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Previous Article | Next Article 
Applied and Environmental Microbiology, August 2004, p. 4419-4423, Vol. 70, No. 8
0099-2240/04/$08.00+0 DOI: 10.1128/AEM.70.8.4419-4423.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Agricultural Biotechnology Center,1
Department of Agricultural Biotechnology and Microbiology, Group of Mycology, Szent István University, Gödöll
, Hungary,4
Institute of Sciences of Food Production, CNR, Bari, Italy,2
Business Unit Biointeractions and Plant Health, Plant Research International, Wageningen, The Netherlands3
Received 8 September 2003/ Accepted 24 February 2004
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ABSTRACT |
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and high-mobility-group (HMG) box sequences from Fusarium avenaceum, F. culmorum, F. poae, and F. semitectum were compared to similar sequences that were described previously for other members of the genus. The DNA sequences of the regions flanking the amplified MAT regions were obtained by inverse PCR. These data were used to develop diagnostic primers suitable for the clear amplification of conserved mating type sequences from any member of the genus Fusarium. By using these diagnostic primers, we identified mating types of 122 strains belonging to 22 species of Fusarium. The box and the HMG box from the mating type genes are transcribed in F. avenaceum, F. culmorum, F. poae, and F. semitectum. The novelty of the PCR-based mating type identification system that we developed is that this method can be used on a wide range of Fusarium species, which have proven or expected teleomorphs in different ascomycetous genera, including Calonectria, Gibberella, and Nectria. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Meiotic recombination can generate and maintain genotypic variation and result in the reassortment of genes that govern traits such as virulence or toxin production (7). The sexual spores (ascospores) produced by some Fusarium species also may function as infectious propagules (11, 19). Although several Fusarium species have a known sexual cycle, i.e., they mate in either a homothallic or heterothallic manner followed by subsequent meiosis and the production of ascospores, important pathogenic species, including Fusarium avenaceum, Fusarium cerealis, Fusarium culmorum, Fusarium equiseti, Fusarium poae, and Fusarium sporotrichioides, have no known sexual stage.
Assessing the potential for mating by toxigenic strains of Fusarium would increase our understanding of the genetic mechanisms that maintain intraspecific diversity and biological and evolutionary species integrity. The frequency of sexual reproduction is also an important parameter for the design of strategies to control plant pathogens, since these strategies are often different for clonally and sexually reproducing organisms. High levels of race-specific resistance can be developed in plant cultivars against clonally reproducing organisms, whereas horizontal resistance could be more effective against pathogens comprising genetically diverse populations as a result of mating and meiotic recombination (16).
The known teleomorphs of Fusarium species belong to the genera Calonectria, Gibberella, and Nectria (5). In heterothallic species, e.g., Gibberella fujikuroi, mating type is controlled by a single locus with two idiomorphic alleles, termed MAT-1 and MAT-2. These alleles contain a conserved box domain and a high-mobility-group (HMG) box domain, respectively. Strains of Gibberella zeae (anamorph of Fusarium graminearum), a homothallic species, carry both the MAT-1 and MAT-2 idiomorphs, closely linked together (27). Strains of Fusarium oxysporum, a species complex with no known sexual stage, also contain transcribed MAT alleles (4, 13, 27). However, the MAT genes have not been studied in any other mitotic holomorph species within the genus Fusarium.
PCR amplification of MAT sequences from various Fusarium species belonging to the G. fujikuroi species complex has been utilized to standardize the mating type terminology for mating populations of this species complex (13) and to develop assays for identifying the presence of the MAT allele without sexual crosses (22, 25). However, the primers used in these previous studies were inadequate for the rest of the genus (13, 22), probably due to sequence divergence that may occur even in conserved MAT sequences of these fungi. The aims of the present study were (i) to demonstrate whether mating type sequences can be found in Fusarium species with no known sexual stage, (ii) to develop a PCR-based technique for the rapid identification of mating types in a wide range of Fusarium species with proven or expected Calonectria, Gibberella, and Nectria teleomorphs, and (iii) to demonstrate the transcription of mating type genes in selected "asexual" Fusarium species during their vegetative growth.
(A preliminary version of this work was presented at the 6th European Conference of Fungal Genetics [A. Moretti, Z. Kerényi, G. Mulé, C. Waalwijk, and L. Hornok, Abstr. 6th Eur. Conf. Fungal Genet., abstr. ECFG6, p. 394-395, 2002].)
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MATERIALS AND METHODS |
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Molecular techniques.
