of C. resinifera : 40 from Williams
Lake, British Columbia, eight from Cranbrook, British
Columbia, 42 from Edson, Alberta, 34 from Big River,
Saskatchewan, 17 from Kirkland Lake, Ontario, 19 from
LaSarre, Quebec, 17 from Saint-Emile, Quebec, ¢ve from
Fore“t Montmorency, Quebec, and 18 from Plaster Rock,
New Brunswick. As well isolates of C. coerulescens were
obtained: one from Merrit, British Columbia, one from
Saint-Emile, and 36 from Cranbrook. Isolates of C. ru¢-
penni from previous collections, two from Prince George,
British Columbia and one from Edmonton, Alberta, were
also analyzed. Further details regarding this collection can
be obtained by request from the authors. Isolates were
stored as mycelial plugs of Oxoid malt extract agar in
20% glycerol at 4‡C for short periods or at 380‡C for
extended periods. Taxonomic characterization of isolates
followed morphological descriptions [3,5] as well as analysis
of rDNA sequences [12,15].
Fig. 1. One most parsimonious tree produced from the alignment of DNA sequences of a fragment of the Ceratocystis L-tubulin gene (tree length = 409,
CI =0.919, RI =0.876). Bootstrap values (1000 replicates) greater than 50% are indicated at the top of the branches. The bar indicates tree scale. Gen-
Bank accession numbers of published sequences: CL13-12 (AY140945), C313 (AY140943), 126-1E1 (AY140935), 0901 (AY140932), 206-291
(AY140937), 9320810 (AY140939), C324 (AY140944), BW2-111 (AY140942), D53-5A3 (AY140946), 123-314 (AY140933), Ai12-33 (AY140940), Ai13-14
(AY140941), 125-214 (AY140934), C609 (AY142242), 92.633.254.7 (AY140938), 152-5-1 (AY140936).
144 P.A. Loppnau, C. Breuil / FEMS Microbiology Letters 222 (2003) 143^147
2.2. PCR reactions and DNA sequencing
DNA for PCR ampli¢cations was extracted as described
previously [16]. A phenol chloroform extraction was performed
prior to isopropanol precipitation. PCR was performed
using a Hybaid Touch Down Thermal Cycler. The
50-Wl reactions contained 200 ng of genomic DNA,
40 pmol of each forward T10 (5P-ACG-ATA-GGT-TCACCT-
CCA-GAC-3P) [17] and reverse BT12 (5P-GTT-GTCAAT-
GCA-GAA-GGT-CTC-3P) primers, 100 WM dNTPs,
and 1U Taq polymerase. A hot start PCR was employed
using: initial denaturation at 94‡C for 4 min, ¢ve cycles of
denaturation at 94‡C for 50 s, primer annealing at 47‡C
for 50 s, and primer extension at 72‡C for 1min. This was
followed by an additional 30 cycles of denaturation at
94‡C for 50 s, primer annealing at 55‡C for 50 s, and
primer extension at 72‡C for 1min . Final chain elongation
was 72‡C for 10 min.
Templates for sequencing reactions were ampli¢ed as
described above and puri¢ed from 1% agarose gels using
a QIAquick Gel Extraction Kit (Qiagen). PCR products
were ligated to pCR-II0 TOPO (Invitrogen). Sequencing
of both strands was carried out using an ABI Prism1
BigDye1 Terminator Cycle Sequencing Ready Reaction
kit (PE Applied Biosystems). Sequencing products were
analyzed at the UBC Nucleic Acid and Protein Service
Laboratory (University of British Columbia, Vancouver,
BC, Canada) on an ABI 373 DNA sequencer (PE Applied
Biosystems). Direct sequencing was performed using the
primers T8 (5P-GAC-CGA-AGA-TGA-AGT-TGT-CG-
การแปล กรุณารอสักครู่..
