Analysis of the mating-type loci of co-occurring and phylogenetically related species of Ascochyta and Phoma

Abstract

Ascochyta and Phoma are fungal genera containing several important plant pathogenic species. These genera are morphologically similar, and recent molecular studies performed to unravel their phylogeny have resulted in the establishment of several new genera within the newly erected Didymellaceae family. An analysis of the structure of fungal mating-type genes can contribute to a better understanding of the taxonomic relationships of these plant pathogens, and may shed some light on their evolution and on differences in sexual strategy and pathogenicity. We analysed the mating-type loci of phylogenetically closely related Ascochyta and Phoma species (Phoma clematidina, Didymella vitalbina, Didymella clematidis, Peyronellaea pinodes and Peyronellaea pinodella) that co-occur on the same hosts, either on Clematis or Pisum. The results confirm that the mating-type genes provide the information to distinguish between the homothallic Pey. pinodes (formerly Ascochyta pinodes) and the heterothallic Pey. pinodella (formerly Phoma pinodella), and indicate the close phylogenetic relationship between these two species that are part of the disease complex responsible for Ascochyta blight on pea. Furthermore, our analysis of the mating-type genes of the fungal species responsible for causing wilt of Clematis sp. revealed that the heterothallic D. vitalbina (Phoma anamorph) is more closely related to the homothallic D. clematidis (Ascochyta anamorph) than to the heterothallic P. clematidina. Finally, our results indicate that homothallism in D. clematidis resulted from a single crossover between MAT1-1 and MAT1-2 sequences of heterothallic ancestors, whereas a single crossover event followed by an inversion of a fused MAT1/2 locus resulted in homothallism in Pey. pinodes.

Figure 1

Schematic representation of the organization of mating‐type loci of the heterothallic Phoma clematidina, P. herbarum, Peyronellaea pinodella and Didymella vitalbina (a) and of the homothallic Pey. pinodes and D. clematidis (b). Mating‐type genes are indicated by dark grey arrows, and other predicted (partial) gene models by light grey arrows. The positions of predicted introns are marked by white boxes. The marker at the bottom indicates a size of 1 kb.

Figure 2

One of the two most parsimonious trees obtained from a heuristic search of the internal transcribed spacer (ITS) sequence alignment. The scale bar shows one change, strict consensus branches are thickened and bootstrap support values from 1000 bootstrap replicates (BS) and posterior probabilities (PP) are shown at the nodes. BS/PP values below 60/0.6 are omitted. Confirmed heterothallic species are indicated in dark grey and confirmed homothallic species in light grey. The tree is rooted with Didymella urticicola GU237761. CBS, Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands; PD, Dutch National Reference Centre of the Plant Protection Service, Wageningen, the Netherlands.

Figure 3

Most parsimonious trees obtained from a heuristic search of the partial MAT1‐1‐1 (a) and MAT1‐2‐1 (b) sequence alignments. The scale bars show the changes; bootstrap support values from 1000 bootstrap replicates (BS) and posterior probabilities (PP) are shown at the nodes. BS/PP values below 60/0.6 are omitted. The trees are rooted with Pyrenophora teres and Pyrenophora graminaea. The first of four most parsimonious trees; the strict consensus branches are thickened. CBS, Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands; PD, Dutch National Reference Centre of the Plant Protection Service, Wageningen, the Netherlands.

Figure 4

Schematic overview of the sequence similarities between the mating‐type loci of the heterothallic Didymella vitalbina and homothallic D. clematidis (a) and between the heterothallic Peyronellaea pinodella and homothallic Pey. pinodes (b). Also depicted are the putative recombination events responsible for the evolution of the homothallic species from heterothallic ancestors. Predicted gene models are indicated by arrows. Putative recombination spots are marked with a large ‘X’ and the putative inversion event is marked by a circular arrow. Sequences of the predicted junctions are indicated below the figure. All MAT1‐1‐derived areas are indicated in light grey; all MAT1‐2‐derived areas are indicated in dark grey; nonidiomorphic areas are indicated in black; sequences at fusion junctions that are shared between MAT1‐1 and MAT1‐2 are indicated in bold black.

Figure 5

Sequence analysis of inversion junctions in Peyronellaea pinodes. (a) Alignment of the 5′ junction of the putative inversion in Pey. pinodes to the reverse complemented (RC) sequence of the 3′ junction. The putative motif is indicated as a shaded box and identical nucleotides are marked in bold and have a larger font size. (b) Alignment of the 5′ and 3′Pey. pinodes junctions to Peyronellaea pinodella MAT1‐1 and MAT1‐2 sequences. The sequences of the 3′ junctions are reverse complemented (RC) for better comparison of the putative motif (shaded box), as also indicated in (a). Identical nucleotides are indicated in bold and nucleotides shared by all three sequences have a larger font size.