Common DNA and RNA manipulation techniques were performed as described by Sambrook et al. (21). PCRs were performed with reaction mixtures containing 1x PCR buffer (MBI Fermentas, Vilnius, Lithuania), 1.5 mM MgCl2, a 0.5 mM concentration of each deoxynucleoside triphosphate, a 0.25 µM concentration of each primer, 1 U of Taq polymerase (MBI Fermentas), and about 20 ng of fungal DNA. Initial denaturation was done at 95°C for 2 min, followed by 30 cycles consisting of 30 s at 94°C, 30 s at 55 to 60°C (depending on the melting temperature of the primers), and 0.5 to 5 min at 72°C (depending on the expected length of the amplicon), and a final elongation step at 72°C for 10 min. The amplification products were separated by electrophoresis in agarose gels, stained with ethidium bromide, and visualized with UV light. Amplicons were cloned into the pBluescript II KS (Stratagene, La Jolla, Calif.) or pGEM-T Easy (Promega, Madison, Wis.) plasmid vector, transformed into Escherichia coli DH5 cells (Clontech, Palo Alto, Calif.), and sequenced by the Sequencing Service of the Agricultural Biotechnology Center. DNA sequences were analyzed with the Lasergene (DNAStar Inc., Madison, Wis.) and Wisconsin (10) software packages, and BLAST searches were done with the GenBank database (2).
PCR amplification of conserved MAT boxes.
Conserved portions of the or HMG box of the MAT-1 or MAT-2 idiomorph were amplified from different Fusarium species by using the degenerate F1 (CGNCCNCTNAAYGSNTTYATG) and F2 (ACYTTNGCNATNAGNGCCCAYTT) primers or the previously described NcHMG1 and NcHMG2 primers (3). Primers F1 and F2 were designed based on deduced amino acid sequence data for MAT-1 idiomorphs known from G. moniliformis (synonym, G. fujikuroi mating population A) (accession no. AF100925), G. zeae (accession no. AF318048), and F. oxysporum (accession no. AB011379) (27). The putative MAT-specific amplicons, identified on the basis of the expected lengths of the fragments, were cloned and sequenced. BLAST comparisons confirmed that these amplicons were indeed MAT-box homologs, i.e., and HMG box homologues. Amplified fragments of the expected sizes were isolated from the gel, cloned, and sequenced.
Cloning entire MAT genes.
Inverse PCR was performed as previously described (23) by using Herculase Taq polymerase (Stratagene) according to the manufacturer's instructions. Amplicons were cloned and sequenced, and the appropriate contigs were assembled by using the SeqMan computer program (DNAStar). To clone intact MAT-1-1 and MAT-2 genes, we designed specific primer pairs (Table 1) based on the sequence information derived from inverse PCR. Using these primers, we amplified, cloned, and sequenced the entire MAT-1-1 and MAT-2 genes from F. avenaceum, F. culmorum, F. poae, and F. semitectum.
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RT-PCR experiments.
Total RNAs were extracted from the mycelia grown on carrot agar plates by use of the TRI reagent (Sigma, St. Louis, Mo.) according to the manufacturer's instructions. The first-strand cDNA reaction (40 µl) contained 1x avian myeloblastosis virus buffer (Promega), a 0.5 mM concentration of each deoxynucleoside triphosphate, 1.7 µM oligo(dT)15 primer, 20 U of RNasin (Promega), 5 U of avian myeloblastosis virus reverse transcriptase (Promega), and about 5 µg of total RNA and was followed by an RQ1 DNase (Promega) treatment. The mixture was incubated at 42°C for 50 min and at 65°C for 10 min to inactivate the reverse transcriptase. Five microliters of the first-strand cDNA reaction was used as a template in a 25-µl standard PCR mixture. Amplifications were done with the same program as described above, except that the number of cycles was increased from 30 to 40.
Nucleotide sequence accession numbers.
The sequences of the amplified regions of F. avenaceum, F. culmorum, F. poae, and F. semitectum were deposited in the EMBL database under accession numbers AJ535625 to AJ535632.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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or HMG boxes were amplified by inverse PCR from F. avenaceum, F. culmorum, F. poae, and F. semitectum, and the resulting fragments were cloned and sequenced. Based on these sequences, new PCR primer pairs were designed for border regions of the MAT-1-1 and MAT-2 genes, and the DNA fragments generated by these primers were cloned and sequenced.
Putative MAT-specific fragments, MAT-1 and MAT-2 genes, and MAT-1-specific box and MAT-2-specific HMG box sequences (Table 2) were identified by comparison with sequences available for the MAT idiomorphs of F. oxysporum, G. fujikuroi, and G. zeae (27). The MAT-1-1 gene identified in F. avenaceum ITEM 859 was 1,218 bp long and encoded a putative protein with an -box motif. The sequences of MAT-1-1 genes from F. culmorum strain 19A1, F. poae TAPO21, and F. semitectum ITEM 3192 were 1,085, 1,203, and 1,129 bp long, respectively, and encoded putative proteins with conserved -box domains. All of these MAT-1-1 gene sequences contained introns at conserved positions (20). No in-frame stop codons were found in these sequences.
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Diagnostic PCR for mating type in Fusarium species.
