Genome sequencing and phylogenetic analyses of common and dwarf bunt of wheat provide insights into their genomic diversity and species boundaries, and enable the development of a detection assay for Tilletia controversa
von Somayyeh Sedaghatjoo
Datum der mündl. Prüfung:2021-09-20
Betreuer:Prof. Dr. Petr Karlovsky
Gutachter:Dr. Wolfgang Maier
Gutachter:Prof. Dr. Andreas von Tiedemann
EnglischThe fungal genus Tilletia is generally recognized by the production of darkly pigmented teliospores, which replace mostly the host ovary. Currently, nearly 200 species of Tilletia species infecting Poaceae are included in this smut genus. Three species, namely T. caries, T. controversa, and T. laevis, cause economically important diseases of wheat in central Europe. Common bunt is caused by T. caries and T. laevis, whereas dwarf bunt is caused by T. controversa. The three species are described based on the morphology of their teliospores. However, they could not be reliably distinguished by using molecular phylogenetic analyses. To obtain deeper insights into the inter- and intraspecies genetic variation and to compare gene contents of these three species we sequenced and functionally and structurally annotated the whole-genomes of four strains of T. caries, five of T. controversa, and two of T. laevis. The obtained data was analysed together with five publicly available genomes (one T. caries, two T. controversa, and two T. laevis strains). In general, our findings demonstrated that the three species were highly similar with regards to genome size and predicted gene content. There was no evidence for expansion or decrease of transposable elements in any of the species. The nine predicted secondary metabolites gene clusters, 84% of the total carbohydrate active enzymes, 72% of secreted proteins, and 50% effector-like proteins were conserved across 16 studied strains. The species-specific proteins made only 0.1% of all predicted proteins, and their function were mainly unknown. In non-repetitive regions, the number of single nucleotide polymorphisms (SNPs) and small insertions or deletions (indels) were lowest within T. laevis (max. 0.52 SNPs/kb and 1.09 bp indels/kb), while they were highest within T controversa (max. 1.47 SNPs/kb and 2.48 indels bp/kb). We also observed extensive sequence identity between the two species of T. caries and T. laevis (0.51 SNPs/kb and 1.04 indels bp/kb on average). At the same time both species showed comparable distances to T. controversa. Accordingly, phylogenomic analysis of 241 protein coding genes revealed two groupings where isolates of T. caries and T. laevis were intermingled in a monophyletic group together, but separated from those of T. controversa, which formed another monophyletic group. Taken together these results suggest that T. caries and T. laevis have either diverged very recently or could be conspecific. These observations also correlate well with the fact that the two species are causing identical disease symptoms, need the same germination conditions, and have similar infection biology. These characteristics are different from those of T. controversa. Dwarf bunt is a quarantine pathogen in several countries. Consequently, its accurate identification is of high priority for plant health as well as to wheat exporters. The current international diagnostic protocol for detection of dwarf bunt in wheat seeds is based on a filtration method and the teliospores morphology. The method is however laborious and requires expert knowledge. To facilitate identification of T. controversa, a loop-mediated isothermal amplification (LAMP) assay was developed. To do this, the generated genomic data were extended further with publicly available genomic data from ten Tilletia isolates in order to identify DNA segments that were conserved in and unique to T. controversa. The developed assay was based on one of these genome regions. The assay specificity was validated against 223 fungal phytopathogens including 11 Tilletia species. The end-point colorimetric based detection LAMP assay had 5 pg limit of detection and showed 100% sensitivity and 97.7% specificity in an interlaboratory test performance study. The majority of smuts are reported to have relatively narrow host ranges including Tilletia species. Tilletia caries and T. controversa however are reported to infect different host species representing several grass genera. To clarify whether they are indeed generalist species with a broad host range or rather represent complexes of cryptic species with narrow host ranges phylogenetic relationships of those species and close relatives were inferred using sequences of three gene regions (ITS rDNA, EF1α, and RPBII). In total 70 specimens were analysed of which 20 specimens were newly produced for this study. In general, the multi locus phylogenetic analysis resolved various species with narrow host ranges parasitizing wild grasses as distinct lineages (i.e., the basal lineages from T. fusca up to T. olida representing 12 species and the crown lineage with T. bromi and T. puccinelliae, respectively). Several small clusters of T. controversa from wild grasses (Thinopyrum intermedium, Bromus marginatus, Agropyron cristatum) and rye (Secale cereale), respectively, clustered as subgroups in a polytomous manner between different clusters of T. caries, T. controversa and T. laevis on wheat. Interestingly, one group of T. controversa sequences obtained from Elymus repens, Th. intermedium, and Agropyron sp. clustered with high support values clearly separate from this polytomous group and together with T. brevifaciens isolates also obtained from Th. intermedium. These representatives of T. controversa with high likelihood represent at least one cryptic species restricted to these wild grasses as hosts and might potentially be conspecific with T. brevifaciens. In the latter case, T. brevifaciens would represent a species with board host range.
Keywords: trimethylamine biosynthesis; Basidiomycota; comparative genomics; species-specific detection; average nucleotide identity; host specificity