ชนิดประจุบวกของระดับ identi ¢โคนิเฟอร์ที่เกี่ยวข้อง ceratocystis sapstain
เชื้อราด้วยว่าใน l-tubulin ยีน ~
ปีเตอร์ . loppnau Colette , บรูล
ภาควิชาวิทยาศาสตร์ไม้มหาวิทยาลัยบริติชโคลัมเบีย 2424 หลักมอลล์ , แวนคูเวอร์ , แคนาดา v6t1z4
ได้รับ 15 พฤศจิกายน 2002 ; ยอมรับ 27 มีนาคม 2003
ตีพิมพ์ครั้งแรกออนไลน์ 17 เมษายน 2003
บทคัดย่อสกุล ceratocystis รวมถึงชนิดที่คล้ายกันจากหลายที่พบบ่อยเป็นตัวแทนของ sapstain ในต้นไม้เขียวชอุ่มเชียว ในบทความนี้เราจะอธิบาย
ง่ายและเชื่อถือได้ การใช้ปฏิกิริยาลูกโซ่โพลีเมอเรสจำกัด fragment length polymorphism ( ว่า ) เทคนิค
ที่ช่วยในการระบุและความแตกต่างของเชื้อราเหล่านี้ PCR ใช้ขยาย 13-kb ชิ้นส่วนของยีนจาก coerulescens l-tubulin
C , C . pinicola , C . douglassi , C . resinifera , C . rufipenni , C . ขัดและ C adiposa . การตรวจและจากตัวแทน
ไอโซเลทคือนี้ ข้อมูลนี้ถูกใช้เพื่อเลือกการสร้างเอนไซม์ที่เฉพาะ - ขยายพันธุ์ : รูปแบบ นี้
วิธีการได้รับการทดสอบในคอลเลกชันของเรากว่า 200 ceratocystis แยกและระบุเชื้อราที่มีระดับสูงของความเชื่อมั่น ลด
เวลาที่จำเป็นในการระบุชนิดเหล่านี้โดยวิธีการคลาสสิก .
2 2003 สหพันธ์สมาคมจุลชีววิทยาในยุโรป จัดพิมพ์โดย Elsevier Science นอกจากนี้สงวนลิขสิทธิ์ .
คำสำคัญ : l-tubulin ;การใช้ปฏิกิริยาลูกโซ่โพลีเมอเรสจำกัด fragment length polymorphism ; ceratocystis ; sapstain ; คราบ
1 สีฟ้า บทนำ
สกุล ceratocystis เซ็นสุ stricto เอลลิสโรมแรมคือ
และกลุ่มเล็ก ๆของ saprobes และปรสิตของในเชิงพาณิชย์
มากมาย พืชเศรษฐกิจที่สำคัญของพืชและต้นไม้ พวกเขาจะแตกต่างจากอื่น ๆโดยเชื้อรา ophiostomatoid
ascomata ที่สุดของพวกเขา askshaped , anamorphs chalara ไว
,ร้อยเรียง [ 1 , 2 ] หลาย ชนิดได้ถูกอธิบายเป็น
เติบโตบนต้นสนโฮสต์และมีความสามารถของการย้อมสี
กระพี้สีฟ้าหรือสีเทา การเจริญเติบโตและการย้อมสีของ
เหล่านี้เชื้อราในไม้อย่างรวดเร็ว และทำให้เกิดการสูญเสีย
มูลค่ามหาศาลเพื่ออุตสาหกรรมผลิตภัณฑ์ไม้จำพวกสน ชนิด เกี่ยวข้อง ceratocystis
เหมือนกันเลยปรากฏครอบครองจาก ดิ ¡
erent นิเวศวิทยาniches [ 3 ] และมี intersterile [ 4 ] พวกเขาแสดงช่วงของ
พฤติกรรมจาก saprobic การเจริญเติบโตของเชื้อโรค . C . coerulescens
ถูกแยกจากทั้งในและ picea สน
ทวีปยุโรปและทวีปอเมริกาเหนือ C . pinicola ได้
ถูกแยกจากสกอตและสนคอร์ซิกาสนในมาก
สหราชอาณาจักร C . douglassi เป็นสปอโรไฟต์ทั่วไปพบในดักลาส ¢
R ไม้ในภาคตะวันตกเฉียงเหนือของอเมริกา [ 5 ] เหล่านี้
สามชนิดตั้งรกรากและคราบก้นของลมเป่า
ต้นไม้และรากหักหรือตัดสดท่อนไม้หรือไม้ [ 6 ] C .
