# Use of plants producing Biological NitrificationInhibitors (BNI) for optimized management of nitrogen in rotation.

Izargi Vega Mas, Aude Mancia

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Izargi Vega Mas, Aude Mancia. Use of plants producing Biological NitrificationInhibitors (BNI) for optimized management of nitrogen in rotation.. GAIN: Gestions innovantes de l'azote et des adventices, Gembloux Agro-Bio Tech, Jan 2025, Gembloux, Belgium. hal-05383981

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Submitted on 26 Nov 2025

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# Use of plants producing Biological Nitrification Inhibitors (BNI) for optimized management of nitrogen in rotation.

Izargi Vega Mas

Postdoctoral Researcher
Plant Physiology and Molecular Biology lab
University of the Basque Country (UPV/EHU)

Aude Mancia

Postdoctoral Researcher

INRAE

ULiege
Nitrogen (N) is essential for crop production

<figure><img src="image_2.png" /></figure>

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By 2050 **food production** needs to increase by 50% to feed the world population.

Higher N inputs do not result in an improved NUE of crops

<figure><img src="image_4.png" /></figure>

Nitrogen Use Efficiency in Europe <65%
<figure><img src="image_5.png" /><figcaption>N input.</figcaption></figure>
HIGH N inputs → N LOSSES FROM THE SOIL: N gas emissions and N leaching

<figure><img src="image_6.png" /></figure>
**GAIN project:** Research and innovation → **nitrogen management**

for sustainable agriculture and mitigation of N losses

<figure><img src="image_7.png" /></figure>
**GAIN project:** Research and innovation → **nitrogen management**

for sustainable agriculture and mitigation of N losses

<figure><img src="image_8.png" /></figure>

### Advantages BNI vs NI:

- Nature-based sustainable solution

- Accessible technology for all farmers and regions

- Reduces economic costs

- Avoid potential toxicity issues
<figure><img src="image_9.png" /><figcaption>First discovery in the tropical grass</figcaption></figure>

*Brachiaria humidicola*

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Is there BNI capacity in cereals?

<figure><img src="image_16.png" /><figcaption>Sorghum bicolor</figcaption></figure>

Grain and biomass production for livestock feeding

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /><figcaption>Rice (Oryza sativa)</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>Maize (Zea mays L.
cv Honey bantam)</figcaption></figure>

<figure><img src="image_20.png" /></figure>
There is **no** BNI activity in ELITE wheat cultivars

<figure><img src="image_21.png" /></figure>

WILD RELATIVES:

Natural occurrence of BNI capacity in
**wheat landraces.**

**Persia** wheat varieties, pervious to “green-revolution”

<figure><img src="image_22.png" /></figure>
**There is no BNI activity in ELITE wheat cultivars**

<figure><img src="image_23.png" /></figure>

**WILD RELATIVES:**

*Leymus racemosus*

Perennial member of the *Triticeae*

<figure><img src="image_24.png" /></figure>

The gene for storage of energy in L.

<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

**Breeding programmes**

Subbarao et al. (2021) PNAS.

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /></figure>

*Leymus racemosus*

<figure><img src="image_29.png" /></figure>

<figure><img src="image_30.png" /></figure>

Chinese spring

F₁ X ELITE wheat lines

<figure><img src="image_31.png" /></figure>

F₂

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F₇ BNI-wheat lines
<figure><img src="image_33.png" /></figure>

Brachiaria

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brachialactona

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Linoleic acid

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α-Linolenic acid

<figure><img src="image_37.png" /></figure>

Oryza sativa (arroz)

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1,9-decanediol

<figure><img src="image_39.png" /></figure>

Acido sirínlico

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Sorghum

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sorgoleone

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MHPP

Methyl 3-(4-hydroxyphenyl) propionate

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sakuranetina

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Zea mays

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Zeanone (1)
[ 2,7-Dimethoxy-
1,4-naphthoquinone ]

<figure><img src="image_46.png" /></figure>

HDMBOA (2)
[ 2-Hydroxy-4,7-dimethoxy-2H-
1,4-benzoxazin-3(4H)-one ]

<figure><img src="image_47.png" /></figure>

α-Linolenic acid

<figure><img src="image_48.png" /></figure>

Triticum aestivum

Not identified
<figure><img src="image_49.png" /></figure>

Incorporation of **BNI producing catch-crops** in rotation with target crops for the stabilization of soil **ammonium pools**.
<figure><img src="image_50.png" /></figure>

SusCrop - ERA-NET
Cofund on Sustainable Crop Production

FACCEJPI

<figure><img src="image_51.png" /></figure>

Funded by
the European Union

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<figure><img src="image_53.png" /></figure>

Determination of N₂O gas emission (Closed chamber method)

<figure><img src="image_54.png" /></figure>

Hensen et al., 2013; Bayabil et al., 2016
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➤ Determination of N₂O gas emission (Closed chamber method)

➤ Determination of soil mineral N pools: NH₄⁺ and NO₃⁻

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<figure><img src="image_57.png" /><figcaption>Soil sampling in the field</figcaption></figure>

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<figure><img src="image_60.png" /><figcaption>Soil sampling from pots</figcaption></figure>

<figure><img src="image_61.png" /></figure>
➤ Determination of N₂O gas emission (Closed chamber method)

➤ Determination of soil mineral N pools: NH₄⁺ and NO₃⁻

➤ Determination of soil **nitrifying microbial populations**.

