Does subjecting plants to water stress enhance biological nitrification inhibition potential of rice?

Biological nitrification inhibition (BNI) is a trait that could improve nitrogen-use efficiency of a crop. We studied varietal differences in BNI potential of rice at the vegetative phase when plants were subjected to water stress. We obtained water-soluble root exudates (RE) and water-extracts of crushed root tissues (RT) from two-week-old seedlings of five rice cultivars grown continuously under adequate water or under water stress and assessed their ability to suppress the activity of Nitrosomonas europaea and the growth of lettuce seedlings. We also investigated how growing rice under continuously saturated (TC) and unsaturated (TU) soil moisture conditions affected potential nitrification rate (PNR) and community structure of ammonia-oxidizing bacteria (AOB) in the rhizosphere compared with bulk soil. We observed that only RT at ≥ 0.50 mg ml−1 suppressed the activity of N. europaea with significant (p < 0.05) differences among rice cultivars. Subjecting rice cultivars PI312777 and PI338046 to water stress significantly (p < 0.05) increased BNI of RT. Inhibition of lettuce root growth by RT was strongly correlated with its BNI (r = 0.83, p < 0.05). In T-RFLP profiles of AOB, the relative abundance of T-RF R205 was lower in TU than in TC in all cultivars except Rexmont. The rice cultivars studied produced compounds that could inhibit ammonia oxidizers, but the degree of BNI differed based on the moisture stress experienced by the crop. Lineages of rice cultivars with known allelopathic potential would be promising candidates to consider for BNI potential.


Introduction
Nearly 15% of global fertilizer nitrogen (N) is directed at a single crop, rice; yet farmers do not realize the full benefit of fertilization because agronomic N use efficiency is typically below 30% (Fixen et al. 2015).Poor N nutrition has been identified as a factor contributing to the highly unpredictable and low yield of aerobicrice (Haefele et al. 2008).The continuing search for plant traits that improve agronomic N use efficiency (NUE) is important for developing more efficient crop varieties.This is particularly beneficial in rice systems where soil remains under unsaturated (aerobic) conditions for more than half of the growing season as a water conservation strategy (Farooq et al. 2022).
Pool sizes of the plant available N forms ammonium (NH 4 + ) and nitrate (NO 3 − ) in soil are affected by nitrification, a biological transformation that occurs rapidly under unsaturated soil conditions yielding NO 3 − .In agricultural soils, nitrification is mainly catalyzed by a diverse group of ammonia oxidizing bacteria (AOB), ammonia oxidizing archaea (AOA), complete ammonia oxidizers (comammox) and nitrite oxidizers.The O 2 availability, NH 4 + concentration and pH like environmental factors mainly govern the dominance of each of these groups and their community structures and relative contribution to nitrification (Briones et al. 2002;Wang et al. 2009;Daims et al. 2015;Zhang et al. 2017).For example, in non-acidic soils under non-limiting NH 4 + supply, such as fluxes after N-fertilizer application, AOB communities can dominate nitrification, whereas AOA can dominate nitrification at low NH 4 + concentration and acidic soil conditions (Wang et al. 2009;Zhang et al. 2017;Farooq et al. 2022).Nitrosomonas spp.and Nitrosospira spp.are examples of as commonly found AOB in paddy soils (Wang et al. 2009;Farooq et al. 2022).It has been predicted that comammox are slow-growing K-strategists adapted to low substrate (i.e.NH 4 + ) concentration and capable of forming microcolonies, flocs and biofilms (Daims et al. 2015).Members of Nitrospira lineage that perform as comammox have been reported in many environments including rice paddy soils (Daims et al. 2015;Pjevac et al. 2017).
Soils undergoing drying and wetting cycles enhance nitrification and denitrification processes, respectively, as occurs during periods of water conservation in rice cropping systems and during rainfed cultivation.Thus, the loss of N through nitrification-denitrification is a major concern that needs to be addressed to build resilient rice systems in the face of climate change (Farooq et al. 2022).The inhibition of nitrification as a method to improve NUE is not a new concept.Synthetic nitrification inhibitors such as nitrapyrin and dicyandiamide (DCD) have been used in upland cereal cropping systems in some parts of the world.Furthermore, their effectiveness in inhibiting nitrification varies widely depending on the community composition of nitrifiers (Matsuba et al. 2003;Papadopoulou et al. 2020).Moreover, synthetic nitrification inhibitors are not widely available or affordable for resource poor farmers in developing nations.
Biological nitrification inhibition (BNI), the suppression of nitrification by biologically active plantderived compounds, is a relatively novel approach for improving NUE in crop production (Subbarao et al. 2006a(Subbarao et al. , 2015)).The BNI potential of different species in the Poaceae, including the Triticeae Tribe, has been studied extensively by Subbarao and co-workers who established positive relationships between shoot N levels and BNI activity of roots (Subbarao et al. 2006a(Subbarao et al. , 2015;;Gopalakrishnan et al. 2009).For species within the Triticeae Tribe, the BNI potential was shown to be a trait that can be genetically transferred via plant breeding approaches (Subbarao et al. 2015(Subbarao et al. , 2021)).BNI-MUNAL is a wheat cultivar that was developed by incorporating the chromosome region that controls BNI production from "wheat grass" Leymus racemosus (Lam.).This modification in the wheat cultivar enhanced suppression of soil nitrifiers, reduced nitrification potential and N 2 O emissions from soil while significantly increasing yield by 10 to 14% under field condition (Subbarao et al. 2021).At the heading stage of wheat, the effectiveness of suppressing nitrification by "BNI-enabled wheat" was found to be comparable to the suppression achieved by applying nitrapyrin at 0.05 kg ha −1 (Subbarao et al. 2006a).The application rate of nitrification inhibitors will vary depending on temperature and soil conditions in the target region (Bronson et al. 1989;Ali et al. 2008).A crop with BNI potential could be a viable means of improving NUE compared with a one-time application of a commercial nitrification inhibitor considering cost, phytotoxic effects, and other environmental concerns related to nitrification inhibitors (Bronson et al. 1989;Subbarao et al. 2006a;Ali et al. 2008).Some rice cultivars have been found to have the BNI trait (Tanaka et al. 2010;Dandeniya 2014;Lu et al. 2019).Recent findings have demonstrated that 1, 9-decanediol, a compound found in rice roots, can inhibit nitrification (Lu et al. 2019;Zhang et al. 2019).Using a known allelopathic rice cultivar (PI312777) in the presence of weeds, Kong et al. (2008) reported reductions in the number of cultivable AOB and total phospholipid fatty acids in the rhizosphere compared with a non-allelopathic rice cultivar.Tanaka et al. (2010) reported that the BNI potential of root exudates was significantly different among rice cultivars based on a study conducted using a recombinant bioluminescent strain of Nitrosomonas europaea.Yet BNI characterization, identification of the genetics associated with BNI production and the possibility of introducing BNI-traits to improved cultivars of rice remains largely unexplored.The rice cultivars with allelopathic traits and those adopted for upland soil conditions (aerobic rice) would be good candidates for investigating BNI traits.
The concentration of biologically active compounds such as allelochemicals in the rhizosphere can vary according to crop growth stage, dilution by soil water, and metabolic activities of root-dwelling microbial communities (Gopalakrishnan et al. 2009;Kaur et al. 2009).Plants under biotic stress from pests, diseases, or weeds, or from abiotic stress such as water limitation are known to have greater root exudation relative to unstressed plants (Bertin et al. 2003;Bi et al. 2007).The composition of these exudates also differs in stressed and unstressed plants.
There is therefore a knowledge gap with respect to whether the BNI potential of rice cultivars change in response to moisture stress of the plant.It should be noted that in a soil-plant system, separating the BNI effect on the activity of nitrifiers from other possible nitrification-suppressing mechanisms is challenging (Verhagen et al. 1994;Nardi et al. 2013Nardi et al. , 2020;;Subbarao et al. 2015;Illarze et al. 2021).Therefore, research on BNI should take a polyphasic approach (Nardi et al. 2020).Studying BNI potentials using laboratory bioassays can generate preliminary information, which can be useful to further design BNI enabled plants (Kaur-Bhambra et al. 2021;Subbarao et al. 2021).
In the research reported herein, we investigated the effect of moisture stress on the BNI potential of rice seedlings using a series of laboratory bioassays and a microcosm study.We first studied the impact of water-soluble, root-derived compounds of rice grown with and without moisture stress on the activity of N. europaea and lettuce (Lactuca sativa L.) seedling growth to assess the relationship between BNI and allelopathy.Secondly, we assessed the effects of rice cultivar and moisture management on ammonium oxidizing archaeal and bacterial (AOA and AOB, respectively) community structure in the rhizosphere compared with that in bulk soil.We included known allelopathic and non-allelopathic rice cultivars developed for paddy rice cultivation and cultivars with unknown allelopathic traits and developed for aerobic rice cultivation for this study.

