Mycologia, 96(3), 2004, pp. 439–451. q 2004 by The Mycological Society of America, Lawrence, KS 66044-8897
Molecular tools for isolate and community studies of Pyrenomycete fungi
Stefan J. Green
lished from sequence analysis of the ITS region, DGGE analysis of the ITS region was found to be more sensitive than DGGE analysis of the 18S rDNA. This study introduces molecular tools for the study of Pyrenomycete fungi by the development of two specific primers, demonstration of the enhanced sensitivity of ITS-DGGE for typing of closely related isolates and application of these tools to environmental samples. Key words: Colletotrichum, DGGE, ITS, nested PCR, PCR Primers, 18S rDNA.
Department of Microbiology and Plant Pathology, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot, Israel, and Institute of Soil, Water and Environmental Sciences, Agriculture Research Organization, The Volcani Center, P.O. Box 6, Bet-Dagan, 50-250, Israel
Stanley Freeman Department of Plant Pathology, Agriculture Research Organization, The Volcani Center, P.O. Box 6, BetDagan, 50-250, Israel
Yitzhak Hadar Department of Microbiology and Plant Pathology, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot, Israel
INTRODUCTION
Pyrenomycete fungi are an important class of the filamentous Ascomycota that include plant pathogens (Freeman et al 2000), decomposers (Samuels and Blackwell 2001), fungal parasites (Spatafora and Blackwell 1993), insect pathogens (Neuveglise et al 1994, Fukatsu et al 1997), human pathogens (Berbee and Taylor 1992) and biocontrol agents (Hermosa et al 2000, Samuels and Blackwell 2001). Pyrenomycetes are defined on the basis of a shared phenotypic characteristic—the formation of a flask-shaped fruiting body (perithecium) (Lumbsch 2000). While it generally is difficult to use fungal morphological features to establish stable phylogenetic associations due to the independent development and loss of such features, the monophyly of the Pyrenomycete fungi previously has been confirmed by analysis of molecular data (Berbee and Taylor 1992, Spatafora and Blackwell 1993, Spatafora 1995, Lumbsch 2000). Numerous molecular techniques have been employed in the detection, identification and phylogenetic analysis of Pyrenomycete and other fungal populations. Fragments of 18S ribosomal DNA (rDNA) have been used as effective markers in genotyping studies when combined with denaturing and temperature gradient gel electrophoresis (DGGE and TGGE, respectively) studies (Hernan-Gomez et al 2000, Vainio and Hantula 2000). In addition, the 28S rDNA ( Jacquot et al 2000), Group I rDNA introns (Neuveglise et al 1997), internal transcribed spacer (ITS) regions ( Jasalavich et al 2000, Viaud et al 2000), intergenic spacer (IGS) regions (Henrion et al 1994), mitochondrial rDNA (Gardes et al 1991), an RNA polymerase II subunit (Liu et al 1999) and
Dror Minz1 Institute of Soil, Water and Environmental Sciences, Agriculture Research Organization, The Volcani Center, P.O. Box 6, Bet-Dagan, 50-250, Israel
Abstract: The Pyrenomycetes, defined physiologically by the formation of a flask-shaped fruiting body present in the sexual form, are a monophyletic group of fungi that consist of a wide diversity of populations including human and plant pathogens. Based on sequence analysis of 18S ribosomal DNA (rDNA), rDNA regions conserved among the Pyrenomycetes but divergent among other organisms were identified and used to develop selective PCR primers and a highly specific primer set. The primers presented here were used to amplify large portions of the 18S rDNA as well as the entire internal transcribed spacer (ITS) region (ITS 1, 5.8S rDNA, and ITS 2). In addition to database searches, the specificity of the primers was verified by PCR amplification of DNA extracted from pure culture isolates and by sequence analysis of fungal rDNA PCR-amplified from environmental samples. In addition, denaturing gradient gel electrophoresis (DGGE) analyses were performed on closely related Colletotrichum isolates serving as a model pathogenic genus of the Pyrenomycetes. Although both ITS and 18S rDNA DGGE analyses of Colletotrichum were consistent with a phylogeny estabAccepted for publication October 20, 2003. 1 Corresponding author. E-mail:
[email protected]
439
440 TABLE I.
MYCOLOGIA PCR primers used for amplification of 18S rDNA and the ITS region of fungi
Primer name ITS1a NS8 PyITS1a PyNS8 ITS4 Py364 FR1b,c NS1 NS1AG GC Clamps 1 2
Primer direction Forward Reverse Forward Reverse Reverse Forward Reverse Forward Forward
Primer sequence (59 → 39) TCC CCG TCC CCG TCC AGG AIC GTA GTA
GTA CAG GTT CTG TCC GTT CAT GTC GTC
GGT GTT GGT GTT GCT AGG TCA ATA ATA
GAA CAC GAA CAC TAT GCT ATC TGC TGC
CCT CTA CCA CAA TGA YGA GGT TTG TTG
GCG CGG GCG CGG TAT CCC AIT TCT TCT
Source White et al 1990 White et al 1990 This paper This paper White et al 1990 This paper Vainio and Hantula 2000 White et al 1990 Modified from White et al 1990
G A G A GC
C CAA AG
CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC G CCC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GCC G
Muyzer and Smalla 1998 Vainio and Hantula 2000
a
GC-clamp number 1 was attached to the 59 end of the primer sequence. GC-clamp number 2 was attached to the 59 end of the primer sequence. c I 5 inosine nucleotides are present at positions 2 and 17. b
genes encoding for histones, beta tubulin and ATPase (Glass and Donaldson 1995, Keeling et al 2000) have been used for population analysis of fungi. The ITS region, or portions thereof, has been used extensively to distinguish between closely related fungal isolates. Because the ITS region is noncoding, its size and sequence are less conserved and more useful for examination of closely related populations (Henrion et al 1994, Pritsch et al 1997). As a result, it has been used to study fungi by restriction enzyme analysis (Rafin et al 1995, Pritsch et al 1997, Freeman et al 2000, Redecker 2000, Viaud et al 2000), terminal restriction fragment length polymorphism (T-RFLP) analysis (Buchan et al 2002), cloning and sequencing (Pritsch et al 2000, Freeman et al 2001), single-strand conformational polymorphism (SSCP) analysis (Gottlieb et al 2000), constant denaturing gel electrophoresis (CDGE) (Kurkela et al 1999), temperature gradient gel electrophoresis (TGGE) (Hernan-Gomez et al 2000) and by denaturing gradient gel electrophoresis (DGGE) (Paavolainen et al 2000). DGGE, the genotyping tool used in this study, is one of several assays, such as SSCP, CDGE and TGGE, that are employed to separate DNA fragments according to properties based on sequence (Muyzer and Smalla 1998, Kurkela et al 1999, Gottlieb et al 2000). This and related techniques provide several advantages for both environmental and pure culture population studies. The separation assays are based on the entire sequence amplified, rather than select regions of restriction sites, and often reduce the effort required for genotyping of strains.