resinifera พบใน picea สนและชนิดของคอนติเนน
ยุโรปและอเมริกาเหนือ นอกจากการเติบโต saprobic
ชนิดนี้ยังได้รับการอธิบายเป็นเชื้อโรคที่อ่อนแอ
เติบโตอย่างรวดเร็ว จากบาดแผลในชีวิตเว็บไซต์ต้นสนนอร์เวย์
C . coerulescens , C . pinicola , C .douglassi
C และ resinifera ทั้งหมดคิดจะไม่กา¢ C ความสัมพันธ์
กับด้วงเปลือกไม้และแมลงพาหะอื่น ๆ sporulate
พร้อมในวัฒนธรรม และผลิตแข็งแรงหอมหอม
อาจดึงดูดเวกเตอร์ของพวกเขา [ 3 , 6 ] .
ของ ceratocystis ปิดฟอร์มความสัมพันธ์ symbiotic
ด้วยเปลือกด้วงอีกสามชนิด ในความสัมพันธ์กับ
ด้วงโจมตีเชื้อราเหล่านี้โรคต้นไม้มีสุขภาพดี .
เชื้อรา sporulate แทบในวัฒนธรรม และไม่ผลิต
กลิ่นผลไม้ ซีรู¢ penni พบในภาคตะวันตกของอเมริกาเหนือ
เป็นเวกเตอร์โดยอเมริกาเหนือต้นสนด้วง dendroctonus
รุ¢เพนนิส และเชื้อโรคของเองเกิลเมิ่นและ
สพรูซสีขาว [ 7 ] C . ขัดพบข้ามยูเรเซียและ
ญี่ปุ่นและ picea ชนิดเวกเตอร์โดย IPS typographus .
เป็นที่รู้จักดีที่สุดในนอร์เวย์ซึ่งเป็นก้าวร้าว
0378-1097 / 03 / $ 22.00 น. 2 2003 สหพันธ์สมาคมจุลชีววิทยาในยุโรป จัดพิมพ์โดย Elsevier Science นอกจากนี้สงวนลิขสิทธิ์ .
ดอย : 10.1016 / s0378-1097 ( 03 ) ผู้ที่ 00256-8
* โทร : 1 ( 604 ) 822-9738 ;
โทรสาร : 1 ( 604 ) 922-9104 .
e - mail address : breuil@interchange.ubc .CA ( C . บรูล )
fems จุลชีววิทยาตัวอักษร 222 ( 2003 ) ได้
www.fems-microbiology 147 . org
เชื้อโรคของนอร์เวย์โก้เก๋ [ 8 ] C . laricicola , นอกจากนี้ยังพบ
ข้ามยูเรเซียและญี่ปุ่นจำกัดลาชและเกี่ยวข้องกับเปลือกด้วง IPS cembrae
.
c adiposa เป็นสัตว์กินเนื้อ ที่เกี่ยวข้อง และจากที่แตกต่างกัน
จากข้างต้นอธิบายชนิด แต่เราจะรวม
ในการศึกษานี้เมื่อมันถูกแยกออกจากเศษไม้และแปรรูปไม้
ในอเมริกาเหนือ [ 9 ] .
ชนิด ceratocystis สามารถ ดิ ดิ ⁄ลัทธิ¡ erentiate .
