PCR-Based Identification of MAT-1 and MAT-2 in the Gibberella fujikuroi Species Complex

Abstract

All sexually fertile strains in the Gibberella fujikuroi species complex are heterothallic, with individual mating types conferred by the broadly conserved ascomycete idiomorphs MAT-1 and MAT-2. We sequenced both alleles from all eight mating populations, developed a multiplex PCR technique to distinguish these idiomorphs, and tested it with representative strains from all eight biological species and 22 additional species or phylogenetic lineages from this species complex. In most cases, either an ϳ800-bp fragment from MAT-2 or an ϳ200-bp fragment from MAT-1 is amplified. The amplified fragments cosegregate with mating type, as defined by sexual cross-fertility, in a cross of Fusarium moniliforme (Fusarium verticillioides). Neither of the primer pairs amplify fragments from Fusarium species such as Fusarium graminearum, Fusarium pseudograminearum, and Fusarium culmorum, which have, or are expected to have, Gibberella sexual stages but are thought to be relatively distant from the species in the G. fujikuroi species complex. Our results suggest that MAT allele sequences are useful indicators of phylogenetic relatedness in these and other Fusarium species.Fusarium isolates in the Gibberella fujikuroi species complex include important fungal pathogens of agricultural crops and trees and may be divided into at least eight different biological species and 32 additional asexual species or phylogenetic lineages (4, 10, 13, 15, 16). All sexually fertile species in the G. fujikuroi species complex are dimictic, i.e., two isolates are cross-fertile if they carry the different mating-type idiomorphs MAT-1 and MAT-2 (4, 6, 9, 13), which share no sequence similarity with respect to either DNA sequence or the proteins encoded (5). The MAT-2 idiomorphs thus far characterized have a conserved HMG (high-mobility-group) domain (3,5,6,9). The MAT-1 idiomorphs have a conserved ␣-domain (5, 25), but no MAT-1 alleles have yet been described from any members of the G. fujikuroi species complex.Our objectives in this study were (i) to sequence conserved portions of the MAT-1 and MAT-2 alleles from the eight known G. fujikuroi mating populations, (ii) to develop a multiplex PCR reaction to be used for the identification of both mating-type idiomorphs within the defined biological species of the G. fujikuroi species complex, (iii) to determine the range of Fusarium species which have Gibberella teleomorphs to which this technique can be successfully applied, and (iv) to test the use of the sequence of the MAT idiomorphs for phylogenetic analyses. This technique eases the identification of strains to be used in crosses to identify new biological species and eliminates the need for sexual crosses to score this trait. MAT sequence variations may be used as phylogenetic characters, providing another marker that can be used to test phylogenies for robustness.

Fusarium ananatum sp. nov. in the Gibberella fujikuroi species complex from pineapples with fruit rot in South Africa

“…In order to determine the mating types of the seven isolates from pineapple in South Africa, the MAT-1 and MAT-2 loci were amplified using PCR, as described by Steenkamp et al (2000). The MAT idiomorphs were amplified with the primer sets GFmat1a (5′-GTTCATCAAAGGGCAAGCG-3′), GFmat1b (5′-TAAGCGCCTC-TTAACGCCTTC-3′), GFmat2c (5′-AGCGTCATT-ATTCG-ATCAAG-3′) and GFmat2d (5′-CTACGTTGAGAGCTGTACAG-3′) (Steenkamp et al 2000).…”

“…The MAT idiomorphs were amplified with the primer sets GFmat1a (5′-GTTCATCAAAGGGCAAGCG-3′), GFmat1b (5′-TAAGCGCCTC-TTAACGCCTTC-3′), GFmat2c (5′-AGCGTCATT-ATTCG-ATCAAG-3′) and GFmat2d (5′-CTACGTTGAGAGCTGTACAG-3′) (Steenkamp et al 2000). The following PCR reaction mixture was used: 1× PCR buffer with MgCl 2 , dNTP (250 μM) each, primers (0.1 μM of each), template DNA (25 ng) and 0.5 U Roche Taq polymerase (Roche Pharmaceuticals).…”

Both mating types in the heterothallic fungus Ophiostoma quercus contain MAT1-1 and MAT1-2 genes

“…For example, comparisons of MAT DNA sequences in different fungi have improved our understanding of the evolution of homothallic and heterothallic mating strategies (Arie et al 1997;Bennett et al 2003;Conde-Ferráez et al 2007;Fraser and Heitman 2004;Li et al 2010;Martin et al 2011;Steenkamp et al 2000;Turgeon 1998). Also, the availability of information on the mating idiomorphs allowed for the assessment of the presence of MAT genes in the genome of apparently asexual species (Foster and Fitt 2003;Mandel et al 2007;Turgeon 1998).…”

“…These DNA-based methods are, however, dependent on the availability of sequence information for the MAT locus, because MAT idiomorph-specific PCR assays exploit the inherent differences in the MAT genes (Dyer et al 2001;Steenkamp et al 2000).…”

Imunoensaios: uma alternativa para a detecção de fungos toxigênicos em alimentos

“…A maioria destes métodos apresenta baixa especificidade e reprodutibilidade, além de consumirem tempo e exigirem pessoal técnico qualificado para a execução (LI; MARQUARDT; ABRAMSON, 2000). Recentemente, estudos de sistemática molecular têm sido realizados na tentativa de facilitar a diferenciação de espécies fúngicas, mas embora possuam boa especificidade, essas metodologias apresentam custo elevado e exigem pessoal técnico qualificado (KERÉNYI et al, 1999;STEENKAMP et al, 2000).…”