PCR amplification serves as the basis for the above mentioned molecular techniques, and a large variety of primers currently exist for the amplification of different regions of fungal DNA (White et al 1990, Glass and Donaldson 1995, Smit et al 1999, Borneman and Hartin 2000). Some of the more general fungal primers target nonfungal populations, and might have sequence mismatches with internal taxonomic groups such as the Pyrenomycete fungi (White et al 1990, Gardes and Bruns 1993, Vainio and Hantula 2000). As a result of such mismatches, internal taxa can be underestimated in environmental or mixed culture analyses. To rectify this situation with regard to the Pyrenomycete fungi and to develop techniques for the rapid analysis of Pyrenomycete populations, several molecular tools were developed and described here for this important taxonomic group. This study had three aims: (i) to design primers specific to the Pyrenomycetes, (ii) to determine the utility of DGGE analysis for genotyping Pyrenomycete isolates and (iii) to demonstrate the applicability of Pyrenomycete-specific PCR and DGGE for environmental analyses. MATERIAL AND METHODS
Primer design and determination of specificity.—Primer sequences used in this study for PCR amplification of fungal DNA are presented in TABLE I. Primer PyITS1 (or PyNS8, the inverse complement of PyITS1), located at the 39 end of the 18S rDNA, was designed by modification of primer ITS1 (White et al 1990) according to sequence analyses indicating two mismatches with Pyrenomycete fungi. An ex-
17 17
3
None None
18–43 18–43
N/A N/A N/A 180 180
58 58
60
18S rDNA primer sets Identification/Sequencing Outer PCR for nested Outer PCR for nested DGGE DGGE 100 105 105 105 105 65 67–63 67–63 57 61 Touchdown Touchdown
1.5 2.5 2.5 4 4
ITS4 ITS4
PyNS8 NS8 PyNS8 FR1 FR1
ITS1 PyITS1
Py364 NS1AG NS1AG NS1 NS1
Nested
Not performed 32.5–37.5 250 6–12
Voltage Temperature (V) (8C) PCR type
PCR conditions
Annealing Elongation [MgCl2] temperature time (mM) (8C) (s) Reverse primer Forward primer
Primer set
PCR and DGGE conditions TABLE II.
PCR amplification and optimization.—PCR amplifications were performed in a Whatman Biometra Tgradient thermal cycler (Biometra, Goettingen, Germany). PCR mixtures contained (per 50 ml of reaction mixture): 5 ml of 103 RedTaq PCR buffer (15 mM Mg21—Sigma, St. Louis, Missouri), 12.5 nmol of each of the four deoxynucleoside triphosphates, 6.25 mg of bovine serum albumin (Roche Applied Science), 25 pmol of each primer, 1.5 U of Taq polymerase (Sigma RedTaq) and 5 to 10 ng of template DNA. Magnesium concentration varied according to primer set, and was adjusted by addition of 25 mM MgCl2 solution (TABLE II). PCR amplifications were conducted with this basic format: initial denaturation at 95 C for 3 min, 35 cycles of denaturation at 94 C for 30 s, annealing at variable temperatures for 30 s, elongation at 72 C for variable times, final elongation at 72 C for 2 min (TABLE II). Environmental samples (described below) were subject to nested PCR amplification with the NS1AG/NS8 and NS1AG/PyNS8 primer sets. These reactions were conducted with a touchdown PCR in which the initial annealing temperature was reduced 0.5 C per cycle for 4 cycles, 0.2 C
Utility
amination of the inferred secondary structure of the 18S rDNA revealed that the two mismatches occurred opposite each other on the stem of a stem-loop structure (FIG. 1). The primer Py364, annealing to a position 364 bases from the 59 end of the 18S rDNA, was configured to incorporate a Pyrenomycete-specific region initially identified by Berbee and Taylor (1992). Primer specificity was determined initially by a BLAST search (NCBI) (Altschul et al 1997) against the primer sequences. Target sequences were identified as Pyrenomycete either directly from information submitted to GenBank or, in cases of indeterminate phylogenetic affiliation, target sequences were submitted to BLAST searches in their entirety and affiliation ascertained by closely related sequences. For the purposes of nested PCR (described below), primer NS1 was modified by addition of four bases to the 39 end to increase its melting temperature. The modified primer, NS1AG, was found to yield more stringent PCR than primer NS1 in reactions with reverse primers NS8 or PyNS8 (data not shown). Primers ITS1, PyITS1 and FR1 were synthesized with a 40 bp GC-rich region (GC clamp) attached to the 59 end of the primer, a technique used to enhance separation during DGGE analysis (Muyzer and Smalla 1998).
Acrylamide Denaturant gradient gradient (%) (%)
DGGE conditions
FIG. 1. Inferred secondary structure of the small subunit rRNA in the region of the ITS1 or PyITS1 primer sequence. The region of ambiguity is located in the stem of a stemloop structure near the end of the 18S rRNA.
