Fusarium Fungi Produce Nitrous Oxide (NO) from Nitrite (NO) in a Model Pot System Simulating the Soybean Rhizosphere.

Nitrous oxide (NO) is a key atmospheric greenhouse gas that contributes to global warming, with anthropogenic NO emissions from agriculture being a particular concern. Among agricultural sources, unknown soil organisms in the legume rhizosphere emit NO from degraded root nodules. To discriminate between fungal and bacterial NO emissions, we adopted an isotopomer ana-lysis, which provides site preference values (the difference in N abundance of the central and terminal N atoms in the NO molecule). The addition of nitrite instead of nitrate to soybean nodulated roots significantly increased SP 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 from 4.2‰ to 13.9‰ with nitrite. These results suggest that nitrite-utilizing NO emissions via fungal denitrification occurred in the model pot system of the soybean rhizosphere. Microscopic observations showed fungal hyphae and crescent spores around NO-emitting nodules. Therefore, we isolated single spores from soybean nodules under a microscope. A phylogenetic ana-lysis 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 NO; however, no NO emissions were noted in the presence of nitrate. A N-nitrite tracer experiment revealed that one NO molecule was derived exclusively from two molecules of nitrite (NO) in the fungal culture. These results suggest that nitrite-utilizing Fusarium fungi mediate NO emissions in the soybean rhizosphere.