Forests soils are highly complex ecosystems and soil microbes are known to have significant effects on plant diversity and productivity . Most trees form a range of mutualistic associations with various filamentous fungi, these root-fungus associations are known as mycorrhizas. Mycorrhizal symbiosis improves plant nutrient acquisition and confers increased resistance to pathogens, while the fungus gains carbohydrates from its host plant . The formation of mycorrhizas affects several aspects of plant physiology and also changes the nutritional and physical properties of the soil. The mycorrhizas and the external mycelia of symbiotic fungi (which together define the mycorrhizosphere) are colonised by bacteria, which may actively influence the growth of external fungal mycelia and mycorrhizal root colonisation. For instance, a group of bacteria known as Mycorrhization Helper Bacteria; MHB  stimulate the formation of mycorrhizas. At the time of writing, numerous bacterial strains from a wide range of major clades have been shown to have MHB-type functions in both arbuscular and ectomycorrhizal symbioses .
Bacteria can facilitate mycorrhization in various ways. In many cases, the positive effects stem from their ability to induce rapid expansion of the fungal mycelium e.g. . Other important mechanisms include the alleviation of soil-mediated stress e.g. [6, 7] and the formation of more extensive plant-fungus contacts by stimulating lateral root formation . However, MHB do not always have positive effects on mycorrhiza formation and can exhibit fungus specificity in promoting symbioses . While the effects of MHB on mycorrhizal fungi have been investigated extensively in vitro, the effects of the fungi on the MHB have largely been neglected. In their seminal work, Frey-Klett et al.  reported that the life span of the Pseudomonas fluorescens strain BBc6R8 was significantly prolonged by exposure to the EM-fungus L. bicolor S238N. This effect was attributed to the fungus because the survival of the bacterial strain was not affected by the presence of non-mycorrhizal roots.
Actinomycetes are frequent colonisers of mycorrhizospheres, rhizospheres and plant roots [10, 11]. They are known for their antagonism against other microbial species [12, 13] and are especially rich sources of antifungal compounds . Depending on the circumstances, they can either inhibit or promote the formation of mycorrhizas reviewed in , and several actinomycete species exhibit MHB activity, Rhodococcus sp. , Streptomyces sp., [16–18]. Among the actinomycete MHB, the strain Streptomyces sp. AcH 505 has drawn most attention, since it forms unique interactions with fungi and plants. The extension of the fungal mycelium is promoted by the AcH 505 metabolite auxofuran , but the fungal biomass is simultaneously reduced due to the thinning of mycelium . Schrey et al.  observed that co-cultivation of MHB Streptomyces sp. AcH 505 with Amanita muscaria and Suillus bovinus increased their rates of mycorrhization. However, co-cultivation with the same strain reduced the in vitro growth of Hebeloma cylindrosporum. This fungus-specificity is due to the differential sensitivity of the ectomycorrhizal fungi to the naphthoquinone antibiotic WS-5995 B, which is produced by AcH 505  in addition to auxofuran. In the host plant, AcH 505 stimulated fine root formation  and facilitated root colonisation by suppressing the plant’s defensive responses . However, while exposure to AcH 505 suppressed defensive responses at the root level, it increased the resistance to the causative agent of grey mould Botrytis cinerea at the leaf level. While previous studies on AcH 505 provided valuable information on its interactions with the host plant and ectomycorrhizal fungi, they were all based on in vitro experiments; to date, no studies on its effects in soil have been conducted.
The discovery of bacteria that promote the establishment and maintenance of mycorrhizas triggered a search for their mechanisms of actions, and a number of publications have described in vitro experiments on MHB-fungus interactions, e.g. [5, 20, 22]. However, much remains to be learned about how MHB-fungus interactions work under natural conditions and how they are affected by the host plant . We therefore investigated the growth responses of AcH 505 and the mycorrhizal fungus Piloderma croceum using a soil-based culture system that was established for studying multitrophic interactions in oaks as part of the TrophinOak collaborative project , see also http://www.trophinoak.de. The pedunculate oak Quercus robur belongs to the Fagaceae family and is obligately ectomycorrhizal under natural conditions. It is host to several symbiotic fungi, including both basidio- and ascomycete species . One of its notable symbiont is Piloderma croceum, which has become a model fungus for studying the formation of oak mycorrhizas . In a preliminary investigation, we observed that AcH 505 promotes the formation of mycorrhizas in oak microcosms. The number of mycorrhizas per microcosm was counted prior to harvesting and was found to be slightly increased by inoculation with AcH 505 according to the test of equal proportions (p = 0.05).
The study conducted herein was conducted to assess i) whether the effects of Streptomyces sp. AcH 505 and the ectomycorrhizal fungus Piloderma croceum on one-another depend on the presence of a host plant, ii) the possible influence of the microbial community on both micro-organisms and iii) how the two micro-organisms influence each other.
For this purpose, AcH 505 and P. croceum were cultivated alone and together under four different culture conditions: in the presence of both the host plant (Q. robur) and soil microbes (represented by a microbial filtrate), in the presence of the host but not soil microbes, in the presence of soil microbes but no host plant, and in the presence of neither soil microbes nor the host. In microcosms including the plant rhizosphere as well as bulk soil samples were taken for quantification analysis. The experimental setup is summarised in Additional file 1.
The abundances of AcH 505 and P. croceum mycelia were estimated by quantitative real-time PCR . Primers were designed to target an intergenic region of the AcH 505 genome, between the gyrA and gyrB genes. The abundance of eukaryotes in environmental samples can be determined using qPCR experiments targeting the highly variable internal transcribed spacer (ITS) regions of rDNA operons [27, 28]. However, fungal genomes contain multiple copies of the ITS-region and the ITS copy number varies between fungal strains . For P. croceum Raidl et al.  estimated about 150 ITS copies per dikaryotic cell. Thus, it can be beneficial to target single copy genes or intergenic regions rather than the ITS when quantifying fungi . To compare the performance of these two approaches in fungal quantification, we designed novel ITS primers, as well as a primer pair that targets an intergenic region between two open reading frames (ORFs) in the P. croceum genome.