441
FUNGI
ITS primer sets DGGE DGGE
PYRENOMYCETE
45 45
TOOLS FOR
61.5 61.5
MOLECULAR
4 4
ET AL:
Run time (h)
GREEN
442
MYCOLOGIA gradients and operating conditions are presented in TABLE II. Gels were stained either with ethidium bromide or GelStar nucleic acid stain (Biowhittaker Molecular Applications, Rockland, Maine) and photographed on a UV transillumination table (302 nm) with a Kodak digital camera (Rochester, New York).
FIG. 2. Results of PCR annealing temperature optimization using the ITS1/ITS4 (top) and PyITS1/ITS4 (bottom) primer sets with DNA of Pyrenomycete and non-Pyrenomycete fungi. Reaction conditions, as described in the text, were held constant with the exception of annealing temperature. Annealing temperatures are marked for each lane. for 10 cycles and maintained at the final annealing temperature (4 C below the initial annealing temperature) for the final 20 cycles. The PCR product generated by these touchdown PCRs was diluted 1:5 with water and used as a template for nested PCR with the NS1/FR1 primer set (PCR conditions are listed in TABLE II). Agarose gel electrophoresis and subsequent staining with ethidium bromide were used to verify the presence and size of all amplification products. Annealing temperatures for primer sets were determined using temperature gradient PCR in which all conditions except annealing temperature were held constant. DNA isolated from four fungal species was used to determine the optimum annealing temperatures for the ITS primer sets, ITS1/ITS4 and PyITS1/ITS4. Saccharomyces cerevisae DNA was used as a target template (no mismatches with either ITS1 or ITS4 primers) for the ITS1/ITS4 primer set, while DNA from Colletotrichum acutatum ALM-US-4 (Freeman et al 2000), Fusarium subglutinans 506/2 (Freeman et al 1999), and Trichoderma sp. (T101) were used as target templates (no mismatches with either PyITS1 or ITS4 primers) for the PyITS1/ITS 4 primer set. Annealing temperatures of 52.9, 55.7, 58.6, 61.4, 64.3 and 67.1 C were tested for both primer sets and each of the four DNA samples (FIG. 2). Primer sets Py364/PyNS8, NS1AG/NS8 and NS1AG/ PyNS8 were similarly optimized (data not shown). Optimized PCR conditions for all primer sets are presented in TABLE II. Using optimized (stringent) PCR conditions, reference DNA extracted from Pyrenomycete and non-Pyrenomycete isolates were analyzed by PCR with four primer sets: PyITS1/ITS4 (Pyrenomycete-specific), ITS1/ITS4 (general fungal), Py364/PyNS8 (Pyrenomycete-specific), and NS1/FR1 (general fungal) (TABLE III). DGGE analysis of ITS and 18S rDNA fragments.—All DGGE gels were 1 mm thick and electrophoresed with the D-Gene System (Bio-Rad) in a 13 TAE (40 mM Tris, 20 mM acetic acid and 1 mM EDTA at pH 8.3) buffer. DGGE analyses of the 18S rDNA fragments were performed using 6% (w/v) acrylamide/bisacrylamide (37.5:1) gels, while DGGE analyses of the ITS fragments were performed using gels containing a 6% (w/v) to 12% acrylamide gradient. Denaturant
Demonstration of primer applicability to environmental samples.—The specificity of the Pyrenomycete primers was demonstrated by applying multiple primer sets to several environmental samples. Fungal populations in a compostamended potting mix and in the rhizosphere of cucumber plants grown in the mix were examined by PCR-DGGE analysis. Total DNA was extracted from the potting mix and from cucumber rhizosphere at 1 and 3 wk post-planting using the UltraClean Soil DNA Isolation Kit (MoBio Laboratories Inc, Solana Beach, California). DNA was PCR amplified with three different primer sets: NS1AG/NS8 nested with NS1/FR1, NS1AG/PyNS8 nested with NS1/FR1, and NS1/FR1 directly. All three PCR reactions yielded identically sized and located fragments, and were analyzed concomitantly by DGGE. Sequence analysis of fungal bands.—Bands were excised from DGGE gels visualized on a Dark Reader transillumination table (Clare Chemical Research Inc., Dolores, Colorado). Using sterile razor blades, acrylamide pieces were excised and placed in 2 ml plastic tubes containing 200 mL of TE and glass beads of approximately 1 mm diam. Tubes were vortexed 10 min, incubated at 37 C for 30 min, and the liquid was used as template for PCR with the NS1/FR1 primers. Generated PCR products were verified to represent the appropriate band by a second DGGE analysis. The pGEMt-T Easy Vector System (Promega, Madison, Wisconsin) was used for cloning of re-amplified, excised DGGE bands. Ligation and transformation reactions were performed as instructed by the manufacturer. Clones were screened by suspending colony material in PCR reaction mixtures, amplifying with the NS1/FR1 primer set and analyzed by DGGE as described before. Plasmids from selected colonies were purified with the WizardTM Plus Miniprep DNA Purification System (Promega, Wisconsin) as instructed by the manufacturer. Clones were sequenced using the Applied Biosystems PRISM Dye Terminator Cycle Sequencing Ready reaction kit supplied with AmpliTaq DNA polymerase. The sequencing products were analyzed with the Applied Biosystems 377 DNA sequencer. Phylogenetic analysis.—ITS sequences of closely related Colletotrichum isolates, comprising species and subspecies level differences, were analyzed. The phylogenetic relationship between these populations was inferred by sequence analysis of the entire ITS regions (ITS1, 5.8S rDNA and ITS2) as described previously (Freeman et al 2001). Analysis of the ITS sequence data using parsimony, distance matrix and maximum-likelihood algorithms yielded trees with identical branching orders. The 18S rDNA sequences recovered from DGGE analysis of potting mix and cucumber rhizosphere (TABLE IV) were aligned to the 18S rDNA database in the phylogenetic package ARB (Strunk et al 1999). A neighborjoining tree was produced using a 50% conservation filter
GREEN
ET AL:
MOLECULAR
TOOLS FOR
(i.e., those positions in which greater than 50% of the sequences were dissimilar were removed from the analysis) to reduce tree artifacts that can result with multiple base changes at a single position. Short sequences (Bands 13 and 15) were inserted into the tree by parsimony analysis. Nucleotide sequence accession numbers.—The partial sequences determined in this study have been deposited in the GenBank database under these accession numbers: AY222613–AY222627 and AY222810 (TABLE IV).
