Identification and characterization of biological nitrification inhibition (BNI) substances in sorghum root


Nitrification, one of several pathways in the soil-N cycle, results in the microbiological conversion of relatively immobile NH4+ into highly mobile NO3-(which is susceptible to losses through leaching [NO3-leaching]), and gaseous N emissions (N2O, NO and N2) by denitrification. The price of nitrogen fertilizer has been rising in recent years. Controlling nitrification through suppression of nitrifier activity is thus critical to improving nitrogen use efficiency (NUE) of agricultural production systems. Suppressing soil nitrification through the release of nitrification inhibitors from plant roots is termed ‘biological nitrification inhibition’ (BNI). This present study aims to characterize BNI function in sorghum, in particular the production of inhibitors, their chemical identity, functionality, and factors regulating their release.

Sorghum roots release two types of nitrification inhibitors: hydrophilic-BNIs and hydrophobic-BNIs. The former were those released into water-based collection medium while  the latter are those released by washing the roots for 30s with dichloromethane (DCM), which has high affinity for hydrophobic compounds.

Three nitrification inhibitors -- MHPP (methyl 3-(4-hydroxyphenyl) propionate), sakuranetin (5,4’-dihydroxy-7-methoxyflavanone) (isolated from hydrophilic BNI activity), and sorgoleone (2-hydroxy-5-methoxy-3-[(8’Z,11’Z)-8’,11’,14’-pentadecatriene]-p-benzoquinone) (isolated from hydrophobic BNI activity) -- were isolated from the inhibitory activity released from sorghum roots (Fig. 1). The release of nitrification inhibitors required the presence of NH4+, whose stimulatory effect lasted 24h, in the root environment (Fig. 2). The release of hydrophilic-BNIs declined at rhizosphere pH > 5.0. Nearly 80% of hydrophilic-BNI released was suppressed at pH ≥7.0 (Fig. 3). A bioluminescence assay using recombinant Nitrosomonas europaea was employed to determine BNI activity. The ED80(effective dose for 80% inhibition Nitrosomonas function) for sakuranetin, sorgoleone, and MHPP was 0.6 µM, 12.0µM, and >120 µM, respectively (Fig. 4).

These results are useful as fundamental knowledge towards utilization research of BNI in sorghum. We should clarify the field conditions in which sorghum BNI is the most efficient and investigate the BNI activity of each substance in the soil. We need to establish reliable screening techniques and selection criteria for breeding.

Figure, table



Japan International Research Center for Agricultural Sciences Crop, Livestock and Environment Division


Administration B

Research project

Utilization of Biological Nitrification Inhibition (BNI) function for the development of breeding materials and application to cropping systems

Program name

Environment and Natural Resource Management

Term of research

FY 2006- FY 2010

Responsible researcher

Subbarao Guntur Venkata ( 生産環境・畜産領域 )

Nakahara Kazuhiko ( 生物資源・利用領域 )

Zakir H. A. K. M. ( 生産環境・畜産領域 )

Ishikawa Takayuki ( 生産環境・畜産領域 )

Yoshihashi Tadashi ( 生産環境・畜産領域 )

Ono Hiroshi ( 食品総合研究所 )

Yoshida Mitsuru ( 食品総合研究所 )

Publication, etc.

Subbarao et al. (2012) Plant Soil

Zakir et al. (2008) New Phytologist, 180:442-451

Japanese PDF

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English PDF

2012_04_A4_en.pdf148.5 KB