In this study, we analysed the microbial communities associated with flowers and leaves of 50 wild flowering plants from different plant families, and found statistically significant impact of biotic and abiotic factors on their structure. We observed distinct microbial compositions between flowers and leaves, with leaves harbouring higher microbial diversity than flowers. We showed that sampling location had significant effect on diversity and community composition. Additionally, soil type had a significant impact on microbial community composition but not on diversity. Altitude showed a significant correlation with increased fungal species richness on flowers and elevated diversity on both leaves and flowers. Family identity effects were explored in a subset of seven plant families, revealing notable influences.
The difference between flowers and leaves in terms of microbial diversity, abundance and community composition
Our results showed that despite of the variations in sampling location, altitude, soil type, and plant species, leaves harboured higher bacterial and fungal diversity compared to flowers. The disparity in microbial diversity between leaves and flowers could be attributed to the shorter lifespan of flowers compared to leaves coupled with the nutrient-rich nature of flowers. Flowers, once open, represent a new, resource-rich niche, where primary colonizers are expected to be R strategists (fast growing microorganisms) due to the limited competition and surplus of resources. The dominance of these faster-growing species together with the short lifespan of flowers, may limit the establishment of diverse microbial communities. In contrast, leaves provides a more stable and prolonged habitat for new microbial colonization. In fact, genera such as Pseudomonas and Pantoea, and Metschnikowia were detected at significantly higher abundant on flowers than on leaves; all of which are recongized as r-strategist genus [56–59]. Another explanation to lower diversity in flower could be due the presence of some floral volatiles such as terpenes, exhibit antibacterial and antifungal properties [60], which may limit flower colonizers to certain microbial taxa that are able to tolerate or metabolize these compounds. Our study revealed distinct microbial composition in leaves and flowers, with significant differences observed in both bacterial and fungal community composition. These differences may be attributed to the unique characteristics of leaves and flowers, including their varying nutritional content, metabolite profiles, and susceptibility to pathogen attack. For instance, flowers often accumulate higher concentrations of secondary metabolites like flavonoids compared to leaves [61], which could potentially influence the microbial composition. Additionally, the susceptibility of flowers to pathogen attack due to their rich nutrient and moisture content, and high frequency of insect visitors [62], may also contribute to the observed differences in microbial composition.
The effect of geographical location on bacterial and fungal communities
Our study revealed distinct responses of fungal and bacterial communities to geographical location. Fungal diversity in both flowers and leaves, and bacterial species richness in flowers, were significantly influenced by location. Bacterial and fungal abundance remained unaffected, while community composition varied across locations. This highlights the potential sensitivity of fungal communities to environmental variations across the landscape. Consistent with findings by Coleine et al. (2018), fungal communities are highly responsive to fluctuations in temperature, humidity, and microclimates. These variations could explain the observed location-specific diversity patterns. For instance, thermophilic fungi at warmer locations or moisture-tolerant fungi in areas with higher humidity might shape unique communities across the landscapes. Additionally, Xiao et al. (2021) suggest that temperature fluctuations can enhance stochasticity within microbial communities, potentially contributing to the observed difference in fungal communities. While bacterial species richness in flowers responded to location, overall diversity patterns remained less pronounced, possibly due to their superior dispersal mechanisms allowing them to colonize diverse locations and potentially exhibit less location-specific diversity patterns [65]. The absence of detailed measurements of specific environmental factors like temperature and soil pH limits conclusive attribution of diversity patterns to specific drivers. The observed abundance pattern suggests that changes in diversity may not necessarily translate directly to changes in abundance. This observation aligns with the regional species pool theory, which posits that local communities are subsets of a larger regional pool, shaped by environmental filters [21]. In our study, the relatively limited environmental heterogeneity within our Carinthia sampling area compared to the broader region might have restricted the influence of location on bacterial diversity, potentially explaining the lack of significant abundance changes.
The effect of soil type on bacterial and fungal communities
Although soil type (carbonate vs. silicate) did not have an effect on fungal and bacterial diversity and abundance, it significantly influence bacterial and fungal community composition. In accordance with established knowledge about plant communities [11], we expected a higher microbial diversity in calcareous soil too. One potential factor might be considered is phosphorus (P) availability, known to be crucial for both plants and microbes [66]. Carbonate and silicate soils have contrasting P profiles: high pH and low P in carbonate vs. neutral pH and moderate P in silicate [67]. These inherent differences could influence microbial communities through mechanisms like differential P utilization by specific taxa or indirect effects on nutrient cycling and plant-microbe interactions favouring P acquisition strategies adapted to distinct P availability. Further research directly linking soil type differences to specific P-related effects on observed microbial composition is needed. Furthermore, studies like Sun et al. (2016) demonstrated how organic matter type could influence soil fungal communities. Similarly, we hypothesize that distinct plant communities associated with carbonate and silicate soils might contribute varying organic matter compositions, indirectly affecting microbial communities on leaves and flowers through resource availability and plant-microbe interactions. While our study relied on geological maps for soil type classification, future investigations directly analyzing soil properties could provide deeper insights into the influence of soil type on these microbial communities. These findings highlight the importance of considering the broader ecological context, including soil characteristics.