RESULTS
Database analysis of Pyrenomycete-specific primers.— The sequences of Pyrenomycete-specific primers PyITS1 and Py364 were checked against sequences in the GenBank database, as described above. BLAST results indicated that the primer PyITS1 (and its reverse complement PyNS8) was matched perfectly by approximately 500 sequences comprising 57 genera distributed throughout the Pyrenomycete class, and that the primer Py364 was matched by approximately 650 sequences comprising 173 genera distributed throughout the Pyrenomycete class. The vast majority of the matching sequences for both primers readily were identifiable as Pyrenomycetes by inspection of information provided with the sequences or by the phylogenetic affiliations of the most closely related sequences (as determined by conducting BLAST searches of entire matching sequences). BLAST search results revealed three sequences that contained the PyITS1 primer but represented non-Pyrenomycete fungi of the class Leotiomycetes (represented by Mycoarthris corallinus and Scleropezicula alnicola, GenBank accession numbers AF128440 and AF141169, respectively). These comprised a small minority of the total number of sequences recovered in the database search. In addition, a small minority of Pyrenomycete fungi contained a mismatch with the Py364 primer. Most of the sequences returned by BLAST analysis conformed to the primer sequence presented here, with a single degeneracy at position 13. However, 20 sequences, representing six Pyrenomycete genera (Sordariomycete, Ambrosiella, Ophiostoma, Cordyceps, Graphium and Pesotum) contained a mismatch with the Py364 primer at position 15 and contained a G instead of an A nucleotide. Only in the genera Ophiostoma (10 of 12 sequences) and Pesotum (two of two sequences) did this mismatch predominate. Although not used here, the Py364 primer may be synthesized with this additional degeneracy. The Py364 primer also matched two nonfungal sequences (a diatom, accession number X85401, and a region of human DNA, accession number AL590290).
PYRENOMYCETE
FUNGI
443
PCR analysis with Pyrenomycete-specific primers.—Using temperature gradient PCR, primer sets ITS1/ ITS4 (ITS general fungal), PyITS1/ITS4 (ITS Pyrenomycete-specific), and Py364/ PyNS8 (18S Pyrenomycete-specific) were optimized for stringent annealing temperatures (TABLE II). An annealing temperature of 61.5 C was determined to be stringent (no amplification of nontarget DNA) for both ITS primer sets based on the results of temperature gradient PCR analysis (FIG. 2). Stringent temperatures were required because amplification of nontarget DNA (i.e., Pyrenomycete fungi with ITS1/ITS4 primers) could be achieved at reduced annealing temperatures. However, under nonstringent conditions, amplification yield of nontarget DNA was reduced significantly with respect to target template (FIG. 2) and, in some cases, no amplification was possible (data not shown). A similar optimization was conducted with the Py364/PyNS8 primers set (data not shown), and a stringent annealing temperature of 65 C was chosen. A variety of Pyrenomycete and non-Pyrenomycete fungi were tested for amplification with the ITS1 and PyITS1 (with ITS4), Py364/PyNS8, and the NS1/FR1 primer sets (TABLE III). The PyITS1/ITS4 primer set did not amplify any non-Pyrenomycete DNA tested, and the ITS1/ITS4 primer, with the exception of weak amplification of C. gloeosporioides AVO-37-4B, did not amplify any Pyrenomycete fungi tested. Most Pyrenomycete fungi (29 of 32) were amplified with the PyITS1/ITS4 primer set, the exceptions being F. oxysporum f.sp. lycopersici and basilici, and Thielaviopsis paradoxa. These organisms also did not amplify with the ITS1/ITS4 primer set but did amplify with both 18S rDNA primer sets. Rhizoctonia solani, a Basidiomycete, also did not amplify with either ITS primer set but did amplify with the general 18S rDNA primer set. The Py364/PyNS8 primer set amplified all 30 of the Pyrenomycete fungi tested and did not amplify any non-Pyrenomycete DNA. 18S rDNA and ITS DGGE analysis of Colletotrichum isolates.—DNA from 15 Colletotrichum isolates (TABLE II) was PCR-amplified with primer sets PyITS1/ITS4 (ITS) and NS1/FR1 (18S rDNA), and the ITS and 18S rDNA fragments generated were analyzed by DGGE, as described. The 15 18S rDNA fragments migrated to three unique positions on the DGGE gel (FIG. 3b). The 18S rDNA fragments of the C. gloeosporioides isolates (lanes 1–3), migrated to a unique position, while the fragments of the deeper-branching group comprised of Colletotrichum ALM-KSH-10 (lane 14) and C. acutatum IMI 345026 (lane 15) migrated together to another unique position. The 10 remaining C. acutatum amplificons (lanes 4–13), mi-
Thielaviopsis 2
1 1 1 2 1 1 2 1 1
Fusarium oxysporum fsp. Albedinis (Foa81)d oxysporum fsp. melonis (FOM 1,2)d oxysporum fsp. melonis (FOM KKI)d oxysporum fsp. lycopersici (FORL C809L)d oxysporum fsp. canariensis (FOC F053)d oxysporum fsp. iris (FOI)c oxysporum fsp. basilici (FOB)d subglutinans 506/2e subglutinans FMS 93c
F. F. F. F. F. F. F. F. F. T. paradoxa
1 1 1 1 1 1 1
harzianum T39c isolate 101c 315c GH-2c Yc 35c 109c
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
PyITS/ITS4 Pyrenomycetes
2
2 2 2 2 2 2 2 2 2
2 2 2 2 2 2 2
1 2 2 2 2 2 2 2 2 2 2 2 2 2 2
ITS1/ITS4 General fungi
ITS primer sets
T. T. T. T. T. T. T.
Trichoderma
Colletotrichum C. gloeosporioides AVO-37-4Bb (AF207792) C. gloeosporioides APL 7b (AF272779) C. gloeosporioides CG 231b (AF272780) C. acutatum NL 12Ab (AF272781) C. acutatum TUT-5954b (AF207794) C. acutatum HV-83Cb (AF272782) C. acutatum STR 3b (AF272784) C. acutatum IMI 223120b (AF272783) C. acutatum IMI 348494b (AF272785) C. acutatum PCN 5b (AF272786) C. acutatum ALM-US-4b (AF207793) C. acutatum APL 2b (AF272787) C. acutatum PCH 8b (AF272788) Colletotrichum ALM-KSH-10b (AF207791) C. acutalum IMI 345026b (AF272789)
Target population
Amplification results of pure culture DNA samples
Pyrenomycetes
Organismsa
TABLE III.
