Previous studies have indicated that fungal endophytes may coexist at very small scales. In this study, niche partitioning between two endophytic species of Microdochium sympatrically colonizing Phragmitis australis was assessed. M. bolleyi and M. phragmitis were found to be significantly segregated for host habitat, but not for host organ and season. However, when additional, unrelated fungi that colonize the same host were also included in the analyses, the latter two factors were also found to contribute to niche partitioning.
Several factors can cause niche differentiation between endophytes, which may attenuate competition and thus allow for a high fungal diversity on the same host species. One factor is space, which is with respect to endophytes hierarchically structured from continent to region, to habitat, to host individual, to host organ, and further down to the level of host cells. Two of these levels, i.e. the habitat type and the host organ, were analyzed. Both, M. bolleyi and M. phragmitis, preferentially colonize the same organ, i.e. roots, confirming an earlier result . Within the limits of detection, nested-PCR assays in this study indicated that M. bolleyi occurs more frequently on roots at dry sites, whereas M. phragmitis occurs more frequently on roots at flooded sites. This suggests spatial niche partitioning at the level of habitat type. Like all molecular assays employing fungal genomic DNA extracted from field samples, the assays from this study cannot distinguish between growing and dormant cells, and thus cannot provide details on metabolic activities or developmental stages. In addition, a possible introduction of bias against rare templates during the first stage of the nested-PCR has to be considered, which would produce false-negative results in case of fungi present at very low abundance. However, if the first step of nested-PCR comprises as many cycles as used here rare templates will be over- not under-amplified, as previously shown . Thus, for assessment of presence-absence data nested-PCR is a highly specific and sensitive method.
Further support for an influence of spatial niche partitioning on the composition of the reed-associated fungal community was obtained when occurrences of three additional species were also considered. Both binomial tests and CCA indicated that all five species were differentiated by host organ and / or habitat. Since P. australis has a vast geographical distribution, it would be interesting to assess the factor space in structuring fungal communities at higher hierarchical levels in the future.
The importance of space in affecting fungal community composition has previously been acknowledged. Much of this information comes from pathogens of agronomically important crops  and from mycorrhizal fungi [14, 32–36]. In addition, endophyte communities seem also to be influenced by the factor space [37–39]. However, in contrast to other types of fungi, little is known about the causes leading to spatial differentiation in endophytes. At the same sites examined in this study an even more distinct preference for the habitat type was previously noted for AM fungi that were not observed at flooded sites at all, whereas at the dry sites, 21 phylotypes were detected at various frequencies . Vertical distribution patterns of reed-associated fungi have been recorded in a brackish tidal marsh, with diverse communities depending on the leaf layer . Site-dependent differences in reed stands are known for Oomycota, where some species preferred either dry or flooded sites . It seems likely that it is not space per se, but rather specific physico-chemical features of the respective sites that cause such differences.
Another factor that can cause niche differentiation between fungal endophytes is time, resolved here at the scale of individual months of the season. The progress of the season drives host developmental processes like the emergence of shoots and leaves in spring and senescence in autumn, and thus dynamically modifies the niches available to plant-associated fungi. The occurrences of M. bolleyi and M. phragmitis were similar for season. Thus, seasonal niche partitioning does not seem to significantly separate Microdochium spp. on common reed. However, binomial tests indicated that time was a factor involved in the separation of the five species analyzed.
The contribution of seasonal variation to fungal community variation has previously been recognized. Endophytic colonization of tropical cacao trees increased with leaf age and partially protected the host against pathogenic Phytophthora sp. . Similarly, endophytic diversity increased during leaf development in Camellia japonica, whereas epiphytic diversity remained stable with season . Seasonal succession was also demonstrated for the mycoflora in a Colorado mountain soil that changed substantially between spring and summer, suggesting functional differentiation . Seasonal variation has been found in an aquatic fungal community decomposing plant debris in streams . In reed stands at Lake Constance, Oomycota populations were shown previously to exhibit seasonal variation . For the reed pathogen Pythium phragmitis, minimal detection in August resembled the decrease of Microdochium spp. during the summer.
Temporal niche differentiation thus contributes to the separation of the five species examined, although to a lesser extent than space. Thus, niche differences resulting from abiotic or biotic attributes seem to separate these fungi and may explain their coexistence on the same host. Temperature was one attribute that distinguished the two Microdochium species in vitro. M. phragmitis, which occurs more frequently at flooded sites, grows faster at lower temperatures, whereas M. bolleyi, which prefers dry sites, grows faster at higher temperatures. For most of the year, based on the in vitro growth rates, temperatures existed in the soil at which M. phragmitis would grow faster than M. bolleyi if additional factors such as competing fungi are not considered. In this context, temperature contributes to the differentiation of other Microdochium species [47, 48].