We designed new degenerate oligonucleotide primers, namely fusALPHAfor (CGCCCTCTKAAYGSCTTCATG), fusALPHArev (GGARTARACYTTAGCAATYAGGGC), fusHMGfor (CGACCTCCCAAY GCYTACAT), and fusHMGrev (TGGGCGGTACTGG TARTCRGG), and defined appropriate PCR conditions. The positions of these primers in the and HMG box sequences of F. avenaceum (AJ535625 and AJ535629) are nucleotides 282 to 302, 456 to 479, 540 to 559, and 775 to 795, respectively. The sizes of the MAT-1- and MAT-2-specific fragments amplified from different species of Fusarium were 200 and 260 bp, respectively (Fig. 1).
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Transcription of MAT genes in Fusarium species with no known sexual stage.
RT-PCR experiments primed with box- or HMG box-specific primers, respectively, were performed to examine the expression of MAT genes in F. avenaceum, F. culmorum, F. poae, and F. semitectum. Electrophoretic separation of the RT-PCR products resulted in the appearance of one characteristic band at the appropriate size, i.e., an 
150-bp and an 200-bp fragment in all samples (Fig. 2). The size differences observed between the amplicons obtained by RT-PCR and fragments generated from the genomic DNAs were due to the presence of an intron in the genomic copies of these MAT boxes. Northern blot analyses of these RT-PCR products, with the appropriate cloned MAT-1-1 or MAT-2 gene as a probe, confirmed the identities of the fragments. Thus, both the MAT-1-1 and MAT-2 genes were transcribed in all asexual Fusarium species involved in this experiment.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and HMG box sequences of four asexual Fusarium species. All Fusarium species involved in this work were found to contain one or the other mating type idiomorph, with the exception of F. graminearum, which was used as a control. The MAT-1- and MAT-2-specific fragments that were amplified from these fungi showed substantial sequence similarities to conserved motifs of the MAT-1-1 and MAT-2 genes from F. oxysporum, G. fujikuroi, and G. zeae, suggesting that these partial sequences represent the mating type idiomorphs in these fungi. Degenerate MAT-specific primers designed by Arie et al. (3) or the G. fujikuroi-specific MAT primers developed in previous studies (13, 22) were unsuitable for generating unambiguous PCR fragments in such diverse Fusarium species (representatives of nine sections) due to sequence differences within the conserved MAT regions of these fungi. Our findings clearly show that conserved MAT-specific sequences are present and expressed in Fusarium species with no known sexual stage. Since the strains of F. acuminatum, F. avenaceum, F. camptoceras, F. cerealis, F. chlamydosporum, F. compactum, F. culmorum, F. equiseti, F. longipes, F. merismoides, F. poae, F. semitectum, F. scirpi, F. sporotrichioides, F. torulosum, F. tricinctum, and F. tumidum all contained only a single MAT allele, presumably they are capable of heterothallic, but not homothallic, mating. These results are consistent with the hypothesis (24) that these fungi may have a cryptic sexual cycle, even though sexual structures have not been identified in field collections and there are no reports of successful forced parings among them in laboratory experiments.
Leslie and Klein (15) explained the absence of sexual reproduction in local populations of the G. fujikuroi species complex by the presence of mutations that concomitantly resulted in female sterility with an increased vegetative propagation capability. Selection for an increased number of asexual propagules can result in a selective accumulation of female sterile strains, which could become prevalent even in large geographic areas. Under such conditions, mating is limited by the absence of normal female fertile partners. The Fusarium species that we examined seem to have functional mating type genes, are aggressive pathogens, and can colonize a wide range of decaying substrates. Populations of these fungi could easily be dominated by successful female sterile clonal lineages that produce more asexual propagules and are therefore not under significant immediate selection pressure to participate in sexual reproduction. The female fertile strains could be such a small minority (<10% in some natural populations [15]) that they are likely to be infrequent, especially under epidemic conditions. Thus, their sexual structures may not be observed in nature simply because of their rarity. Alternatively, the purportedly asexual species also may require environmental conditions for sexual reproduction that are uncommon when disease epidemics occur or that are difficult or unusual conditions to mimic in the laboratory.
The PCR method that we developed for the mating type assessment of these Fusarium species facilitates the recognition of potentially compatible strains that could be used in crossing experiments to obtain teleomorphic structures. This approach could increase our knowledge of reproductive strategies in these fungi and allow a realistic evaluation of the potential for generating strains with new pathotypes and/or altered mycotoxin-producing abilities and could be used to assess disease control strategies that presume that limited genotypic variation and rearrangement occur within the pathogen population.
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ACKNOWLEDGMENTS |
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We are indebted to E. Barta for his advice on phylogenetic comparisons.
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FOOTNOTES |
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, Hungary. Phone: 36 28 526100. Fax: 36 28 526131. E-mail: hornok{at}abc.hu. 
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REFERENCES |
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