PUB - Publikationen an der Universität Bielefeld

In recent decades, farmers had to cope with changing climatic conditions, competition for arable land and the expectation to produce high-yielding and high-quality products. To manage these difficulties, farmers use high-efficient plants and perform pest management, including the use of fungicides. These Fungicides contain one or more active ingredients on which they are categorised, regulated, and approved for organic or conventional farming. Despite the usefulness of fungicides, they have been shown to accumulate in the environment and cause direct harm to non-target organisms. When fungicides are applied to flowers, nectar-inhabiting microbes get in direct contact with the substance and contaminate floral rewards for pollinators, thus directly impairing the pollinators’ health. Also, the treated plants can be affected by the fungicide, changing their metabolism and/or photosynthesis. It can therefore be expected that the plant-pollinator interaction may be altered due to fungicide-elicited changes in the plants. Moreover, fungicides may impact taste-relevant metabolites, thus impacting the fruit quality. However, there is a lack of studies which investigate these fungicide impacts, as existing studies often focus on individual impacts instead of considering the interrelationships between all associated factors.

This thesis aimed to investigate the effects of two fungicides on fruit traits and interactions with pollinators. To do this, I treated strawberry plants (Rosaceae: *Fragaria* × *ananassa*) either with a fungicide approved for organic farming (Cuprozin® progress) or one for conventional farming (SWITCH®) and compared them with untreated plants. On those plants, I investigated the influence of these fungicides on different flower attributes, the foraging behaviour of floral visitors in the greenhouse and field, and taste-relevant metabolites in the fruit.

In **Chapter II**, I focused on the influence of two fungicides on attractiveness-related floral attributes and the concomitant effect on bumblebees (Apidae: *Bombus terrestris*). Plant-pollinator interactions are highly complex and shaped by various factors, including fungicide treatments. To better understand these interactions, I analysed floral volatiles, pollen weight and pollen protein from greenhouse-grown plants and observed the foraging behaviour of bumblebees towards these flowers, under controlled conditions. Additionally, I analysed the pollen viability and nectar fungi composition from treated field-grown plants. Cuprozin® progress resulted in a lower floral volatile emission and a slightly lower pollen protein content. However, this did not change the latency until the first flower visit of bumblebees but did reduce the overall flower visit frequency. In contrast, SWITCH® increased the floral volatile emission and delayed the first flower visit by bumblebees. These findings indicate that depending on the active ingredient, fungicides can alter the attraction towards a pollinator species. Additionally, flowers of untreated plants were visited more often than those of fungicide-treated plants. These changed behaviours could be caused by the reduced pollen quality or an off taste, triggered by the fungicides.

To further investigate the potential impacts of fungicides on flower visitors, I assessed their abundance on treated plants, in the field, in **Chapter III**. On those plants, I monitored the composition and diversity of flower visitors in 2021 and 2022. In 2022, I separately related the abundance of honeybees (Apidae: *Apis mellifera*) and other taxa to the number of open flowers. The composition of flower visitors differed in dependence of the fungicide treatment in both years. However, this did not impact the diversity or visitation frequency of the total number of flower visitors. Although non-*A. mellifera* visits were significantly correlated with the number of fungicide-treated open flowers, and the flower visits of honeybees were not affected by the fungicides. Both fungicides had no impact on the number of open flowers, but the amount of open flowers promoted the insect visitation. Both, the different handling of the plants during the fungicide treatment and the different time intervals from the last fungicide treatment to the observation start, may explain the differences in the composition of potential pollinators. These two factors may have also influenced nectar microbes, leading to changes in the foraging behaviour of visitors.

In **Chapter IV**, I investigated the effects of fungicides on the fruit metabolism from three strawberry “types” (two cultivars and the wild strawberry), to assess the potential impact on the fruit taste. At the harvest, I determined the potential yield and mean fruit weight per plant. Using different analytic platforms, I analysed fruit extracts for their primary metabolites: total phenolics by photometric quantification, amino acids by HPLC, sugars and (in)organic acids by GC-MS, and their specialised (or secondary) metabolites i.e., aroma volatiles by thermal desorption (TD-GC-MS). The potential yield and mean fruit weight per plant were not impacted by the fungicides but differed significantly between the three strawberry types. In contrast, the total phenolic content in fruits was affected by the interaction of the fungicide treatment and strawberry type, while the leaf content only differed between strawberry types. In addition, the total amino acid concentration and the content of the main sugars were significantly affected by the fungicide treatment. Furthermore, the sum of all aroma volatile metabolites was affected by the interaction of the fungicide treatment and strawberry type. However, the response of individual metabolites depended on the used fungicide and plant type. Given that many of the aroma volatile metabolites derive from primary metabolites, fungicides-induced changes in the primary metabolites may impact the fruit taste and may influence the quality of the fruits.

Finally, in **Chapter V**, I conducted a review of studies in the Web of Science™ database which handled the topic of fungicide effects on floral scent, pollen attributes, pollination, and fruit metabolism. Subsequently, I compared my studies from **Chapters II-IV** with the collected literature of my search. The review highlights the need for more research on various active ingredients and plant species, as the fungicide-induced responses are strongly dependent on the used active ingredients and the plant species.

In conclusion, this thesis highlights that fungicides affect treated plants on various levels. By altering different floral attributes, fungicides can alter the attractiveness of flowers towards floral visitors and may affect pollination efficiency. The impact on pollination, together with the fungicide-induced changes in fruit metabolism, affects the quality and potentially the taste of fruits. Future research should further address the potential impacts of fungicide treatments on the foraging behaviour of pollinators, to disentangle the complexity of visual and olfactory cues. This could be tested, by analysing the UV reflectance of treated plants and by pollinator choice tests, with floral extracts of the treated flowers (see discussion in **Chapter V**). Moreover, taste tests should be carried out to further assess the fungicide effects on the strawberry taste.