• Marker gene for ammonia oxidizing **bacteria** (AOB) and **archaea** (AOA) → *amoA*

<figure><img src="image_62.png" /></figure>

<figure><img src="image_63.png" /></figure>

<figure><img src="image_64.png" /></figure>
## FIELD STUDIES. Spain

<figure><img src="image_65.png" /></figure>

Universidad
del País Vasco

Euskal Herriko
Unibertsitatea

<figure><img src="image_66.png" /></figure>

Grupo AN
DESDE 1960

### 1º rotation (2021): Sorghum

<figure><img src="image_67.png" /></figure>

### 2º rotation (2022): Wheat (cv García)

<figure><img src="image_68.png" /></figure>

Selection of optimum Sorghum cultivars is necessary to improve
wheat yield in rotation systems

<figure><img src="image_69.png" /></figure>

<figure><img src="image_70.png" /></figure>

<figure><img src="image_71.png" /></figure>

ic
TO extend:

Other examples from the field

- Sorhgum. Under drought conditions.

- Crop rotations with Brachyaria...
FIELD STUDIES. Spain

Sorghum culture decreased soil AOB
population only in 2017

Sorghum would increase its BNI activity
under drought conditions

**Greenhouse study:** Is there BNI capacity in sorghum under moderate drought conditions?

<figure><img src="image_72.png" /></figure>

During the drier 2017 season, aerobic conditions would favor nitrification and increase the efficiency of sorghum BNI activity under drought conditions.

<figure><img src="image_73.png" /></figure>
**Main objective:** To develop sustainable tools for ammonium fertilizer management, mitigating soil nitrogen losses and advancing towards efficient and full-organic farming systems.

1. Identify adequate organic N fertilizers to be stabilized with synthetic nitrification inhibitors

2. To assess the potential of rhizospheric soil from BNI-producing crop genotypes to effectively reduce nitrification and mitigate N₂O emissions from organic amendment
**Main objective:** To develop sustainable tools for ammonium fertilizer management, mitigating soil nitrogen losses and advancing towards efficient and full-organic farming systems.

1. Identify adequate organic N fertilizers to be stabilized with synthetic nitrification inhibitors

2. To assess the potential of rhizospheric soil from BNI-producing crop genotypes to effectively reduce nitrification and mitigate N₂O emissions from organic amendment

## MICROCOSMS INCUBATION EXPERIMENT:

soil + fertilizer + synthetic nitrification inhibitor DMPP

<figure><img src="image_74.png" /></figure>

Under controlled conditions:
60 % WFPS
21/24 °C

<figure><img src="image_75.png" /></figure>

DMPP (3, 4-Dimethylpyrazole Phosphate)

<figure><img src="image_76.png" /></figure>
1 synthetic fertilizer and 4 types of organic fertilizers

(100 mg N/ kg dry fertilizer)

- **Ammonium sulphate** (mineral, solid)

<figure><img src="image_77.png" /></figure>

- **DIGESTATE** (Liquid)

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<figure><img src="image_79.png" /></figure>

<figure><img src="image_80.png" /></figure>

<figure><img src="image_81.png" /></figure>

From Hemapriya & Ganesh, 2024
## 1 synthetic fertilizer and 4 types of organic fertilizers

(100 mg N/ kg dry fertilizer)

* **Ammonium sulphate** (mineral, solid)

100 % $NH_4^+$

<figure><img src="image_82.png" /></figure>

* **DIGESTATE** (Liquid)

46 % $NH_4^+$

<figure><img src="image_83.png" /></figure>

* **CATTLE SLURRY** (Solid)

31 % $NH_4^+$

<figure><img src="image_84.png" /></figure>

* **ORGAMINE pellets** (Solid)

31 % $NH_4^+$

<figure><img src="image_85.png" /></figure>

* **CHICKEN MANURE** (Solid)

13 % $NH_4^+$

<figure><img src="image_86.png" /></figure>

<figure><img src="image_87.png" /></figure>

<figure><img src="image_88.png" /></figure>
Non-destructive

Monitoring of N₂O emissions.

