Moriuchi Makoto, Kuzunuki Keiichi, Ikenishi Fumio, Sameshima Reiko, Nakagiri Akira, Toyoda Sakae, Katsuyama Chie, Kakizaki Kaori, Itakura Manabu, Yoshida Naohiro, Suwa Yuichi, Minamisawa Kiwamu
Graduate School of Life Sciences, Tohoku University.
Department of Chemical Science and Engineering, Institute of Science Tokyo.
Microbes Environ. 2025;40(2). doi: 10.1264/jsme2.ME24092.
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.
一氧化二氮（N₂O）是一种关键的大气温室气体，会导致全球变暖，农业产生的人为N₂O排放尤其令人担忧。在农业来源中，豆科植物根际中未知的土壤生物会从降解的根瘤中排放N₂O。为了区分真菌和细菌的N₂O排放，我们采用了同位素异构体分析，该分析可提供位点偏好值（N₂O分子中中心氮原子和末端氮原子的氮丰度差异）。在盆栽系统中，向大豆根瘤添加亚硝酸盐而非硝酸盐，显著提高了位点偏好值，从-3.5‰提高到4.2‰。此外，共生慢生根瘤菌中nirK基因（编码异化亚硝酸盐还原酶）的突变，在添加亚硝酸盐的情况下，使位点偏好值从4.2‰显著提高到13.9‰。这些结果表明，在大豆根际的模型盆栽系统中，通过真菌反硝化作用利用亚硝酸盐的N₂O排放发生了。显微镜观察显示，在排放N₂O的根瘤周围有真菌菌丝和新月形孢子。因此，我们在显微镜下从大豆根瘤中分离出单个孢子。系统发育分析表明，所有12个真菌分离株均为镰刀菌属物种，存在于大豆田间土壤中。当这些分离株在添加硝酸盐或亚硝酸盐（1 mM）的甘油-蛋白胨培养基中培养时，12个分离株中有11个能强烈地将亚硝酸盐转化为N₂O；然而，在有硝酸盐存在的情况下未观察到N₂O排放。一项N-亚硝酸盐示踪实验表明，在真菌培养物中，一个N₂O分子仅来源于两个亚硝酸盐分子（NO₂⁻）。这些结果表明，利用亚硝酸盐的镰刀菌真菌介导了大豆根际的N₂O排放。
