Browsing by Subject "Molecular phylogeny"

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    Species of the Diaporthe/Phomopsis Complex (DPC) in European soybean and establishment of quadruplex Real-Time PCR for diagnosis
    (2022) Hosseini, Behnoush; Vögele, Ralf
    Diaporthe seed decay is among the most disruptive soybean diseases around the world, which cause significant yield losses and affect soybean quality. Different Diaporthe species cause this disease, while Diaporthe longicolla is considered the main causal agent. The species of this fungal complex (genus Diaporthe is also called the Diaporthe/Phomopsis Complex / DPC) have to be accurately identified for epidemiological studies of the disease and for optimal control measures. To identify the major causal agents of seed decay in Europe, DPC-damaged soybean seeds of various cultivars, that were collected from different fields in Germany, France, and Austria were tested by seed plating. 32 Diaporthe isolates could be obtained. The isolates were morphologically identified by the colors and shape of the colony, conidia dimensions, and by whether pycnidia with α- and/or β-conidia or perithecia with ascospores are formed. To corroborate morphological identification, sequences of the internal transcribed spacer (ITS), translation elongation factor 1-α (TEF1), and beta-tubulin (TUB) sequences were obtained. From the results of both morphological and molecular analyses it became clear that all isolates belong to one of the four species D. longicolla, D. caulivora, D. eres, and D. novem. The pathogenicity of all strains on soybean was tested. Molecular phylogenies were calculated and based on the above results updated species descriptions were created. This study identified these four species as the main Diaporthe pathogens for soybean in central Europe. A sensitive and accurate method for quick detection of these pathogens was developed based on multiplex real-time PCR. Specific TaqMan primer-probe sets for the four species were designed based on TEF1 sequences. The primer-probe sets were tested for specificity and efficiency using PCR products and genomic DNA from the four Diaporthe species and several other soybean pathogens. These primer-probe sets reliably distinguish the different species and they can be used to detect them in the same reaction by quadruplex real-time PCR. DNA from different soybean plant materials including healthy and infected seeds or seed coats, stems, and leaves was used to test the quadruplex real-time PCR assay. Application of the assay was extended to quantify the pathogens. Standard curves for the four species were created from serial dilutions of genomic DNA diluted with DNA from soybean tissue. An additional standard curve was created from serial dilutions of soybean DNA diluted with ddH2O. To gain the ratio of fungal DNA per plant DNA (ng/ng), DNA samples from soybean tissues can now be examined in the new assay and a parallel SYBR® Green-based real-time PCR. The assay was first applied to six soybean seed lots with putative Diaporthe contamination. In all seed lots seeds contaminated with Diaporthe species and even some seeds infected with more than one Diaporthe species were found, while other seeds were free of the pathogens. The load of fungal biomass varies strongly between individual seeds.