Synergism between the feremycorrhizal fungus Austroboletus occidentalis and soil free-living nitrogen-fixing bacteria

In feremycorrhizal (FM) symbiosis, the Australian native fungus Austroboletus occidentalis improves growth and nutrient (e.g. N and P) acquisition of different host plants, including agricultural crops, without colonizing their roots. A controlled environment (CE) trial was conducted to explore the possible synergistic interactions between the FM fungus A. occidentalis and soil free-living N-fixing bacteria (diazotrophs). Wheat (Triticum aestivum) plants were grown under N deficiency conditions in a field soil (treatment control), , or supplied with a consortium containing four free-living diazotroph isolates (treatment diazotrophs), A. occidentalis inoculum (treatment FM), or both diazotrophs and A. occidentalis inoculums (dual inoculation). After seven weeks of growth, significantly greater shoot biomass was observed in plants inoculated with diazotrophs (by 25%), A. occidentalis (by 101%), and combined inoculums (by 106%), compared to the uninoculated control. All inoculated plants also had higher shoot nutrient content (including that of N, P, K, Mg, Zn, and Mn) than the control treatment. Compared to control and diazotrophs treatments, significantly greater shoot N content were observed for the FM treatment (i.e. synergism between the FM fungus and soil native diazotrophs) and dually-inoculated plants (i.e. synergism between the FM fungus and native/inoculated diazotrophs). Furthermore, dually-inoculated plants had the highest levels of shoot nutrient contents (e.g. N, P, K, Zn and Mn), and soil total N (13-24%) across treatments. Root colonization by soil indigenous arbuscular mycorrhizal (AM) fungi declined in all inoculated plants compared to the control, presumably due a shift in plant-fixed carbon allocation from AM fungi towards the microbial-driven Nitrogen compounds.. Non-metric multidimensional scaling (NMDS) analysis of the bacterial 16S rRNA gene amplicons revealed that the growth and N nutritional benefits conferred to wheat plants by the FM fungus (in both FM and dual treatments) were accompanied by modification of the entire soil microbiome. in vitro study showed that A. occidentalis could not grow on substrates containing lignocellulosic materials or sucrose, while being able to growon media supplemented with simple sugars such as glucose and fructose, supporting the findings of our CE trial showing that improved N nutrition of plants inoculated with A. occodentalis was primarily due to its interaction with N-related microorganisms rather than through organic matter decomposition. This results provide insight into N nutrition of plants involved in the FM symbiosis via revealing synergistic interactions between the FM fungus and free-living diazotrophs, proposing a potential to boost microbial N fixation for non-legume crops.