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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, Bet-Dagan, 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
Dror Minz 1
Institute of Soil, Water and Environmental Sciences, Agriculture Research Organization, The Volcani Center, P.O. Box 6, Bet-Dagan, 50-250, Israel
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ABSTRACT |
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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 established 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
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), 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 Black-well 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 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.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. Primer PyITS1 (or PyNS8, the inverse complement of PyITS1), located at the 3' 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 examination 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 5' 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 3' 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 5' end of the primer, a technique used to enhance separation during DGGE analysis (Muyzer and Smalla 1998).
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). 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).
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). 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).
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. 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). 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 compost-amended 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 ampli-fied 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 µL 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 pGEM®-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 neighbor-joining tree was produced using a 50% conservation filter (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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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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).
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), migrated to a third location, but no separation between these fragments was observed in the DGGE analysis.
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). 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 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 deep-branching 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 Pyr-enomycete-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 5' 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 amplification 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.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) contains two mismatches with the Pyrenomycete fungi. 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 non-Pyrenomycete 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 nonstrin-gent 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 non-stringent 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 particular 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 compost-amended 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) 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 States-Israel Binational Agriculture Research and Development Fund.
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FOOTNOTES |
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1 Corresponding author. E-mail: minz{at}volcani.agri.gov.il
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