1
1 nt 1 1 1 1 1 1 1
1 nt 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Py364/PyNS8 Pyrenomycetes
1
1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
NS1/FR1 General fungi
18S primer sets
444 MYCOLOGIA
Continued
Cucumis
Phytophthora
b
a
Amplification results symbols: 2, no product; 1, positive amplification; nt 5 not tested. Isolates sequenced by Freeman et al 2001. c DNA obtained from isolates by S. Green, S. Freeman, Y. Hadar and D. Minz. d DNA obtained from isolates by Freeman & Maymon 2000. e DNA obtained from isolates by Freeman, Maymon & Pinkas 1999.
Cucumis sativus
P. cactorum
2
2
2
2
2
2
1
1
2
1
1
1
ITS1/ITS4 General fungi
2
2
2
2
2
2
Py364/PyNS8 Pyrenomycetes
1
1
1
1
1
1
NS1/FR1 General fungi
TOOLS FOR
Plants
Oomycetes
Rhizoctonia
Tuber
Saccharomyces
Botrytis
PyITS/ITS4 Pyrenomycetes
18S primer sets
MOLECULAR
R. solani
T. melanosporum
S. cerevisiae
B. cinerea
Target population
ITS primer sets
ET AL:
Basidiomycetes
Non-Pyrenomycete Ascomycetes
Organismsa
TABLE III.
GREEN PYRENOMYCETE FUNGI
445
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
AY222810 AY222613 AY222614 AY222615 AY222616 AY222617 AY222618 AY222619 AY222620 AY222621 AY222622 AY222623 AY222624 AY222625 AY222626 AY222627
Potting Mix Potting Mix Potting Mix Rhizosphere, Rhizosphere, Rhizosphere, Potting Mix Potting Mix Potting Mix Rhizosphere, Rhizosphere, Rhizosphere, Rhizosphere, Potting Mix Potting Mix Rhizosphere, wk wk wk wk
3 wk
1 1 3 3
3 wk 3 wk 3 wk
Sample Name Heteromita globosa Heteromita globosa Heteromita globosa Heteromita globosa Heteromita globosa Uncultured fungus clone BAQA254 Papulosa amerospora Pseudallescheria ellipsoidea Nigrosabulum globosum Pseudallescheria ellipsoidea Petriella setifera Trichoderma viride Pseudallescheria ellipsoidea Coccidioides immitis Saccobolus sp. ARON 2131 Saccobolus sp. ARON 2131
NS1/FR1 NS1/FR1 NS1/FR1 NS1/FR1 NS1/FR1 NS1/FR1 NS1AG/PyNS8 NS1AG/PyNS8 NS1AG/PyNS8 NS1AG/PyNS8 NS1AG/PyNS8 NS1AG/PyNS8 NS1AG/PyNS8 NS1AG/NS8 NS1AG/NS8 NS1AG/NS8
U42447 U42447 U42447 U42447 U42447 AJ130849 AF064050 U43911 AF096180 U43911 U43908 AF525230 U43911 X58571 U53393 U53393
Accession No.
Nearest relative (BLAST) Primer set
* See selected bands in Fig. 4 and phylogenetic tree in Fig. 5.
Band Band Band Band Band Band Band Band Band Band Band Band Band Band Band Band
Band Sequence identification* accession No.
TABLE IV. Eukaryotic sequences recovered from compost-amended cucumber systems
97 95 89 93 93 96 95 97 99 98 97 99 96 97 97 97
% similarity
Cercozoa Cercozoa Cercozoa Cercozoa Cercozoa Unknown Pyrenomycetes Pyrenomycetes Pyrenomycetes Pyrenomycetes Pyrenomycetes Pyrenomycetes Pyrenomycetes Eurotiomycetes Pezizomycetes Pezizomycetes
Phylogeny
446 MYCOLOGIA
GREEN
ET AL:
MOLECULAR
TOOLS FOR
PYRENOMYCETE
FUNGI
447
FIG. 3. (a) Neighbor-joining tree of Colletotrichum ITS sequences. The phylogenetic analysis was performed on 430 bases of aligned ITS sequences. Similar branching was achieved with all methods tested (see text). Colletotrichum isolates are numbered 1–15 in parentheses after their names, as described in the text. Scale bar indicates estimated 1% sequence divergence. (b) DGGE analysis of the 18S rDNA fragment (NS1/FR1) of closely related Colletotrichum populations (as shown in FIG. 3a). The fragment size is approximately 1650 bp. (c) DGGE analysis of the complete ITS rDNA (PyITS1/ITS4) fragment (ITS1, 5.8S rDNA, and ITS2) of the same closely related Colletotrichum populations. The fragment size is approximately 600 bp.