ตัวอย่างเช่น C coerulescens , C . resinifera และ C . ru ¢ penni
ซ้อนในโฮสต์และทางภูมิศาสตร์ที่หลากหลาย แม้ว่าบาง
สัณฐานวิทยา ดิ ¡ erences อยู่ เช่น ขนาดและความยาวฐาน perithecial
ascospore มีจํานวนมาก
เชื้อราด้วยว่าใน l-tubulin ยีน ~
ปีเตอร์ . loppnau Colette , บรูล
ภาควิชาวิทยาศาสตร์ไม้มหาวิทยาลัยบริติชโคลัมเบีย 2424 หลักมอลล์ , แวนคูเวอร์ , แคนาดา v6t1z4
ได้รับ 15 พฤศจิกายน 2002 ; ยอมรับ 27 มีนาคม 2003
ตีพิมพ์ครั้งแรกออนไลน์ 17 เมษายน 2003
บทคัดย่อสกุล ceratocystis รวมถึงชนิดที่คล้ายกันจากหลายที่พบบ่อยเป็นตัวแทนของ sapstain ในต้นไม้เขียวชอุ่มเชียว ในบทความนี้เราจะอธิบาย
ง่ายและเชื่อถือได้ การใช้ปฏิกิริยาลูกโซ่โพลีเมอเรสจำกัด fragment length polymorphism ( ว่า ) เทคนิค
ที่ช่วยในการระบุและความแตกต่างของเชื้อราเหล่านี้ PCR ใช้ขยาย 13-kb ชิ้นส่วนของยีนจาก coerulescens l-tubulin
C , C . pinicola , C . douglassi , C . resinifera , C . rufipenni , C . ขัดและ C adiposa . การตรวจและจากตัวแทน
ไอโซเลทคือนี้ ข้อมูลนี้ถูกใช้เพื่อเลือกการสร้างเอนไซม์ที่เฉพาะ - ขยายพันธุ์ : รูปแบบ นี้
วิธีการได้รับการทดสอบในคอลเลกชันของเรากว่า 200 ceratocystis แยกและระบุเชื้อราที่มีระดับสูงของความเชื่อมั่น ลด
เวลาที่จำเป็นในการระบุชนิดเหล่านี้โดยวิธีการคลาสสิก .
2 2003 สหพันธ์สมาคมจุลชีววิทยาในยุโรป จัดพิมพ์โดย Elsevier Science นอกจากนี้สงวนลิขสิทธิ์ .
คำสำคัญ : l-tubulin ;การใช้ปฏิกิริยาลูกโซ่โพลีเมอเรสจำกัด fragment length polymorphism ; ceratocystis ; sapstain ; คราบ
1 สีฟ้า บทนำ
สกุล ceratocystis เซ็นสุ stricto เอลลิสโรมแรมคือ
และกลุ่มเล็ก ๆของ saprobes และปรสิตของในเชิงพาณิชย์
มากมาย พืชเศรษฐกิจที่สำคัญของพืชและต้นไม้ พวกเขาจะแตกต่างจากอื่น ๆโดยเชื้อรา ophiostomatoid
ascomata ที่สุดของพวกเขา askshaped , anamorphs chalara ไว
,ร้อยเรียง [ 1 , 2 ] หลาย ชนิดได้ถูกอธิบายเป็น
เติบโตบนต้นสนโฮสต์และมีความสามารถของการย้อมสี
กระพี้สีฟ้าหรือสีเทา การเจริญเติบโตและการย้อมสีของ
เหล่านี้เชื้อราในไม้อย่างรวดเร็ว และทำให้เกิดการสูญเสีย
มูลค่ามหาศาลเพื่ออุตสาหกรรมผลิตภัณฑ์ไม้จำพวกสน ชนิด เกี่ยวข้อง ceratocystis
เหมือนกันเลยปรากฏครอบครองจาก ดิ ¡
erent นิเวศวิทยาniches [ 3 ] และมี intersterile [ 4 ] พวกเขาแสดงช่วงของ
พฤติกรรมจาก saprobic การเจริญเติบโตของเชื้อโรค . C . coerulescens
ถูกแยกจากทั้งในและ picea สน
ทวีปยุโรปและทวีปอเมริกาเหนือ C . pinicola ได้
ถูกแยกจากสกอตและสนคอร์ซิกาสนในมาก
สหราชอาณาจักร C . douglassi เป็นสปอโรไฟต์ทั่วไปพบในดักลาส ¢
R ไม้ในภาคตะวันตกเฉียงเหนือของอเมริกา [ 5 ] เหล่านี้
สามชนิดตั้งรกรากและคราบก้นของลมเป่า
ต้นไม้และรากหักหรือตัดสดท่อนไม้หรือไม้ [ 6 ] C .
