Phosphorus (P) is the second most important macronutrient for plant growth after nitrogen. The majority of P in soils is unavailable for plants. Fertilizers are the only way of providing P for plants. However, only small amount of this P is plant available. In addition, environmental pollution by P fertilizers and increasing price of P fertilizers because of the scarcity of the rock phosphate (as the main source of producing P fertilizer) have created interest in new strategies as substitute or supplement for P fertilizers. One of these strategies is applying phosphate-solubilizing microorganisms (PSM) as an ecofriendly option to make P available for plants. Recently, few different products have been introduced and marketed claiming to increase availability of P for plants. One of them is Jumpstart based on Penicillium bilaii produced by Novozymes A/S. It has been shown that P. bilaii can release P from inorganic phosphate pools through the colonization of root system and producing organic acid exudates and thereby improve the plant growth. However, the challenges are to obtain robust and reliable performance, to gain low year to year variation, and to limit the influence from environmental conditions. It has been shown that mycorrhizal helper bacteria presenting in mycorrhizal fungi could stimulate fungal growth, promote establishment of root-fungus symbiosis and enhance plant production. But it is unknown if the comparable relationship exist between the non-mycorrhizal fungus P. bilaii and its hyphae associated bacteria. In the current PhD thesis, we assumed that hyphae-associated microbiome of P. bilaii might harbor helper bacteria with ability to improve fungal growth and P solubilization performance. Therefore, we aimed to isolate bacteria associated with the P. bilaii hyphae and identify the fungal growth stimulating bacteria with the perspective of promoting efficiency of Jumpstart in soil – plant system. For this purpose, most of the work within the current project was carried out by development of suitable model systems by mimicking the natural soil habitat to reach to the reliable performance in soil. In manuscript I, the objective was to recover bacterial community associating with P. bilaii hyphae in a close-to-natural soil system and characterize the bacterial community composition to determine whether P. bilaii can select specific bacteria from the whole soil bacterial community. Therefore, a novel microcosm system was established to isolate bacteria from the hyphae living in the soil thereby mimicking the natural habitat for the fungal-bacterial interaction. A patent application has been filed for this novel isolation technique. Bacterial colonization of the hyphae was confirmed by fluorescence microscopy. Furthermore, the hyphae-associated microbiome of P. bilaii was characterized comprehensively by 16S rRNA gene-targeted amplicon sequencing. Comparison between amplicon sequencing of the 16S rRNA genes from soil bacterial community and hyphae associated bacterial microbiom indicated that the fungal hyphae selected for a specific part of the soil bacterial community, as only four phyla in the soil were represented in the hyphosphere community and the dominant phylum was Proteobacteria. At the genus level, Burkholderia was dominating along with Pseudomonas and Massilia. In manuscript II, we investigated the functional properties of the hyphae associated bacteria isolated from P. bilaii. Initially, we established a confrontation assay including P. bilaii and bacteria of a strain collection of 200 hyphae associated bacteria to distinguish “good”, “bad” and “neutral” bacteria. In this assay, 19% of the isolated strains stimulated fungal growth, 56% inhibited the growth while 25% were neutral. Five bacterial strains, all belonging to the Bacillus genus, with pronounced fungal growth stimulation were selected for further functional assays in relation to fungal growth parameters and P solubilization performance. All five Bacillus strains could significantly increase spore germination on water agar plates while only three of them could increase spore germination on artificial root exudate medium. Furthermore, all five strains promoted fungal growth on artificial root exudate medium while four of them increased growth of P. bilaii on water agar plates showing the importance of carbon status on the interactions between P. bilaii and the selected Bacilli. In addition, three out of five strains showed positive effect on the fungal outgrowth on organic and inorganic P containing media. Four bacterial strains showed positive effect on organic P solubilization by the fungus while three of them could promote inorganic P solubilization by fungus. However, none of bacteria were able to solubilize organic and inorganic P on their own. Bacterial effect on fungal growth in gamma-sterilized and non-sterilized soil systems were measured by qPCR and the results indicated that two out of five selected bacteria belonging to Bacillus simplex could increase fungal biomass in the soil. We also showed that all five selected bacteria were able to colonize the hyphae in gamma-sterilized soil. In addition, inoculation of helper bacteria with P. bilaii on oilseed rape increased plant biomass significantly (P<0.05) in pot trials compared to P. bilaii inoculation and helper bacteria alone, supporting a synergistic interaction between helper bacteria and P. bilaii. In manuscript III, we analyzed the complete genome sequence of fungal growth promoting Bacillus simplex strain 313. We found several predicted genes and/or operons that are potentially important for the interactions between B. simplex strains 313 and P. bilaii, including genes involved in colonization and biofilm formation (motility and chemotaxis, surfactin and phenazine production), auxin biosynthesis, iron acquisition and metabolism, acetoin and butanediol metabolism, dimethyl sulfide production, acid phosphatase and alkaline phosphatase, and B-vitamins (biotin, thiamin, pyridoxine and riboflavin) biosynthesis. Based on the results obtained in this PhD project, it can be concluded that P. bilaii can strongly select a specific part of the soil bacteria, which include beneficial bacteria with positive effect on the fungal growth and function in pure culture, and with a potential for application in more realistic systems as soil and plant models. These positive interactions between the fungus and bacteria could be considered as a promising tool to obtain reliable results after applying the bio-fertilizers in the soil system in combination with plants.