Whole-genome sequencing elucidates the species-wide diversity and evolution of fungicide resistance in the early blight pathogen Alternaria solani.

Early blight of potato is caused by the fungal pathogen Alternaria solani and is an increasing problem worldwide. The primary strategy to control the disease is applying fungicides such as succinate dehydrogenase inhibitors (SDHI). SDHI-resistant strains, showing reduced sensitivity to treatments, appeared in Germany in 2013, shortly after the introduction of SDHIs. Two primary mutations in the SDH complex (SdhB-H278Y and SdhC-H134R) have been frequently found throughout Europe. How these resistances arose and spread, and whether they are linked to other genomic features, remains unknown. For this project, we performed whole-genome sequencing for 48 A. solani isolates from potato fields across Europe to better characterize the pathogen's genetic diversity in general and understand the development and spread of the genetic mutations that lead to SDHI resistance. The isolates can be grouped into seven genotypes. These genotypes do not show a geographical pattern but appear spread throughout Europe. We found clear evidence for recombination on the genome, and the observed admixtures might indicate a higher adaptive potential of the fungus than previously thought. Yet, we cannot link the observed recombination events to different Sdh mutations. The same Sdh mutations appear in different, non-admixed genetic backgrounds; therefore, we conclude they arose independently. Our research gives insights into the genetic diversity of A. solani on a genome level. The mixed occurrence of different genotypes, apparent admixture in the populations, and evidence for recombination indicate higher genomic complexity than anticipated. The conclusion that SDHI tolerance arose multiple times independently has important implications for future fungicide resistance management strategies. These should not solely focus on preventing the spread of isolates between locations but also on limiting population size and the selective pressure posed by fungicides in a given field to avoid the rise of new mutations in other genetic backgrounds.

Quantitation of Toxic Steroidal Glycoalkaloids and Newly Identified Saponins in Post-Harvest Light-Stressed Potato (Solanum tuberosum L.) Varieties.

Although domesticated potatoes contain a large variety of steroidal glycoalkaloids (SGAs) and saponins, in the past, many research projects mainly focused on the two major SGAs, α-solanine and α-chaconine. This study investigates the quantitative changes, induced by post-harvest LED light exposure, of six SGAs and four saponins in 12 potato cultivars at three different time points (1, 7, and 16 days), by using ultra-performance liquid chromatography tandem mass spectrometry. Altogether, SGA contents of 3.0-17.1 mg/100 g fresh weight (FW) could be observed in the analyzed tubers with potato varieties highly exceeding the newly discussed safety limit of 10 mg/100 g. The overall contents of 0.1-5.4 mg/100 g FW of the so far barely studied saponins, like protoneodioscin or barogenin-solatrioside, highly differed between the assayed potato cultivars. Furthermore, cultivar-specific regulations of SGAs and saponins could be observed due to light exposure.

Steroidal Saponins─New Sources to Develop Potato (Solanum tuberosum L.) Genotypes Resistant against Certain Phytophthora infestans Strains.

Plant pathogens such as Phytophthora infestans that caused the Irish Potato Famine continue to threaten local and global food security. Genetic and chemical plant protection measures are often overcome by adaptation of pathogen population structures. Therefore, there is a constant demand for new, consumer- and environment-friendly plant protection strategies. Metabolic alterations induced by P. infestans in the foliage and tubers of six different potato cultivars were investigated. Using a combination of untargeted metabolomics, isolation techniques, and structure elucidation by MS and 1D/2D-NMR experiments, five steroidal glycoalkaloids, five oxylipins, and four steroidal saponins were identified. As the steroidal saponins showed antioomycete but no hemolytic activity, they may thus be considered as probably safe target substances for enrichment in breeding programs for disease resistance and as chemical lead structures for the production of nature-derived synthetic antioomycetes.

Biosynthesis of α-solanine and α-chaconine in potato leaves (Solanum tuberosum L.) - A 13CO2 study.

α-Solanine and α-chaconine are the major glycoalkaloids (SGAs) in potatoes, but up to now the biosynthesis of these saponins is not fully understood. In planta13CO2 labeling experiments monitored by nuclear magnetic resonance spectroscopy (NMR) and high-resolution mass spectrometry (HRMS) unraveled the SGA biosynthetic pathways from CO2 photosynthates via early precursors to the SGAs. After a pulse of ~ 700 ppm 13CO2 for four hours, followed by a chase period for seven days, specific 13C-distributions were detected in SGAs from the leaves of the labeled plant. NMR analysis determined the positional 13C-enrichments in α-solanine and α-chaconine characterized by 13C2-pairs in their aglycones. These patterns were in perfect agreement with a mevalonate-dependent biosynthesis of the isopentenyl diphosphate and dimethylallyl diphosphate precursors. The 13C-distributions also suggested cyclization of the 2,3-oxidosqualene precursor into the solanidine aglycone backbone involving a non-stereoselective hydroxylation step of the sterol a mixture of 25S-/25R-epimers of the SGAs.

Occurrence of sdh Mutations in German Alternaria solani Isolates and Potential Impact on Boscalid Sensitivity In Vitro, in the Greenhouse, and in the Field.

The fungus Alternaria solani is the main pathogen causing early blight on potatoes (Solanum tuberosum L.). An increase in the development of resistance to the succinate dehydrogenase inhibitor (SDHI) boscalid, one of the main active ingredients for the control of early blight, has been reported. For this study, monitoring data from Germany were collected between 2013 and 2016 and an increase in the occurrence of A. solani succinate dehydrogenase (SDH) mutant isolates was observed. In addition to the known point mutations in sdh complex II, a new mutation in subunit C was found in German isolates (SdhC-H134Q). SDHI fungicide sensitivity testing was performed in the laboratory, greenhouse, and field. Reduced boscalid sensitivity was shown for mutant isolates (SdhB-H278Y and SdhC-H134R) both in vitro and in vivo. In addition, field trials with artificial inoculation were performed in 2016 and 2017. In both years, fungicide efficacy was significantly reduced after mutant inoculation compared with wild-type inoculation.

Influence of inoculum and climatic factors on the severity of Fusarium head blight in German spring and winter barley.

Fusarium head blight (FHB) of small cereals is a disease of global importance with regard to economic losses and mycotoxin contamination harmful to human and animal health. In Germany, FHB is predominantly associated with wheat and F. graminearum is recognised as the major causal agent of the disease, but little is known about FHB of barley. Monitoring of the natural occurrence of FHB on Bavarian barley revealed differences for individual Fusarium spp. in incidence and severity of grain infection between years and between spring and winter barley. Parallel measurement of fungal DNA content in grain and mycotoxin content suggested the importance of F. graminearum in winter barley and of F. langsethiae in spring barley for FHB. The infection success of these two species was associated with certain weather conditions and barley flowering time. Inoculation experiments in the field revealed different effects of five Fusarium spp. on symptom formation, grain yield and mycotoxin production. A significant association between fungal infection of grain and mycotoxin content was observed following natural or artificial infection with the type B trichothecene producer F. culmorum, but not with the type A trichothecene-producing species F. langsethiae and F. sporotrichioides. Trichothecene type A toxin contamination also occurred in the absence of significant damage to grain and did not necessarily promote fungal colonisation.