resinifera พบใน picea สนและชนิดของคอนติเนน
ยุโรปและอเมริกาเหนือ นอกจากการเติบโต saprobic
ชนิดนี้ยังได้รับการอธิบายเป็นเชื้อโรคที่อ่อนแอ
เติบโตอย่างรวดเร็ว จากบาดแผลในชีวิตเว็บไซต์ต้นสนนอร์เวย์
C . coerulescens , C . pinicola , C .douglassi
C และ resinifera ทั้งหมดคิดจะไม่กา¢ C ความสัมพันธ์
กับด้วงเปลือกไม้และแมลงพาหะอื่น ๆ sporulate
พร้อมในวัฒนธรรม และผลิตแข็งแรงหอมหอม
อาจดึงดูดเวกเตอร์ของพวกเขา [ 3 , 6 ] .
ของ ceratocystis ปิดฟอร์มความสัมพันธ์ symbiotic
ด้วยเปลือกด้วงอีกสามชนิด ในความสัมพันธ์กับ
ด้วงโจมตีเชื้อราเหล่านี้โรคต้นไม้มีสุขภาพดี .
เชื้อรา sporulate แทบในวัฒนธรรม และไม่ผลิต
กลิ่นผลไม้ ซีรู¢ penni พบในภาคตะวันตกของอเมริกาเหนือ
เป็นเวกเตอร์โดยอเมริกาเหนือต้นสนด้วง dendroctonus
รุ¢เพนนิส และเชื้อโรคของเองเกิลเมิ่นและ
สพรูซสีขาว [ 7 ] C . ขัดพบข้ามยูเรเซียและ
ญี่ปุ่นและ picea ชนิดเวกเตอร์โดย IPS typographus .
เป็นที่รู้จักดีที่สุดในนอร์เวย์ซึ่งเป็นก้าวร้าว
0378-1097 / 03 / $ 22.00 น. 2 2003 สหพันธ์สมาคมจุลชีววิทยาในยุโรป จัดพิมพ์โดย Elsevier Science นอกจากนี้สงวนลิขสิทธิ์ .
ดอย : 10.1016 / s0378-1097 ( 03 ) ผู้ที่ 00256-8
* โทร : 1 ( 604 ) 822-9738 ;
โทรสาร : 1 ( 604 ) 922-9104 .
e - mail address : breuil@interchange.ubc .CA ( C . บรูล )
fems จุลชีววิทยาตัวอักษร 222 ( 2003 ) ได้
www.fems-microbiology 147 . org
เชื้อโรคของนอร์เวย์โก้เก๋ [ 8 ] C . laricicola , นอกจากนี้ยังพบ
ข้ามยูเรเซียและญี่ปุ่นจำกัดลาชและเกี่ยวข้องกับเปลือกด้วง IPS cembrae
.
c adiposa เป็นสัตว์กินเนื้อ ที่เกี่ยวข้อง และจากที่แตกต่างกัน
จากข้างต้นอธิบายชนิด แต่เราจะรวม
ในการศึกษานี้เมื่อมันถูกแยกออกจากเศษไม้และแปรรูปไม้
ในอเมริกาเหนือ [ 9 ] .
ชนิด ceratocystis สามารถ ดิ ดิ ⁄ลัทธิ¡ erentiate .