Other attributes may be involved in spatial niche differentiation for habitat type observed for Microdochium spp. Carbon usage patterns of the two species were found to overlap significantly more than expected by chance, although certain substrates, including compounds of the central carbon metabolism, secondary sugars, and sugar alcohols, are utilized differentially. In P. australis site-dependent variations for central metabolites were reported . Basal culm internodes from flooded sites had higher total amino acid and lower total carbohydrate contents than those from dry sites. Several metabolites were individually recorded in that study, but none of those varying for habitat type could explain the contrasting habitat preferences of the two Microdochium species when considering the results of the BIOLOG experiments.
Earlier studies have noted that host-derived carbohydrates might affect the occurrences of plant-associated fungi. Root-associated endophytes grew better in vitro on low concentrations of certain carbohydrates than rhizosphere and soil fungi retrieved from the same Austrian grassland . It was suggested, "that plant sugars or sugar alcohols may constitute signals that facilitate adaptation of certain fungi to a specific host plant". Some of such compounds are differentially utilizable by Microdochium spp. Another study reported that Neotyphodium endophytes were inhibited in vitro by high concentrations of hexose and were incapable of utilizing xylose and arabinose . These findings were supported by results showing that Neotyphodium lolii grows more slowly in varieties of its host Lolium perenne bred for intrinsically high sugar concentrations . For AM fungi, it was suggested that competition for the same carbon sources present in the same niche caused differential colonization . A report comparing ericoid and orchid mycorrhizal fungi found that carbon source utilization was generally quite similar in vitro except for distinct differences for tannic acid and certain amino acids . These publications indicate that the quality and the quantity of carbon sources available in the host may be one of the attributes influencing the composition of the associated fungal community.
Although the BIOLOG system provides interesting insights in the capacity of fungi to utilize various carbon sources, the difference in growth conditions in vitro compared to in planta should be considered. Single carbon sources are tested in vitro, whereas in planta many different sources are present. For the moment, it is not clear whether the carbon sources differentially used by Microdochium spp. in vitro are available at contrasting levels in roots or whether they have physiological importance for the fungi. Furthermore, competition with other endophytes for carbon sources may also influence their occurrences in the field. Thus, the challenging task remains to prove that differential utilization of carbon sources in vitro contributes to the coexistence of endophytes in planta.
Interactions between species implied by positive or negative co-occurrence was the third factor examined with respect to the differential colonization of the roots of common reed by Microdochium spp. Although spatial niche partitioning between M. bolleyi and M. phragmitis was significant, it was not perfect. Since none of the comparisons assessed by Fisher's Exact test exhibited any negative co-occurrence, a direct antagonism between these two species is unlikely. Moreover, in 8.4% of the samples both species were detected which may suggest "true" coexistence. Otherwise, reduced competition for space or carbon (or other essential compounds and ions) may explain this finding. This could occur if colony sizes were much smaller than sample sizes or if the two species used different resources. However, the two Microdochium species constitute only a small part of the entire fungal community colonizing common reed. Thus, antagonism or synergism might be indicated when considering additional fungi. This is already seen when including the data available for three more species. Whereas the EcoSim analysis suggests an overall signature of negative co-occurrence, Fisher's Exact test indicates negative and positive co-occurrences for certain species pairings. It is noteworthy that none of the three additional species exhibited negative co-occurrence with M. bolleyi and M. phragmitis in the total data set. Instead, M. bolleyi generally co-occurred significantly more frequently with Ms7Mb4 and Ms43Mb21 than expected by chance. Such a positive co-occurrence may appear when the conditions that are conducive for one species are also favorable for another species. Alternatively, positive co-occurrence may result from synergism. On the other hand, there existed an overall negative co-occurrence between Stagonospora sp. and Ms7Mb4, significantly preferring leaves  and roots , respectively. This could have resulted from strongly contrasting niche preferences, severe competition for the same substrates or from the secretion of toxins (antagonism). Our results suggest that it is rather unlikely that antagonism by any of the other three fungi is responsible for the differential colonization of roots by Microdochium spp. Since the fungal community on common reed is larger than addressed here, we cannot rule out that other endophytes may exert such influences.