Increased hyphal branching and growth of ectomycorrhizal fungus Lactarius rufus by the helper bacterium Paenibacillus sp.

Paenibacillus sp. EJP73 has been previously demonstrated as a mycorrhization helper bacterium (MHB) for the Lactarius rufus-Pinus sylvestris symbiosis in both laboratory and glasshouse experiments. In the present study, the effect of Paenibacillus sp. EJP73 metabolites on L. rufus EO3 pre-symbiotic growth was tested in two agar plate-based systems. Specifically, volatile metabolites were investigated using a dual plate system, in which the presence of strain EJP73 resulted in a significant negative effect on L. rufus EO3 hyphal radial growth but enhanced hyphal branching and reduced internode distance. Soluble metabolites produced by strain EJP73 were tested on L. rufus EO3 growth in single-agar plate assays by incorporating bacterial cell-free whole or molecular weight fraction spent broth into the agar. Whole spent broth had a negative effect on hyphal growth, whereas a low molecular weight fraction (100-1,000) promoted colony radial growth. Headspace and spent broth analysis of strain EJP73 cultures revealed 2,5-diisopropylpyrazine to be the most significant component. Synthesised 2,5-diisopropylpyrazine and elevated CO2 (2,000 ppm) were tested as specific volatile metabolites in the dual plate system, but neither produced the response shown when strain EJP73 was present. Increased pre-symbiotic hyphal branching leading to increased likelihood of plant infection may be an important MHB mechanism for strain EJP73. Although the precise signal molecules could not be identified, the work suggests a number of metabolites may work synergistically to increase L. rufus root colonisation.