General morphology of representative strains of each of the lineages (arrhizus = CBS

330.53, delemar = CBS 390.34) is depicted in Fig. 5 and Fig. 6. In main traits the varieties have closely similar features. One of the measurable variables was spore size, but frequently variability of this parameter was large even in a single strain. Zygospores were observed only in three out of 166 contrasts. Two out of the three successful matings were obtained at condition (iii) using SNA for precultivation and spores suspensions as inoculum. The third successful mating was obtained at condition (i) using MEA media. One of these strain pairs (CBS 148.22 × CBS 346.36) represents positive mating within arrhizus, while two pairings (CBS 372.63 × CBS 346.36 and CBS 131498 × CBS 346.36) represented positive mating between arrhizus (CBS 346.36) and strains Pexidartinib clinical trial belonging to the basal ITS type C cluster[19] of delemar. CBS 346.36 is a sexually highly competent GSK-3 inhibitor strain, crossing with representatives of both lineages. The number of zygospores produced in the three contrasts was very low and zygospore formation was restricted to a small area that was not positioned in the contact zone of the two strains. In all cases the number of zygospores that did not complete their development distinctly exceeded the number of mature zygospores. In the intra-arrhizus

contrast (CBS 148.22 × CBS 346.36) several preliminary CYTH4 stages and two mature orange brown zygospores were produced (Fig. 7) that were crushed during slide preparation (size of the crushed zygospores including warts: (i) 156 (172) μm in diam, (ii) 140 (152) × 132 (148) μm. The contrast CBS 131498 ×  CBS 346.36 resulted in several (approx. 20) zygospores in different developmental stages, most of them remaining orange

and small while two became mature reflected by a larger size [104 (116) × 92 (104) μm and 116 (136) × 108 (128) μm] and a deeper color (Fig. 7f). The zygospores formed in the second arrhizus-delemar mating (CBS 346.36 × CBS 372.63) stayed small and less intensively colored. In agreement with Abe et al. [19] our multi-locus study recognized the arrhizus and delemar lineages as two phylogenetically separate entities. The distinction matched with differences in the production of organic acids: arrhizus possesses two genes for lactate dehydrogenase, ldhA and ldhB, which are responsible for the production of lactic acid. Strains of delemar lack the ldhA gene resulting in the production of fumaric and malic acid.[19, 31] We were unable to detect any additional phenotypic difference between arrhizus and delemar. The two entities are very close to each other in ITS sequence data, and each show further intra-group differentiation matching with subtypes A–D of Abe et al. [19] No differences in their ecology, distribution and pathogenicity could be detected in our data.