The effect of altitude on bacterial and fungal communities
Our study revealed distinct responses for bacterial and fungal communities in flowers to the increasing altitude. While fungal diversity significantly increased with altitude, bacterial diversity was not affected. However, both communities displayed altered community composition and a slight decline in abundance across the altitudinal gradient. Lower temperatures and decreased oxygen availability at higher elevations might favour specific psychrophilic and hypoxia-adapted fungal communities [69] potentially leading to increased fungal richness and diversity. Additionally, the diverse abiotic conditions along altitudinal gradients might create more niches for various fungal species to occupy [70]. While bacterial species richness remained stable, we observed shifts in community composition and a slight decline in abundance. Lindow & Brandl (2003) suggest carbon-source availability as a major constraint for bacterial growth in the phyllosphere. The observed decline in abundance for both fungi and bacteria could be attributed to combined effects of temperature, UV radiation [72], and potential resource limitations at higher altitudes [71]. Interestingly, only fungal Shannon diversity increased in leaves with altitude. This pattern aligns with the mechanisms mentioned for flowers, suggesting similar selection pressures and niche availability favouring specific fungal groups. Resource limitations at higher altitudes, particularly nitrogen availability [73], could explain the lack of change in overall fungal and bacterial diversity despite potential functional adaptations. The observed changes in community composition for both fungi and bacteria in leaves aligns with the findings by Sundqvist et al. (2013) who demonstrated that altitudinal gradients significantly impact soil bacterial composition through associated changes in temperature and precipitation. As leaves directly interact with this abiotic environment, similar selection pressures favouring specific adaptations to these changing conditions might explain the observed shifts in leaf bacterial communities.
Our analysis of the fungal core microbiome defined as classes present in at least 70% of sampled flowers and leaves across altitudes reveals distinct patterns (Fig. 6). Consistent with the temperature selection hypothesis, we observed a decline in Dothideomycetes and an increase in Leotiomycetes observed on both flowers and leaves. Dothideomycetes are often associated with warmer climates, limits their suitability at higher elevations, while Leotiomycetes, particularly the Helotiales order, harbor psychrophilic fungi found in alpine glaciers [23]. While temperature plays a crucial role, other factors might influence the core microbiome. Plant nitrogen availability linked to Dothideomycete presence [75], could be influenced by altitude. Additionally, Dothideomycetes are known for their tolerance to harsh conditions [76], suggesting potential adaptations beyond just temperature.
The bacterial core microbiota also displays notable patterns (Fig. 6). The dominance of Gammaproteobacteria on flowers and Alphaproteobacteria on leaves suggests niche specialization based on plant organ function. Gammaproteobacteria, known for their roles in plant growth promotion and pathogen protection [77] align with their prevalence in the flower microenvironment that is crucial for reproduction and seed development. Alphaproteobacteria, with their diverse metabolic capabilities [78], efficiently utilize and cycle nutrients on leaves. Their ability to maintain activity under environmental fluctuations is potentially advantageous in the leaf environment [79]. Interestingly, both bacterial classes exhibited contrasting trends with altitude. Alphaproteobacteria decline on leaves, potentially due to their lower tolerance for colder temperatures at higher altitudes [80], putting them at a disadvantage compared to psychrophilic Gammaproteobacteria [81], whose increased abundance aligns with this potential advantage. Additionally, Alphaproteobacteria might be sensitive to nitrogen limitations known to occur at higher altitudes [73], further contribute to their decline.
The effect of plant family identity on microbial diversity, abundance and community composition
Our study observed a significant bacterial species richness and a trend towards fungal Shannon diversity on flowers. In contrast, leaves only showed significant effect in bacterial and fungal abundance, but no changes in diversity and community composition. This aligns with previous findings by Redford et al. (2010) and Knief et al. (2010), suggesting that different plant families create distinct niches that favour specific microbial taxa. The observed diversity on flowers could be attributed to floral specificity where plant families likely possesses unique phytochemical profiles and nutrient content in their exudates [84, 85]. This aligns with studies demonstrating how host genotype shapes exudate profiles [86, 87], suggesting a link between family identity, exudate composition, and microbial communities. Our findings suggest that, lowest bacterial abundance in Rosaceae coincides with their known richness in hydrolysable tannins with antimicrobial and antifungal properties [88, 89]. The presence of these tannins in Rosaceae exudates could be suppressing microbial growth, contributing to their lower abundance compared to other families. Additionally, the presence of cyanogenic glycosides [90] might create a less favourable environment for some bacterial taxa [91], contributing to lower abundance. The observed abundance patterns across families with Asteraceae exhibiting the highest bacterial abundance on both leaves and flowers further highlights the potential influence of differential exudate profiles. Further studies characterizing specific phytochemical profiles and their antimicrobial properties across families are crucial to confirm this mechanism. In contrast to flowers, leaves exhibited lack of diversity and community composition despite the change in microbial abundance with family identity. This could be due to the higher baseline diversity and stronger environmental pressures experienced by leaves [34] might mask subtle diversity effects driven by family identity. This aligns with the observation that host genotype effects and physiological traits may not always align with plant phylogeny [39], potentially explaining why family identity, while influencing abundance, had a weaker effect on community composition compared to other studies [31, 33]. Our findings also agree with Junker et al. (2011), suggesting that microbial communities might be more organ-specific than species-specific, which is further supported by the stronger effect of family identity on flowers compared to leaves in our study. While family-level analysis provides valuable insights, it is crucial to acknowledge phenotypic variations within species [93] and genotype-specific effects on microbial communities [94] can exist within families. Therefore, investigating species-level data within each family could provide a more clear understanding. Additionally, the regional species pool could also indirectly influence observed patterns [95, 96]. Differences in pollen and nectar availability across habitats could contribute to the observed abundance patterns, as suggested by Russell et al. (2019).