ตัวอย่างเช่น C coerulescens , C . resinifera และ C . ru ¢ penni
ซ้อนในโฮสต์และทางภูมิศาสตร์ที่หลากหลาย แม้ว่าบาง
สัณฐานวิทยา ดิ ¡ erences อยู่ เช่น ขนาดและความยาวฐาน perithecial
ascospore มีจํานวนมาก
การแปล กรุณารอสักครู่..
ภาษาอื่น ๆ
การสนับสนุนเครื่องมือแปลภาษา: กรีก, กันนาดา, กาลิเชียน, คลิงออน, คอร์สิกา, คาซัค, คาตาลัน, คินยารวันดา, คีร์กิซ, คุชราต, จอร์เจีย, จีน, จีนดั้งเดิม, ชวา, ชิเชวา, ซามัว, ซีบัวโน, ซุนดา, ซูลู, ญี่ปุ่น, ดัตช์, ตรวจหาภาษา, ตุรกี, ทมิฬ, ทาจิก, ทาทาร์, นอร์เวย์, บอสเนีย, บัลแกเรีย, บาสก์, ปัญจาป, ฝรั่งเศส, พาชตู, ฟริเชียน, ฟินแลนด์, ฟิลิปปินส์, ภาษาอินโดนีเซี, มองโกเลีย, มัลทีส, มาซีโดเนีย, มาราฐี, มาลากาซี, มาลายาลัม, มาเลย์, ม้ง, ยิดดิช, ยูเครน, รัสเซีย, ละติน, ลักเซมเบิร์ก, ลัตเวีย, ลาว, ลิทัวเนีย, สวาฮิลี, สวีเดน, สิงหล, สินธี, สเปน, สโลวัก, สโลวีเนีย, อังกฤษ, อัมฮาริก, อาร์เซอร์ไบจัน, อาร์เมเนีย, อาหรับ, อิกโบ, อิตาลี, อุยกูร์, อุสเบกิสถาน, อูรดู, ฮังการี, ฮัวซา, ฮาวาย, ฮินดี, ฮีบรู, เกลิกสกอต, เกาหลี, เขมร, เคิร์ด, เช็ก, เซอร์เบียน, เซโซโท, เดนมาร์ก, เตลูกู, เติร์กเมน, เนปาล, เบงกอล, เบลารุส, เปอร์เซีย, เมารี, เมียนมา (พม่า), เยอรมัน, เวลส์, เวียดนาม, เอสเปอแรนโต, เอสโทเนีย, เฮติครีโอล, แอฟริกา, แอลเบเนีย, โคซา, โครเอเชีย, โชนา, โซมาลี, โปรตุเกส, โปแลนด์, โยรูบา, โรมาเนีย, โอเดีย (โอริยา), ไทย, ไอซ์แลนด์, ไอร์แลนด์, การแปลภาษา.
- แต่ละประเทศเป็นศูนย์กลางด้านเศรษฐกิจ ที่
- Introduction: This study tests a concept
- . Adaptation through learning is inheren
- ฉันกำลังชี้แจงเกี่ยวกับงานให้ทุกคนทราบทา
- guards
- เพื่อนฉันร้ายมาก
- ด้วยพื้นที่เหมาะสำหรับการพักผ่อนริมทะเล
- เหล็กดัดฟัน
- One of the most essential ways to protec
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- ขอโทษที่ฉันไม่ได้ตอบข้อความของคุณเลย
- อาจมีทั้งดีและไม่ดี
- ฉันไม่เชื่อว่าคุณคิดถึงฉัน เพราะคุณหล่อ
- The subjects in the aerobic group walked
- ฉันเรียนวิชาการเขียน
- Nine
- ฉันเอาขวดที่ใช้แล้ว ไปใส่ในถังขยะที่แยกไ
- The blackmailing of children has emerged
- Bath and large bathThe Japanese are very
- แม่มีปากกา10ด้าม แบ่งให้พ่อ5ด้าม
- Brown
- เข่งปลาทู
- 응원
- One of the most essential ways to protec
