Tilletia controversa causing dwarf bunt of wheat is a quarantine pathogen in several countries. Therefore, its specific detection is of great phytosanitary importance. Genomic regions routinely used for phylogenetic inferences lack suitable polymorphisms for the development of species-specific markers. We therefore compared 21 genomes of six Tilletia species to identify DNA regions that were unique and conserved in all T. controversa isolates and had no or limited homology to other Tilletia species. A loop-mediated isothermal amplification (LAMP) assay for T. controversa was developed based on one of these DNA regions. The specificity of the assay was verified using 223 fungal samples comprising 43 fungal species including 11 Tilletia species, in particular 39 specimens of T. controversa, 92 of T. caries and 40 of T. laevis, respectively. The assay specifically amplified genomic DNA of T. controversa from pure cultures and teliospores. Only Tilletia trabutii generated false positive signals. The detection limit of the LAMP assay was 5 pg of genomic DNA per reaction. A test performance study that included five laboratories in Germany resulted in 100% sensitivity and 97.7% specificity of the assay. Genomic regions, specific to common bunt (Tilletia caries and Tilletia laevis together) are also provided.

The fungal genus Tilletia includes a large number of plant pathogens of Poaceae. Only a few of those cause bunt of wheat, but these species can lead to significant yield losses in crop production worldwide. Due to quarantine regulations and specific disease control using appropriate seed treatments for the different disease agents, it is of high importance to distinguish Tilletia caries and Tilletia laevis as causal agents of common bunt accurately from Tilletia controversa, the causal agent of the dwarf bunt. Several studies have shown that matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is a useful tool to differentiate closely related fungal species. The aim of this study was to assess whether MALDI-TOF MS analysis is able to distinguish specimens of the three closely related pathogens T. caries, T. laevis, and T. controversa and whether it may constitute an alternative method to the morphology-based identification or germination tests. Spectral data are available via ProteomeXchange with identifier PXD030401. Spectra-based hierarchical cluster analysis (HCA) and discriminant analysis of principal components (DAPC) of the obtained mass spectra showed two main clusters. One cluster included specimens of T. controversa, whereas the second cluster comprised T. laevis and T. caries specimens. Even though main spectral profiles (MSPs) for species identification are missing, MALDI-TOF MS has proven to be a useful method for distinguishing between T. controversa and the two causal agents of common bunt, using direct analysis of teliospores, but was unable to separate T. caries and T. laevis species. Key points • MALDI-TOF MS was developed to classify Tilletia species causing bunt of wheat. • Best results were achieved when combining HCA and DAPC analysis. • The method resulted in an accuracy of 98.51% testing 67 Tilletia specimens. Graphical abstract

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ABSTRACT:Interest in the use of cover crops for soil health improvement and soil amelioration with organic matter is increasing. In addition to more general benefits, cover crops have also long been implemented for biological nematode suppression. For example for more than three decades, Heterodera schachtii has been managed by cultivating nematode-resistant brassica cover crops in Central Europe and more recently in North America. Production systems may require additional cover crop species when brassica cover crops are undesirable because of the potential increase of other soil-borne diseases, e.g., club root, that could negatively impact brassica cash crops. Alternative plant species fit for nematode suppression are needed. The tropical legume Crotalaria juncea has been successfully implemented for the suppression of Meloidogyne spp. in climates most typical to its growth requirements. This plant combines the benefits of nitrogen fixation with nematode antagonistic properties. Crotalaria juncea poses no consumption risk for ruminant animals in contrast to other species of this genus. The current study determined the C. juncea host status to five cyst nematodes, and examined the vegetation period requirements of the crop of exemplary two commercial cultivars. Both cultivars were resistant to the nematode species tested, and grew most vigorously when planted in May or June when soil temperatures were conducive for germination. This widely known nematode-suppressing plant can be grown in Central Europe. Further field studies will need to address the implementation of this plant species.

Tilletia caries and T. laevis, which are the causal agents of common bunt, as well as T. controversa, which causes dwarf bunt of wheat, threaten especially organic wheat farming. The three closely related fungal species differ in their teliospore morphology and partially in their physiology and infection biology. The gene content as well as intraspecies variation in these species and the genetic basis of their separation is unknown. We sequenced the genome of four T. caries, five T. controversa, and two T. laevis and extended this dataset with five publicly available ones. The genomes of the three species displayed microsynteny with up to 94.3% pairwise aligned regions excluding repetitive regions. The majority of functionally characterized genes involved in pathogenicity, life cycle, and infection of corn smut, Ustilago maydis, were found to be absent or poorly conserved in the draft genomes and the biosynthetic pathway for trimethylamine in Tilletia spp. could be different from bacteria. Overall, 75% of the identified protein-coding genes comprising 84% of the total predicted carbohydrate utilizing enzymes, 72.5% putatively secreted proteins, and 47.4% of effector-like proteins were conserved and shared across all 16 isolates. We predicted nine highly identical secondary metabolite biosynthesis gene clusters comprising in total 62 genes in all species and none were species-specific. Less than 0.1% of the protein-coding genes were species-specific and their function remained mostly unknown. Tilletia controversa had the highest intraspecies genetic variation, followed by T. caries and the lowest in T. laevis. Although the genomes of the three species are very similar, employing 241 single copy genes T. controversa was phylogenetically distinct from T. caries and T. laevis, however these two could not be resolved as individual monophyletic groups. This was in line with the genome-wide number of single nucleotide polymorphisms and small insertions and deletions. Despite the conspicuously different teliospore ornamentation of T. caries and T. laevis, a high degree of genomic identity and scarcity of species-specific genes indicate that the two species could be conspecific.

Table of Contents iii 3. Chapter 3. Development of a loop-mediated isothermal amplification assay for the detection of Tilletia controversa based on genome comparison .