Development of a loop-mediated isothermal amplification assay for the detection of Tilletia controversa based on genome comparison

Sedaghatjoo, Somayyeh; Förster, Manuel; Niessen, Ludwig; Karlovský, Petr; Killermann, Berta; Maier, Wolfgang

doi:10.1038/s41598-021-91098-2

Cited by 11 publications

(10 citation statements)

References 61 publications

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“…The genomes of the three Tilletia species appeared to be largely syntenic as up to 94% of non-repetitive DNA regions could be aligned in a pairwise manner. Furthermore, we detected more than 98.7% average nucleotide identity in one-to-one aligned DNA regions, which was in general agreement with Nguyen et al ( 2019 ) and Sedaghatjoo et al ( 2021 ). The high genomic synteny observed between the three species can be explained by their close phylogenetic relationship (Carris et al 2007 ).…”

Section: Discussionsupporting

confidence: 92%

Comparative genomics reveals low levels of inter- and intraspecies diversity in the causal agents of dwarf and common bunt of wheat and hint at conspecificity of Tilletia caries and T. laevis

et al. 2022

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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.

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Section: Discussionsupporting

confidence: 92%

Comparative genomics reveals low levels of inter- and intraspecies diversity in the causal agents of dwarf and common bunt of wheat and hint at conspecificity of Tilletia caries and T. laevis

et al. 2022

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“…Two specimens, T. caries (AL15) and T. controversa (OL16), were selected in this study to implement and optimize the method. A single germinated teliospore of these Tilletia specimens was transferred to M-19 agar media (Trione 1964 ) and cultivated, maintained, and lyophilized as described by Sedaghatjoo et al ( 2021a ) to obtain enough biomass for MALDI-TOF MS analyses. Lyophilized mycelium of specimens AL15 and OL16 were stored at the Federal Research Centre for Cultivated Plants (JKI, Braunschweig, Germany).…”

Section: Methodsmentioning

confidence: 99%

Discrimination of Tilletia controversa from the T. caries/T. laevis complex by MALDI-TOF MS analysis of teliospores

Förster

Sedaghatjoo

Maier

et al. 2022

Appl Microbiol Biotechnol

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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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“…Although the full programme includes automatic design of PCR primers, only the fucs (find unique core sequences) entry point was used in the current study, which provides multiple output sequences specific to the respectively queried genomes. RUCS has been applied previously to determine unique sequences for LAMP primer design by Sedaghatjoo et al (2021). At the time of this study, for the majority of the GTD‐related fungi, including the three targeted species FMED, PCH and PMI, only one genome per species was available in the GenBank database, which increases the risk of retrieving output sequences with a high degree of intra‐specific variation.…”

Section: Discussionmentioning

confidence: 99%

Detection of Esca‐associated fungi in grapevine trunks using loop‐mediated isothermal amplification (LAMP) assays

Marek,

Eberl,

Otba

et al. 2023

Annals of Applied Biology

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Esca is a grapevine trunk disease (GTD) that is caused by filamentous fungi. It is responsible for considerable economic losses in viniculture on a global scale. Despite many unknown factors contributing to the development of symptoms in affected plants, Phaeoacremonium minimum (PMI), Phaeomoniella chlamydospora (PCH), and Fomitiporia mediterranea (FMED) are generally considered as the main causative fungal species. Early detection and specific identification of these pathogens therefore play an important role in disease control and evaluation of suitable countermeasures. In this study, loop‐mediated isothermal amplification (LAMP) assays were developed for each of the three pathogens. A genome‐based approach was applied for detection and selection of unique target DNA sequences. The designed primer sets showed overall good specificities, with some observed cross‐reactions towards closely related Phaeoacremonium species for the PMI primer set. The developed assays had detection limits of 100 pg (FMED, PMI) and 1 pg (PCH) per reaction (corresponding to 1,460 (FMED); 1,950 (PMI); 342 (PCH) genome copies per reaction). The application of the assays to field samples was demonstrated by testing individual infected grapevine trunks from two European viticultural regions using crude DNA obtained in a rapid sample preparation step. LAMP assay results matched those of PCR following a conventional DNA extraction protocol. The study showed that LAMP‐based rapid molecular detection of major Esca agents can serve as a useful tool for further research and surveillance of a highly devastating grapevine disease.The application of computer‐based whole genome comparison between target and non‐target species for the identification of unique target sequences as the basis for LAMP (or PCR) primer design was demonstrated to be a useful approach in species for which scarce sequence information is available. Moreover, the developed method for rapid DNA preparation from grapevine trunks may potentially be adapted to the DNA‐based detection also of other fungal species that cause grapevine trunk diseases.This article is protected by copyright. All rights reserved.

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Development of a loop-mediated isothermal amplification assay for the detection of Tilletia controversa based on genome comparison

Cited by 11 publications

References 61 publications

Comparative genomics reveals low levels of inter- and intraspecies diversity in the causal agents of dwarf and common bunt of wheat and hint at conspecificity of Tilletia caries and T. laevis

Comparative genomics reveals low levels of inter- and intraspecies diversity in the causal agents of dwarf and common bunt of wheat and hint at conspecificity of Tilletia caries and T. laevis

Discrimination of Tilletia controversa from the T. caries/T. laevis complex by MALDI-TOF MS analysis of teliospores

Detection of Esca‐associated fungi in grapevine trunks using loop‐mediated isothermal amplification (LAMP) assays

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