GENERAL DISCUSSION AND PERSPECTIVES
With this work, I present one of the first comprehensive studies dealing with the bacterial community composition (BCC) on submerged macrophytes in freshwater and brackish water. My aim was to expand the scarce knowledge on heterotrophic biofilms on natural aquatic surfaces, especially submerged macrophytes, and the factors determining the BCC. With cultivation-dependent and -independent techniques (DGGE, FISH, clone library), I described the BCC on different macrophytes in a freshwater (Lake Constance) and a brackish water habitat (Schaproder Bodden). We investigated the influence of habitat, plant species and plant part (age), plant chemistry and environmental factors (conductivity, water temperature, water level, pH) on the BCC.
In almost all samples analyzed, we found a BCC dominated by bacteria of the CFB group with fluctuating abundance, followed by alpha- and betaproteobacteria. Only on Potamogeton perfoliatus, the abundance of gammaproteobacteria was slightly higher compared to the other substrates. Planctomycetes were most abundant on brackish water plants and only seldom occurred on freshwater plants. Actinomycetes were of minor abundance on all substrates analyzed. I found that differences in BCC on submerged macrophytes depend on habitat, substrate (plant species) and in part also on plant age (plant part). These findings are in accordance with other studies on freshwater biofilms. There, also alpha- and betaproteobacteria, and bacteria of the CFB-group were most abundant (Brümmer et al. 2000, Schweitzer et al. 2001, Grossart et al. 2008). In my samples, I seldom found gammaproteobacteria, only associated with senescing P. perfoliatus. This is probably related to the higher release of organic compounds and nutrients during senescence and in accordance with other studies (Wagner et al. 1993, Brachvogel et al. 2001). The BCC found in the field was confirmed by the initial colonization of axenic Myriophyllum spicatum in an outdoor mesocosm. After an initial dominance of CFB bacteria, betaproteobacteria dominated the biofilm. This indicates that macrophytes are colonized very quickly and the BCC is very constant with regard to major bacterial groups.
In all our community analyses, we found a distinct heterogeneous BCC for freshwater M. spicatum apices compared to all other samples. We hypothesized that the distinct BCC of M. spicatum apices might be related to the high total phenolic content in these samples. I used detailed statistical analyses (BIO-ENV) relating plant chemistry and environmental factors to the relative abundance of bacterial groups identified by FISH and the DGGE banding pattern of BCC on different substrates.
With DGGE data, we did not get significant correlations except for the leaf section, where conductivity explained some of the BCC variability. This was surprising, since the community analysis with DGGE gives a finer resolution since ideally individual strains are detected compared to FISH, where we used six probes to detect major bacterial groups. Probably the lower replicate number of DGGE (n = 13) may have lead to insignificant results. In contrast, the BIO-ENV analysis performed with the FISH data set resulted in significant effects. Both environmental factors, especially water level and temperature, conductivity and pH as well as plant chemistry, i.e., plant carbon and total phenolic content, explained most of the BCC variability.
Similar factors have also been found to influence the BCC of attached and free-living bacteria in other freshwater habitats (Lindström et al. 2005, Allgaier & Grossart 2006).
The different outcome of and conclusions drawn from DGGE and FISH analyses show that it is very important how determinants for BCC are calculated. It also shows that community analyses should always be done with two or more complementary methods. This is also supported by our finding that M. spicatum apices had a distinct biofilm based on cluster analysis of the banding pattern in DGGE. If the BCC was analysed with FISH, the difference between the substrates were not as obvious. This can also be explained by the probes we chose for FISH.
They are designed for major bacterial groups, which comprise many ecologically and physiologically different strains. Thus, the information of single strain shifts during the season or between different substrates gets lost, if only general probes are applied. The high variance of CFB bacteria in our samples is probably also related to the probe chosen. It detects only 38% of this group, but presently no better probes are
available (Loy et al. 2003). To avoid this bias, one could use a combined set of probes for CFB-bacteria to minimize detections errors.
Seasonal dynamics of the bacterial groups were observed for alpha- and betaproteobacteria as determined by FISH, while cluster analysis of the DGGE banding pattern did not reveal distinct seasonal changes. On lake snow, a distinct succession of different subgroups within the betaproteobacterial community was detected with only three probes for close relatives of this group (Schweitzer et al.
2001), although the total abundance of the betaproteobacteria did not change. Thus, I propose that further community analysis should be performed with more specific probes for the most abundant groups to get are detailed community analysis and to follow distinct successions more precisely.
The BCC data combined with the total cell counts of the substrates show that the plant chemistry might have an impact on bacteria, but not necessarily a negative one.
Total cell counts were higher on M. spicatum than on P. perfoliatus. This could be a consequence of higher nutrient leakage or leaf structure. Feathery, finely dissected leaves tend to support higher densities of epiphytes compared to laminar ones (Lalonde & Downing 1991). Flagellates also might influence the BCC and bacterial numbers (Jürgens & Matz 2002). Maybe the high polyphenolic content of M. spicatum negatively affected bacterivorous flagellates, which could increase total cell counts.
Lethal and sublethal effects of M. spicatum exudates on invertebrates have been shown recently (Linden & Lehtiniemi 2005). Unfortunately, due to time constraints, we did not evaluate flagellate abundances.
If the total phenolic content really influences the BCC can only be determined in controlled experiments with substrates releasing defined concentrations and types of phenolic plant secondary metabolites (see below).
Exuded plant compounds or the quality of the DOC in a water body may influence the BCC, as has been shown previously (Eiler et al. 2003, Huss & Wehr 2004, Tadonléké 2007). Sphagnum mosses, for example, harbour a unique epiphytic
community (Opelt et al. 2007), most likely caused by the extreme habitats they live in.
