BackgroundEarly blight, caused by the fungus Alternaria solani, occurs on potato mainly in the south-eastern part of Sweden, but also in other parts of the country. The aim of this study was to investigate the genetic diversity of A. solani populations from different potato growing regions in south-eastern Sweden using AFLP marker analysis. In addition, the cultured isolates were examined for substitutions in the gene encoding cytochrome b, associated with loss of sensitivity against QoI fungicides.ResultsNei's gene diversity index for the Swedish populations of A. solani revealed a gene diversity of up to 0.20. Also genetic differentiation was observed among populations of A. solani from different locations in south-eastern Sweden. The mitochondrial genotype of the isolates of A. solani was determined and both known genotypes, GI (genotype 1) and GII (genotype 2), were found among the isolates. The occurrence of the F129L substitution associated with a loss of sensitivity to strobilurins was confirmed among the GII isolates. In vitro conidial germination tests verified that isolates containing the F129L substitution had reduced sensitivity to azoxystrobin and, at a lower extent, to pyraclostrobin.ConclusionsGenetic diversity was relatively high among isolates of A. solani in south-eastern part of Sweden. F129L substitutions, leading to reduced sensitivity to strobilurins, have been established in field populations, which may have implications for the future efficacy of QoI fungicides.

Early blight, caused by the fungus Alternaria solani, is a common foliar disease in potato. Quinone outside inhibitor (QoIs) fungicides have commonly been used against A. solani. To avoid or delay development of fungicide resistance it is recommended to alternate or combine fungicides with different modes of action. Therefore, we compared two different fungicide programs against early blight in field trials and studied within season changes in the pathogen population. An untreated control was compared with treatments using azoxystrobin alone and with a program involving difenoconazole followed by boscalid and pyraclostrobin combined. Isolates of A. solani were collected during the growing season and changes in the population structure was investigated. We also screened for the amino acid substitution in the cytochrome b gene and investigated changes in sensitivity to azoxystrobin. Treatment with azoxystrobin alone did not improve disease control in 2014 when the disease pressure was high. However, lower severity of the disease was observed after combined use of difenoconazole, boscalid and pyraclostrobin. The efficacy of both fungicide treatments were similar during the field trial in 2017. Two mitochondrial genotypes (GI and GII) were found among isolates, where all isolates, except two, were GII. All GII isolates had the F129 L substitution while the two GI isolates were wild type. Population structure analysis and principal component analysis (PCA) of amplified fragment length polymorphisms (AFLP) data revealed within season changes in the A. solani populations in response to fungicide application. Isolates with the F129 L substitution had reduced sensitivity to azoxystrobin in vitro and their sensitivity tended to decrease with time.

The incidence of the pathogenic fungus Alternaria solani on potato during the years 2009 to 2016 was analysed and the distribution of the two genotypes of cytochrome b (genotype 1, GI and genotype 2, GII) determined. Potato leaflets with lesions resembling early blight were collected in August and September 2009-2016 in the southeastern part of Sweden and during 2010 and 2012-2014 in the central part of Sweden. In total, 2042 potato leaflet samples were analysed using diagnostic PCR methods. Alternaria solani was more commonly found in the samples collected later in the growing seasons. The occurrence of substitutions in the gene encoding cytochrome b, associated with loss of sensitivity towards strobilurins, was determined by sequencing a segment in the target gene in all confirmed A. solani samples. The proportion of A. solani GI decreased in relation to GII over the years. All except five GI samples were non-mutated. Genotype 2 was already present in the first year of sampling but only one sample possessed the F129 L substitution. A distinct shift from non-mutated GII to GII F129 L was observed between the season of 2011-2012 and a similar shift occurred in the pathogen population from GI to GII between 2012 and 2013. Identification of Alternaria alternata was performed during 2009-2013. The species was most often in co-occurrence with A. solani. Most of the samples investigated possessed the G143A substitution associated with strobilurin resistance.

Fungicide resistance has become a challenging problem in management of Septoria tritici blotch (STB), caused by Zymoseptoria tritici, the most destructive disease of winter wheat throughout western and northern Europe. To ensure the continued effectiveness of those fungicides currently used, it is essential to monitor the development and spread of such resistance in field populations of the pathogen. Since resistance to the key families of fungicides used for STB control (demethyalation inhibitors or azoles, succinate dehydrogenase inhibitors or SDHIs and Quinone outside Inhibitors or QoIs) is conferred through target-site mutations, the potential exists to monitor resistance through the molecular detection of alterations in the target site genes. As more efficient fungicides were developed and applied, the pathogen has continuously adapted through accumulating multiple target-site alterations. In order to accurately monitor these changes in field populations, it is therefore becoming increasingly important to completely sequence the targeted genes. Here we report the development of a PacBio assay that facilitates the multiplex amplification and long-read sequencing of the target gene(s) for the azole (CYP51), SDHI (Sdh B, C, and D), and QoI (cytochrome b) fungicides. The assay was developed and optimised using three Irish Z. tritici collections established in spring 2017, which capture the range of fungicide resistance present in modern European populations of Z. tritici. The sequences obtained through the PacBio assay were validated using traditional Sanger sequencing and in vitro sensitivity screenings. To further exploit the long-read and high throughput potential of PacBio sequencing, an additional nine housekeeping genes (act, BTUB, cal, cyp, EF1, GAPDH, hsp80-1, PKC, TFC1) were sequenced and used to provide comprehensive Z. tritici strain genotyping.

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