Molecular characterization of Cercospora beticola and its relatives

The genus Cercospora is one of the largest and most heterogeneous genera of hyphomycetes and contains many important plant pathogens. Generally species of Cercospora are considered to be host-specific at the level of the plant genus or family, and this concept has led to the description of more than 3 000 species. Currently only 600-700 species of Cercospora are recognized, while an additional 281 species are treated under C . apii sensu lato , which is the oldest name for a large complex of morphologically indistinguishable Cercospora species occurring on a wide range of host plants. Cercospora beticola, a species also belonging to the C. apii complex, causes Cercospora leaf spot on sugar beet and has a huge impact on the yield and quality of Beta vulgaris (sugar beet) production worldwide. Cercospora apii , which is morphologically identical to C. beticola , causes Cercospora leaf spot on Apium graveolens (celery) and it has been proposed that C. beticola and C. apii are synonymous. Multilocus sequence data, amplified fragment length polymorphism (AFLP) analyses and cultural characteristics were used to identify and characterize morphologically similar Cercospora species that occur on celery and sugar beet. During this study, an undescribed Cercospora species was isolated from celery and it was shown that these three species, although morphologically very similar, are distinct functional species that should be retained as separate entities. A PCR-based diagnostic protocol was developed to distinguish the three Cercospora species. This thesis illustrated that C. apii and C. beticola can be isolated from each others' host and that the third species of Cercospora is thus far only known from celery. This third species was formally described as C. apiicola.For most Cercospora species, including C. beticola , the sexual stage is unknown. Phylogenetic analyses of a variety of Cercospora species have placed them as a well-defined clade in the teleomorph genus Mycosphaerella . Therefore, if a sexual stage does exist for C. beticola , it would be a species of Mycosphaerella . Degenerate primers were developed and used to isolate the mating type genes of several Cercospora species. The MAT1‑1‑1 and MAT1-2 genes were characterized and both genes were found to be present in C. beticola, C. zeae-maydis and C. zeina .   As only one of the two genes was found to be present in an isolate it is proposed that these Cercospora species are heterothallic. The mating types were approximately evenly distributed in field populations of these three species, a phenomenon that is often observed for actively sexually reproducing species.Previous studies showed that genetic variation exists within C. beticola,but it was not known whether this variation was due to chromosomal rearrangements, asexual or sexual recombination. Data obtained from AFLP analyses conducted during this study showed that the genetic variation observed in this species is most likely caused by recombination events occurring in natural populations. The high level of genetic variation, high number of distinct genotypes and the equal distribution of the mating types within C. beticola populations are clear indications that sexual recombination events most likely play an important role in the reproductive cycle of this genetically diverse and heterogeneous species. The development and testing of additional molecular markers (microsatellites and SNPs) on C. beticola populations are also included in this thesis. These markers were highly polymorphic and showed high levels of genetic variation as well as a high number of haplotypes. The markers can therefore be used for future studies to quantify the genetic diversity within C. beticola .Two relatives of C. beticola, namely Dothistroma septosporum and D. pini, cause red band needle blight on pines. A sexual stage is known but rarely found, and therefore it has been assumed that sexual reproduction seldom occurs. The MAT1-1-1 and MAT1-2 genes were isolated from both of these species using the degenerate primers that were used to isolate the mating type genes of Cercospora species. Dothistroma mating type-specific primers were developed during this study and these primers can distinguish between the two Dothistroma species as well as identify which mating type is present.This thesis covers a broad range of techniques, from classical morphological and cultural studies to the development of novel molecular tools. In order to obtain a better understanding of the taxonomy, reproductive strategies, intra- and inter-species variation of members of the C. apii complex, all data sets were required to delineate the various operational species involved.