grated to a third location, but no separation between these fragments was observed in the DGGE analysis. The 15 ITS fragments migrated to four different positions on the DGGE gel (FIG. 3c). The ITS fragments of the three C. gloeosporioides and the two deeper-branching Colletotrichum (C. ALM-KSH-10 and C. acutatum IMI 345026) migrated, similarly to the 18S rDNA fragments, to two distinct positions in the gel. The ITS fragments of the remaining 10 C. acutatum isolates, however, migrated to two positions in the gel. The ITS fragments of C. acutatum strain NL 12A (lane 4) comigrated with those of strains IMI
348496 (lane 9), PCN 5 (lane 10), ALM-US-4 (lane 11), APL 2 (lane 12) and PCH 8 (lane 13). ITS fragments representing C. acutatum TUT-5954 (lane 5), HV-83C (lane 6), STR 3 (lane 7), and IMI 223120 (lane 8) migrated together to a fourth unique position. The phylogenetic relationship between the Colletotrichum isolates was inferred by sequence analysis of the ITS region. Using 430 bases of aligned sequences, a neighbor-joining tree of the 15 isolates was produced (FIG. 3a). Based on this analysis, three broad groups among the 15 isolates were identified. These
448
MYCOLOGIA
FIG. 4. DGGE analysis of general and Pyrenomycete fungal populations detected in a compost-amended plant potting mix and in the rhizosphere at 1 and 3 wk of cucumber plants grown in the potting mix. Samples of DNA extracted from potting mix (A), cucumber rhizosphere at 1 wk (B), and cucumber rhizosphere at 3 wk (C) were amplified with general fungal primers (NS1/FR1), Pyrenomycete-specific fungal primers (NS1AG/PyNS8 nested with NS1/FR1), and general fungal primers not targeting Pyrenomycete fungi (NS1AG/NS8 nested with NS1/FR1). Select bands taken for sequencing are labeled (Bands 1–16) and sequencing results are presented in TABLE IV.
groups consisted of the C. gloeosporioides (isolates 1– 3), the deep-branching C. acutatum (isolates 14 and 15), and the 10 remaining C. acutatum (isolates 4– 13). Within the C. gloeosporioides and the deepbranching C. acutatum groups, less than 1% sequence difference was detected. Within the 10 intermediate C. acutatum (isolates 4–13), three additional groups were detected: isolates 9–13, isolate 4, and isolates 5–8. Isolate C. acutatum NL12A (isolate 4) was closer phylogenetically to, and migrated in the ITS DGGE gel to the same position as, the C. acutatum group composed of isolates 9–13. 18S rDNA analysis of environmental samples.—DNA extracted from three environmental samples (potting mix and cucumber rhizospheres from 1 and 3 wk of plant growth) was profiled with three primers sets: NS1/FR1, NS1AG/PyNS8 nested with NS1/FR1, and NS1AG/NS8 nested with NS1/FR1. Because the final PCR product from each reaction was the NS1/FR1 fragment, the samples were analyzed concurrently (FIG. 4). Sequencing of dominant bands was conducted to demonstrate that bands appearing in Pyrenomycete-specific PCR indeed were derived from Pyrenomycete fungi and to identify dominant potting mix and rhizosphere fungal populations. Results of the sequence analyses are presented in TABLE IV. A neighbor-joining phylogenetic tree based on an analysis of 679 bases downstream of the 59 end of the 18S rDNA was produced with the recovered DGGE sequences and their most closely related GenBank sequences (TABLE IV and FIG. 5). Direct PCR amplifi-
FIG. 5. Neighbor-joining tree of 18S rDNA sequences recovered from DGGE analysis of cucumber rhizospheres. The phylogenetic analysis was performed on 679 bases of aligned 18S rDNA sequences as described in the text. Band numbers refer to bands isolated from the DGGE analysis in FIG. 4. Specific information regarding each sequence is presented in TABLE IV. Scale bar indicates estimated 10% sequence divergence.
cation of the three samples with the primer set NS1/ FR1 yielded population profiles primarily composed of eukaryotes most closely related to the bacteriovorus flagellate Hetermoita globosa (Bands 1–5, FIGS. 4, 5 and TABLE IV). In addition, several other non-Pyrenomycete fungi were identified with either direct NS1/FR1 PCR amplification or by nested PCR amplification of the NS1AG/NS8 primer set (Bands 6, 14–16, FIGS. 4, 5 and TABLE IV). No Pyrenomycete fungi were detected with either the NS1AG/NS8 (nested with NS1/FR1) or the NS1/FR1 primer sets. Using Pyrenomycete-specific PCR (NS1AG/PyNS8 nested with NS1/FR1), Pyrenomycete fungi were detected exclusively with the exception of weak amplification of cucumber DNA in the two rhizosphere samples (Bands 7–13, FIGS. 4, 5 and TABLE IV). A total of seven sequences, representing a wide variety of Pyrenomycete fungi, were recovered by this approach. DISCUSSION
The purpose of this study was to develop molecular tools for the identification and analysis of Pyrenomycete fungi in pure culture and environmental samples. The results of this study indicate that Pyrenomycete fungi, common and important plant pathogens and biocontrol agents, may be underestimated in environmental samples by application of general fungal primers. We demonstrate here that the commonly used fungal primer ITS1 (White et al 1990) contains two mismatches with the Pyrenomycete fun-
GREEN
ET AL:
MOLECULAR
TOOLS FOR
