Diazotrophic bacteria reside inside Suillus tomentosus/Pinus contorta tuberculate ectomycorrhizae

Culturable bacteria from Suillus tomentosus / Pinus contorta tuberculate ectomycorrhizae (TEM) tissues were evaluated to identify possible causative agents of TEM-associated nitrogenase activity in a naturally regenerating pine forest. Bacteria from internal and external TEM tissues were isolated over a 3 year sampling period and representative colony types were identified using gas chromatograph fatty acid methyl ester and 16S rDNA sequence analyses. Eighteen species of bacteria belonging to at least eight genera (Pseudomonas, Bacillus, Xanthomonas, Xanthobacter, Sphingobacterium, Burkholderia, Curtobacterium, and an actinomycete) were isolated from the TEM surface. In contrast, four bacterial species belonging to three genera (Paenibacillus, Methylobacterium, and Micrococcus) distinct from those found on the TEM surface were isolated from surface-sterilized TEM. None of the TEM surface isolates were diazotrophic but three of the four species from the TEM interior (Paenibacillus amylolyticus, Paenibacillus pabuli, and Methylobacterium mesophilicum) possessed the nifH gene and were capable of some acetylene reduction. Our findings indicate that nitrogen fixing bacteria also reside within TEM and should be considered as possible causative agents of TEM-associated nitrogenase activity on lodgepole pine.