Polyphenols have been shown to be detrimental to bacteria by chelating proteins, iron and nutrients (Scalbert 1991). Defined synthetic polyphenols may inhibit biofilm formation (Huber et al. 2003). Thus, exuded polyphenols from M. spicatum might even inhibit quorum sensing biofilm formation. We successfully isolated three strains that are capable of degrading the exuded polyphenols of M. spicatum (this study, (Müller et al. 2007)), providing further evidence that this plant selects towards a defined bacterial community. Very special among those isolates is Matsuebacter sp.
FB 25. This betaproteobacterium is able to degrade polyphenols constitutively and almost nothing is known about its ecology so far.
We tested if this bacterium would influence the impaired growth of Acentria ephemerella larvae when fed M. spicatum. Epiphytic bacteria are inevitably taken up during feeding and thus may interact with the herbivore (Dillon & Dillon 2004). We designed two no-choice feeding assays to compare larval growth on M. spicatum with different bacterial biofilms. Unfortunately, larvae fed with mesocosm plants exhibited a high mortality. Most likely this was caused by the higher leaf toughness due to a higher ash content of these plants. Thus, we can presently only conclude that Matsuebacter sp.-colonized M. spicatum did not enhance the growth of A. ephemerella compared to axenic M. spicatum. We expected an influence of this bacterium either by attenuating the impact of polyphenols by degrading them, influencing the gut microbiota or as additional carbon or nitrogen source, thus raising the food quality of the plants. That the effect of the bacteria was rather negligible might also be caused by the short gut passage of the larvae that accounts for the transient nature of most gut bacteria. Alternately, Matsuebacter sp. might not be able to gain dominance or even co-exist in a gut already colonized by other gut bacteria. The knowledge on the gut microbiota of adults and larvae is still scarce. To further investigate the impact of bacteria and food quality on larval growth, we could use Pantoea agglomerans, isolated from the biofilm of M. spicatum and frequently found in herbivore guts
(Dillon et al. 2002). To elucidate the impact of M. spicatum derived tannins and associated bacteria on the growth it would be necessary to raise A. ephemerella larvae on an artificial diet. Thus, it would be possible to exclude other factors such as food quality. But all attempts to feed those larvae with an artificial diet failed (Choi et al.
2002, Erhard et al. 2007).
We also studied the initial colonisation of axenic M. spicatum with selected bacterial isolates. Matsuebacter sp. was a strong colonizer in single species set-ups but not in combination with Agrobacterium vitis. Interestingly, Matsuebacter sp. was able to inhibit P. agglomerans in such a way that it could not dominate the biofilm if A.
vitis was present. The mechanism of this interaction remains unclear so far. We assume that Matsuebacter sp. may be able to degrade bacteriostatic compounds P.
agglomerans releases against A. vitis. This would facilitate the biofilm formation of A.
vitis. Or competition for resources (space, nutrients) affects the interaction, e.g., the differences in the constitutive capacity to degrade plant polyphenols. In preliminary investigation, we also tested if those strains produce N–acylhomoserinlactones, but all tests were negative even though quorum sensing activities have been described before for A. vitis and P. agglomerans (Holden et al. 1999, Wang et al. 2008). We think that the interactions of those strains are an ideal model for further investigations.
With our new experimental set-up, we are able to analyze the complex interactions between the three strains. This assay allows also investigating the reciprocal impact of the bacteria on the plant and vice versa.
Perspectives
For a better understanding of the processes shaping biofilm formation, succession and interactions of bacteria within the biofilm, but also with the host or herbivores, further investigations are needed.
I suggest that the biofilm on the investigated macrophytes should be analyzed with more specific probes and to use a combination of methods such as MICRO-FISH or stable isotope probing to obtain detailed data on bacterial abundance and activity.
The inclusion of more microscopic methods such as scanning electron microscopy (SEM) or confocal laser scanning microscopy (CLSM) should allow answers to the spatial distribution of bacteria in the biofilm. Green fluorescent protein labelling of certain bacterial strains would facilitate the biofilm research, since FISH is very laborious and the complete detachment of the bacterial biofilm always has a certain uncertainty. Strains that do not need to be stained would facilitate the analysis.
Cyanobacteria, eukaryotic algae, fungi and flagellates should also be included into further analysis, since they are important components of the biofilms on aquatic surfaces.
The final evidence that the polyphenols of M. spicatum have a crucial influence on the BCC is still lacking. The proof for this can only be given experimentally, since field work includes too many unknown or uncontrolled factors that would also influence the BCC. Field work would also require an immense sampling number.
Thus, we need an experimental set-up with natural lake water flow-through and a substrate that can be manipulated with respect to the provided carbon source. The first trials showed that due to the high chemical reactivity of tannic acid and other polyphenols, experiments are difficult and susceptible to artefacts.
To investigate the interactions of bacterial isolates and their impact on the plant, not only the standard plant chemistry (total phenolic content, chlorophyll) should be analysed but also the reactions of the plant on the genomic (real time PCR) or protein level (SDS-PAGE). If the strains induce plant responses, they could further be manipulated with respect to degradation pathways, biofilm formation or single
pathways to elucidate, which physiological traits are responsible for the interactions with the plants or other bacteria.
Thus, aquatic biofilms on aquatic macrophytes and the allelochemical interactions of aquatic plants and biofilm bacteria remains an interesting and exciting field of research. With my PhD thesis, I elucidated some of the open questions but still, many more studies have to be done to understand biofilms and their ecological impact on macrophytes.
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