gi. To rectify this problem and to develop tools specifically for the study of Pyrenomycetes, two regions of the 18S rDNA diagnostic for the Pyrenomycete fungi were identified. These regions were used here as primers for PCR amplification of Pyrenomycetes but may also be used as DNA or RNA probes. Using a combination of sequence analysis, PCR amplification of the 18S rDNA and the ITS region from a limited set of pure culture fungal DNA and multiple primer set and sequence analysis of environmental samples, we have demonstrated the specificity of these sequences for the Pyrenomycete fungi. In addition, BLAST searches and PCR analyses indicated that the primers target a negligible number of nonPyrenomycete sequences, particularly when the two Pyrenomycete primers are used together. Both Pyrenomycete-specific primers Py364 and PyITS1 (or PyNS8) are located in regions of the 18S rDNA that are inferred to code for stem-loop structures of the 18S rDNA, and both contain multiple mismatches with non-Pyrenomycete 18S rDNA. These mismatches might be the result of mutational events coupled with a compensatory mutation to maintain secondary structure in the stem of the rDNA (Berbee and Taylor 1992). Such locations are fortuitous for the design of specific primers due to the presence of multiple base-pair changes between target and nontarget sequences. Despite the multiple mismatches between Pyrenomycete primers and non-Pyrenomycete DNA (and vice-versa), PCR amplification of DNA with mismatches with either primer can be achieved with nonstringent annealing temperatures, particularly with primer set ITS1 (or PyITS1)/ITS4 (two mismatches with nontarget DNA). This, combined with the extensive use of the ITS1 primer for PCR, cloning and sequencing purposes, may explain the presence of numerous Pyrenomycete sequences mistakenly matching the ITS1 primer (data not shown). Under nonstringent conditions, the primer sequence becomes incorporated into copies of the nontarget templates and then may be wrongly included in submissions to sequence databases. For example, a sequence of Trichoderma koningii (GenBank accession number AF218790) matched the ITS1 primer sequence, while all other (nine) T. koningii or Hypocrea koningii sequences reported so far that contain the primer site, matched the PyITS1 primer sequence (data not shown). Our PCR analyses of numerous Trichoderma isolates and detection of Trichoderma from cucumber rhizosphere, suggest that Trichoderma contain the PyITS1 primer sequence, not that of the ITS1 primer. We believe that the specificity of the PyITS1 primer (and concomitantly, the presence of two mismatches with Pyrenomycetes of the ITS1 primer) is of partic-
PYRENOMYCETE
FUNGI
449
ular concern in any mixed culture or environmental application of the primers. In a mixture of Pyrenomycete and non-Pyrenomycete fungal DNA, the ITS1/ITS4 primer set is unlikely to amplify Pyrenomycete DNA. This is evidenced by the weak amplification of mismatched template PCR (FIG. 2), and by the absence of Pyrenomycete sequences in the DGGE analyses of DNA amplified with the NS1AG/NS8 primer set (TABLE IV and FIG. 4). Other fungal primers, such as ITS5 (based on the sequence of Neurospora crassa), FR1 (Vainio and Hantula 2000), EF4 and fung5 (Smit et al 1999), do target the Pyrenomycete fungi. However, none of these primers target the Pyrenomycetes exclusively, and also can target nonfungal organisms. In our analysis of fungal populations detected in a compostamended potting mix and from rhizospheres of cucumbers grown in the potting mix, populations most closely affiliated with the heterotrophic soil flagellate Hetermita globosa, frequently were recovered with the NS1/FR1 primer set (TABLE IV). Using DGGE analysis combined with sequencing of excised bands, we demonstrated that PCR of environmental DNA with the NS1AG/PyNS8 primer set generated amplification products of only Pyrenomycete fungi and weak amplification of cucumber DNA. In contrast, no Pyrenomycete fungi were detected in the general fungal analyses, presumably due to the dominant presence of flagellate DNA (NS1/FR1) and sequence mismatches (NS1AG/NS8). Using the PyITS1/ITS4 primer set, DNA fragments containing the entire ITS region of closely related Colletotrichum isolates were resolved successfully using DGGE analysis. In DGGE analysis of both the ITS and 18S rDNA fragments from Colletotrichum isolates, DNA fragments migrated in a pattern consistent with the phylogeny established by sequence analysis of the entire ITS region (FIG. 3a). However, an additional level of resolution between isolates of C. acutatum was provided with the ITS-DGGE analysis relative to the 18S rDNA-DGGE. While sequence analysis is the most informative method of typing isolates, it is also time consuming and expensive relative to DGGE analysis. We demonstrate here that DGGE can be used effectively in such analyses, and that application of DGGE to the ITS is more sensitive than DGGE analysis of the 18S rDNA. Furthermore, DGGE analysis of the ITS proved to be consistent with a phylogeny established by sequence analysis. As demonstrated in this work, the Pyrenomycete primer sets can be used to (i) putatively identify a fungal isolate as a Pyrenomycete (or non-Pyrenomycete) by stringent PCR, (ii) provide a large fragment of the 18S rDNA or the entire ITS region for sequence and thereafter phylogenetic analysis, (iii)
450
MYCOLOGIA
provide a mechanism for typing isolates by identifying differences in DNA sequence via DGGE analysis, (iv) analyze community structure of Pyrenomycete fungi in environmental samples and (v) verify the isolation of relevant fungal populations from environmental samples. The importance of the Pyrenomycete fungi as pathogens and biocontrol agents suggests that these molecular tools will prove useful. This research was supported by research grant number US-3108-99 from BARD, the United StatesIsrael Binational Agriculture Research and Development Fund. LITERATURE CITED