<figure><img src="image_89.png" /></figure>

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Day 0

Day 7

Day 15

Day 48

<figure><img src="image_103.png" /><figcaption>Infrared gas
analyzer</figcaption></figure>

<figure><img src="image_104.png" /></figure>

LIÈGE université

Gembloux Agro-Bio Tech

Biosystems Dynamics and Exchanges

<figure><img src="image_105.png" /></figure>
<figure><img src="image_106.png" /></figure>

Soil sampling for destructive analyses :

- Mineral nitrogen

- Total and dissolved organic carbon

- pH

- microbial DNA
Stabilization of organic N fertilizers with synthetic nitrification inhibitors

<figure><img src="image_107.png" /><figcaption>Incubation Day</figcaption></figure>

<figure><img src="image_108.png" /></figure>

DMPP effectively reduces N₂O emission from both inorganic/organic fertilizers

<figure><img src="image_109.png" /></figure>

Reduction in N₂O emmsion by DMPP
DMPP stabilizes ammonium fraction of the fertilizers delaying it's consumption by the inhibited ammonia oxidizing bacteria (AOB) population

<figure><img src="image_110.png" /></figure>

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→ **OBJECTIVE :** Assessing if rhizospheric soil from BNI-producing crop genotypes can decrease nitrification and subsequent N₂O emissions

Selected of species/genotypes :

<figure><img src="image_116.png" /><figcaption>Triticum aestivum (wheat)</figcaption></figure>

→Persia 44

<figure><img src="image_117.png" /><figcaption>Sinapis alba (white mustard)</figcaption></figure>

→ 2 genotypes (SA4 and SA9)
## Selection of species/genotypes :

<figure><img src="image_118.png" /></figure>

*Results from previous screening experiments*

<figure><img src="image_119.png" /><figcaption>Hydroponic growth of crops</figcaption></figure>

<figure><img src="image_120.png" /><figcaption>Collection of root exudates</figcaption></figure>

<figure><img src="image_121.png" /><figcaption>Nitrosomonas bioassay with root exudates</figcaption></figure>

## System previously optimised by the team in CATCH BNI project :

Jauregui, I., Vega-Mas, I., Delaplace, P., Vanderschuren, H., & Thonar, C. (2023). An optimized hydroponic pipeline for large-scale identification of wheat genotypes with resilient biological nitrification inhibition activity. *New phytologist*, 238(4), 1711-1721.
## Selection of species/genotypes :

<figure><img src="image_122.png" /></figure>

Results from previous screening experiments

<figure><img src="image_123.png" /></figure>
Experimental set-up : Short-term (3 weeks) incubation experiment

<figure><img src="image_124.png" /></figure>

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20°C, 60% WFPS
Total N₂O emissions

<figure><img src="image_127.png" /></figure>

NT Soil not treated

SDMPP Soil + DMPP

P44 Rhizospheric soil *T.aestivum* Persia 44

SA4 Rhizospheric soil *S.alba* SA4

SA9 Rhizospheric soil *S.alba* SA9

<figure><img src="image_128.png" /></figure>

<figure><img src="image_129.png" /></figure>
<figure><img src="image_130.png" /></figure>

# Stabilization of organic N fertilizers with biological nitrification inhibitors (BNIs)

<figure><img src="image_131.png" /><figcaption>## NO₃⁻-N concentrations</figcaption></figure>

<figure><img src="image_132.png" /></figure>

<figure><img src="image_133.png" /></figure>

<figure><img src="image_134.png" /></figure>
* Involvement in two new european projects:

## FERTIGO

<figure><img src="image_135.png" /></figure>

<figure><img src="image_136.png" /></figure>

UNIVERSITÉ
LIDRE
DE BRUXELLES

<figure><img src="image_137.png" /></figure>

IPK
LEIBNIZ INSTITUTE

<figure><img src="image_138.png" /></figure>

John Innes Centre
Unicasting. Nature's Diversity

<figure><img src="image_139.png" /></figure>

<figure><img src="image_140.png" /></figure>

To determine the effect of *Plantago lanceolata* and other P mobilising species on N/P cycles in several context:

- temporary grassland and cover crops mixture in Ireland

- maize intercropped with companion plants in Germany

- stabilization of fertilizers in Belgium

## PLANTAGO

<figure><img src="image_141.png" /></figure>

UNIVERSITÉ
LIDRE
DE BRUXELLES

FiBL

To acquire fundamental knowledge on the potential of *P. lanceolata* to minimize N losses from agroecosystems:

- Identify genotypes with proven BNI potential

- find molecular markers associated with BNI release

- decipher mode of action of BNI metabolites

- evaluate effect of plantain on functional microbial communities
Thank you for listening

<figure><img src="image_142.png" /></figure>

https://www.suscrop.eu/projects-second-call/catch-bni

This project has received funding from the European Union's Horizon 2020 research and innovation programme, under the SUSCrop-ERA-Net COFUND programme (grant agreement No [771134]).

<figure><img src="image_143.png" /></figure>

<figure><img src="image_144.png" /></figure>

SusCrop - ERA-NET

Cofund on Sustainable Crop Production

FACCEJPI

<figure><img src="image_145.png" /></figure>

This project has been found by the SPW, Project D31-1378-S1-GAIN

<figure><img src="image_146.png" /></figure>
NH₄⁺-N concentrations

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