Mascha Hoffmeister

Alternaria solani, the causal agent of early blight, may dramatically reduce yield in potato production on a global scale. Quinone outside inhibitors (QoIs), succinate dehydrogenase inhibitors (SDHIs), and demethylation inhibitors (DMIs) represent three major fungicide groups that are used to control early blight on potato. Early blight has acquired adaptation to QoIs and SDHIs, making DMI fungicides the prime class of chemistries in early blight control. We investigated the current DMI sensitivity status of field isolates of A. solani . The combination of two mutations leading to the amino acid exchanges L143F and G446S was detected in the DMI target gene CYP51 in some isolates. Both mutations occurred only in combination and were linked to reduced DMI sensitivity. To test for the role of these mutations in DMI adaptation, we generated strains with single L143F or G446S mutations, as well as L143F + G446S double mutations by targeted mutagenesis. Single mutations caused slightly lower DMI sensitivity; this was more pronounced with the mutation combination L143F + G446S. Importantly, in vivo infection assays performed in the greenhouse revealed significantly lower competitiveness of the double mutants compared to wild-type isolates, indicating that the L143F + G446S haplotype is connected with lower fitness. However, CYP51 haplotypes do not differ in spore morphology or quantity. Furthermore, CYP51 haplotypes generated by targeted mutagenesis are all able to infect tomato plants in the greenhouse. IMPORTANCE: This study investigates the mutations in the CYP51 gene of the fungus Alternaria solani , which causes early blight. By understanding the effect of these mutations, researchers can better manage fungicide resistance, ensuring effective disease control. This study provides biotechnological methods suitable for investigating single-site mutations and their individual effect on fungicide sensitivity.

Cercospora leaf spot disease (CLS) caused by the ascomycete Cercospora beticola is the most widespread fungal leaf disease in sugar beet. Fungicides of two active ingredient classes, quinone-outside inhibitors (QoIs) and demethylation inhibitors (DMIs), were important tools for CLS control. Over the years, C. beticola has become resistant to QoIs and a sensitivity shift has been reported for DMIs. In this study the mechanisms causing variation in DMI sensitivity in C. beticola isolates from Europe were analyzed. The CYP51 mutations I387M, Y464S, and L144F were detected in many isolates and most isolates carried the L144F in combination with mutation I309T. Furthermore, single isolates with other mutation combinations have been found. Wildtype isolates were found in low frequency in all European countries. Isolates that contained L144F showed higher EC₅₀ values than those without L144F. Ranges of EC₅₀ values of different CYP51 haplotypes were overlapping, an indication that other resistance mechanisms are present. Mutation L144F is more frequently encoded by codon TTC (96%) than by TTT (4%), and the usage of codon TTC was correlated with increased EC₅₀ values, this being more pronounced for difenoconazole than for mefentrifluconazole. In addition, it could be observed that the usage of codon GAG for E at amino acid position 170, instead of GAA, was more frequently found in isolates with a higher adaptation compared with haplotypes that did not contain L144F. Overall, GAA was present in 67% of all isolates and GAG in 33%, with an unequal distribution within the haplotypes. These data indicate that target site mutations, especially L144F haplotypes, influence DMI sensitivity, and that in L144F haplotypes, L144F codon usage might be responsible for variations within L144F haplotypes. The codon usage for E170 may influence sensitivity and increase EC₅₀ variation of wildtype isolates and isolates with “weak” mutations, but not in L144F haplotypes.

Succinate dehydrogenase inhibitors (SDHIs) are an important group of fungicides (FRAC Group 7) for the control of Venturia inaequalis. Mechanisms which lead to reduced SDHI sensitivity in fungi are based on mutations in the SDH-subunits B, C, or D of the target gene. Monitoring studies have shown that most of the European population is still sensitive to SDHIs. Single cases of SDHI resistance have been detected shortly after market introduction although mainly at trial sites. The underlying resistance mechanisms have been identified as target site mutations (B-T258I, C-N85S and C-H151R). However, these were single findings which did not increase in frequency or even disappeared when the same field was analyzed one or more seasons later. This is in contrast to findings on other resistance cases which are stable over several seasons even without selection pressure, such as QoI and benzimidazole resistance. High frequencies of target site mutations for QoIs and benzimidazoles (G143A and E198A, respectively) have been detected in a 2022 survey, whereas no SDHI target site mutation exceeded the detection limit of our molecular detection assay in any sample.

BIO Web of Conferences, open access proceedings in biology, life sciences and health

Colletotrichum lentis is an important pathogen of lentils in Canada causing the disease anthracnose. Fungicide applications are an important tool for the control of this disease and, historically, QoI fungicides have been used intensively. Reduced field efficacy of QoI fungicides on C. lentis has been reported recently and could be linked to the presence of QoI resistant isolates. In this study, the cytochrome b gene of C. lentis was sequenced, and the mutation G143A was identified as the causal resistance mechanism. Furthermore, a sensitive and reliable molecular biological assay was developed for the quantitative detection of the G143A mutation by pyrosequencing in field samples.

Downy mildew of common sage (Salvia officinalis), caused by Peronospora salviae-officinalis, has become a serious problem in sage production worldwide. The effect of temperature was determined for conidia germination and disease development. In vitro, conidial germination rate was highest at temperatures between 10 °C and 20 °C and was strongly reduced at temperatures above 25, but conidia were also able to germinate at 2 °C. Temperatures between 15 and 20 °C were most favourable for infection and disease progress in infection experiments in climate chambers, with highest sporulating leaf area observed at 15 and 20 °C, and highest symptomatic leaf area at 20 °C. P. salviae-officinalis is still able to infect sage plants at 5 °C, but sporulation was only observed at higher temperatures. Oospores developed 14 days after inoculation at 15 °C, and 8 days after inoculation at 20 and 25 °C. The infection trials also showed that dark incubation is not a prerequisite for successful infection of sage. Furthermore, P. salviae-officinalis needs two events of leaf wetness or high humidity to complete its asexual life cycle. First, a leaf wetness event of at least three hours is needed for conidial germination and penetration of the host. Second, high humidity of at least 90% is needed at the end of the infection cycle for sporulation. Seed washing revealed the presence of P. salviae-officinalis oospores in seed lots of sage suggesting that infested seeds might play a major role in the fast spread of sage downy mildew, which is an important finding for phytosanitary or quarantine measures.