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389–3402. Berbee ML, Taylor JW. 1992. Convergence in ascospore discharge mechanism among Pyrenomycete fungi based on 18S ribosomal RNA gene sequence. Mol Phylogenet Evol 1:59–71. Borneman J, Hartin RJ. 2000. PCR primers that amplify fungal rRNA genes from environmental samples. Appl Environ Microbiol 66:4356–4360. Buchan A, Newell SY, Moreta JIL, Moran MA. 2002. Analysis of internal transcribed spacer (ITS) regions of rRNA genes in fungal communities in a southeastern U.S. salt marsh. Micro Eco 43:329–340. Freeman S, Maymon M, Pinkas Y. 1999. Use of GUS transformants of Fusarium subglutinans for determining etiology of mango malformation disease. Phytopathology 89:456–461. , Minz D, Jurkevitch E, Maymon M, Shabi E. 2000. Molecular analyses of Colletotrichum species from almond and other fruits. Phytopathology 90:608–614. , Maymon M. 2000. Reliable detection of the fungal pathogen Fusarium oxysporum f.sp. albedinis, causal agent of Bayoud disease of date palm, using molecular techniques. Phytoparasitica 28:341–348. , Minz D, Maymon M, Zveibil A. 2001. Genetic diversity within Colletotrichum acutatum sensu Simmonds. Phytopathology 91:586–592. Fukatsu T, Sato H, Kuriyama H. 1997. Isolation, inoculation to insect host, and molecular phylogeny of an entomogenous fungus Paecilomyces tenuipes. J Invertebr Pathol 70:203–208. Gardes M, White TJ, Fortin JA, Bruns TD, Taylor JW. 1991. Identification of indigenous and introduced symbiotic fungi in ectomycorrhizae by amplification of nuclear and mitochondrial ribosomal DNA. Can J Bot 69:180– 190. , Bruns TD. 1993. ITS primers with enhanced specificity for basidiomycetes—application to the identification of mycorrhizae and rusts. Mol Ecol 2:113–118. Glass NL, Donaldson GC. 1995. Development of primer sets designed for use with the PCR to amplify conserved
genes from filamentous ascomycetes. Appl Environ Microbiol 61:1323–1330. Gottlieb AM, Ferrer E, Wright JE. 2000. rDNA analyses as an aid to the taxonomy of species of Ganoderma. Mycol Res 104:1033–1045. Henrion B, Chevalier G, Martin F. 1994. Typing truffle species by PCR amplification of the ribosomal DNA spacers. Mycol Res 98:37–43. Hermosa MR, Grondona I, Iturriaga EA, Diaz-Minguez JM, Castro C, Monte E, Garcia-Acha, I. 2000. Molecular characterization and identification of biocontrol isolates of Trichoderma spp. Appl Environ Microbiol 66: 1890–1898. Hernan-Gomez S, Espinosa JC, Ubeda JF. 2000. Characterization of wine yeasts by temperature gradient gel electrophoresis (TGGE). FEMS Microbiol Lett 193:45–50. Jacquot E, van Tuinen D, Gianinazzi S, Gianinazzi-Pearson V. 2000. Monitoring species of arbuscular mycorrhizal fungi in planta and in soil by nested PCR: application to the study of the impact of sewage sludge. Plant and Soil 226:179–188. Jasalavich CA, Ostrofsky A, Jellison J. 2000. Detection and identification of decay fungi in spruce wood by restriction fragment length polymorphism analysis of amplified genes encoding rRNA. Appl Environ Microbiol 66: 4725–4734. Keeling PJ, Luker MA, Palmer JD. 2000. Evidence from beta-tubulin phylogeny that microsporidia evolved from within the fungi. Mol Biol Evol 17:23–31. Kurkela T, Hanso M, Hantula J. 1999. Differentiating characteristics between Melampsoridium rusts infecting birch and alder leaves. Mycologia 91:987–992. Liu YJ, Whelen S, Hall BD. 1999. Phylogenetic relationships among ascomycetes: evidence from an RNA polymerse II subunit. Mol Biol Evol 16:1799–1808. Lumbsch HT. 2000. Phylogeny of filamentous ascomycetes. Naturwissenschaften 87:335–342. Muyzer G, Smalla K. 1998. Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie van Leeuwenhoek 73:127–141. Neuveglise C, Brygoo Y, Vercambre B, Riba G. 1994. Comparative-analysis of molecular and biological characteristics of strains of Beauveria brongniartii isolated from insects. Mycol Res 98:322–328. , , Riba G. 1997. 28s rDNA group-I introns: a powerful tool for identifying strains of Beauveria brongniartii. Mol Ecol 6:373–381. Paavolainen L, Kurkela T, Hallaksela AM, Hantula J. 2000. Pleomassaria siparia is composed of two biological species, both of which occur in dead twigs of Betula pendula and B. pubescens. Mycologia 92:253–258. Pritsch K, Boyle H, Munch JC, Buscot F. 1997. Characterization and identification of black alder ectomycorrhizas by PCR/RFLP analyses of the rDNA internal transcribed spacer (ITS). New Phytologist 137:357–369. , Munch JC, Buscot F. 2000. Identification and differentiation of mycorrhizal isolates of black alder by sequence analysis of the ITS region. Mycorrhiza 10:87– 93.
GREEN
ET AL:
MOLECULAR
TOOLS FOR
Rafin C, Brygoo Y, Tirilly Y. 1995. Restriction analysis of amplified ribosomal DNA of Pythium spp. isolated from soilless culture systems. Mycol Res 99:277–281. Redecker D. 2000. Specific PCR primers to identify arbuscular mycorrhizal fungi within colonized roots. Mycorrhiza 10:73–80. Samuels GJ, Blackwell M. 2001. Pyrenomycetes—fungi with perithecia, p. 221–255. In: McLaughlin DJ, McLaughlin EG, Lemke PA, eds. The Mycota VII Part A. SpringerVerlag, Heidelberg. Smit E, Leeflang P, Glandorf B, van Elsas JD, Wernars K. 1999. Analysis of fungal diversity in the wheat rhizosphere by sequencing of cloned PCR-amplified genes encoding 18S rRNA and temperature gradient gel electrophoresis. Appl Environ Microbiol 65:2614–2621. Spatafora JW, Blackwell M. 1993. Molecular systematics of unitunicate perithecial ascomycetes—the ClavicipitalesHypocreales connection. Mycologia 85:912–922. . 1995. Ascomal evolution of filamentous ascomy-
PYRENOMYCETE
FUNGI
451
cetes—evidence from molecular data. Can J Bot 73: S811–S815. Strunk O, Gross O, Reichel B, May M, Hermann S, Stuckmann N, Nonhoff M, Lenke T, Ginhart T, Vilbig A, Ludwig T, Bode A, Schleifer KH, Ludwig W. 1999. ARB: a software environment for sequence data. Munich, Germany: Department of Microbiology, Technische Universitat Munchen. Vainio EJ, Hantula J. 2000. Direct analysis of wood-inhabiting fungi using denaturing gradient gel electrophoresis of amplified ribosomal DNA. Mycol Res 104:927–936. Viaud M, Pasquier A, Brygoo Y. 2000. Diversity of soil fungi studied by PCR-RFLP of ITS. Mycol Res 104:1027–1032. White TJ, Bruns T, Lee S, Taylor JW. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis M, Gelfand DH, Sninsky JJ, White TJ, eds. PCR protocols—a guide to methods and applications. San Diego, California: Academic Press. p 315–322.
