Fusarium Fungi Produce Nitrous Oxide (N₂O) from Nitrite (NO₂^–) in a Model Pot System Simulating the Soybean Rhizosphere

Abstract

Nitrous oxide (N₂O) is a key atmospheric greenhouse gas that contributes to global warming, with anthropogenic N₂O emissions from agriculture being a particular concern. Among agricultural sources, unknown soil organisms in the legume rhizosphere emit N₂O from degraded root nodules. To discriminate between fungal and bacterial N₂O emissions, we adopted an isotopomer ana­lysis, which provides site preference values (the difference in ¹⁵N abundance of the central and terminal N atoms in the N₂O molecule). The addition of nitrite instead of nitrate to soybean nodulated roots significantly increased SP_N2O from –3.5‰ to 4.2‰ in a pot system. Moreover, a mutation of the nirK gene (encoding dissimilatory nitrite reductase) in symbiotic bradyrhizobia significantly increased SP_N2O from 4.2‰ to 13.9‰ with nitrite. These results suggest that nitrite-utilizing N₂O emissions via fungal denitrification occurred in the model pot system of the soybean rhizosphere. Microscopic observations showed fungal hyphae and crescent spores around N₂O-emitting nodules. Therefore, we isolated single spores from soybean nodules under a microscope. A phylogenetic ana­lysis revealed that all 12 fungal isolates were Fusarium species, which exist in soybean field soil. When these isolates were cultivated in glycerol-peptone medium supplemented with nitrate or nitrite (1‍ ‍mM), 11 of the 12 isolates strongly converted nitrite to N₂O; however, no N₂O emissions were noted in the presence of nitrate. A ¹⁵N-nitrite tracer experiment revealed that one N₂O molecule was derived exclusively from two molecules of nitrite (NO₂^–) in the fungal culture. These results suggest that nitrite-utilizing Fusarium fungi mediate N₂O emissions in the soybean rhizosphere.