Planting material
The five rice cultivars used in the experiments included two allelopathic cultivars (PI312777 and PI338046), two non-allelopathic cultivars (PI502968 and IR55423), and cultivar IR80508-B-57-3-B which has not been tested for allelopathic effects.The allelopathic potential of these cultivars has been defined based on their ability to interfere with weed seed germination and growth (Khanh et al. 2007;Kong et al. 2008).Seeds of cultivars PI312777 and PI338046 were obtained from the Dale Bumpers National Rice Research Center, AR, USA.Seeds of cultivars IR55423 (Apo) and IR80508-B-57-3-B (ApCr) were obtained from the International Rice Research Institute, Los Baños, The Philippines.Seeds of PI502968 (Rexmont) were obtained from the National Small Grain Collection, USDA-ARS, Idaho, USA.Rice seeds used in the experiments were surface-sterilized with 1% sodium hypochlorite (NaClO) for 30 min and soaked in sterilized distilled water for 24 h (Bi et al. 2007).

Extraction of rice-root derived compounds
Rice cultivars PI312777, PI338046, Apo, and ApCr were tested for their BNI potential.Rice was grown in a capillary mat system to generate large quantities of living root tissues as described previously (Czarnota 2001;Bertin et al. 2003).Two watering regimes were used as treatments: (a) continuous watering (CW), and (b) water stressed (NW).Three replications per rice cultivar by watering regime treatment combination were used (SI Appendix, Study 1).The capillary mat system was constructed by placing wet double layer cheesecloth (12 × 55 cm) (Krackeler Scientific, Inc., Albany, NY) along the length on a bright aluminum window screen (28 × 43 cm).One hundred surface sterilized and soaked rice seeds were placed in between two layers of wet cheese cloth covering an area of 25 cm 2 per cultivar per replicate.The screens were placed sandwiched between VattexP capillary mat (Hummert International, Earth City, MO), and a Weed-X weed mat (Dalen Products, Inc., Knoxville, TN).One edge of the VattexP capillary mat, the Weed-X weed mat, and the cheesecloth were draped into a water trough to facilitate capillary watering.The entire system was covered with 4 mm clear plastic to provide the seeds with a humid environment.The capillary mat systems were placed in a controlled environment chamber with a temperature of 25 ± 1ºC and a photoperiod of 14/10 h light/dark.The capillary mat system was kept moistened with Milli-Q water until 14 days after germination (DAG) for the CW treatment; whereas watering was discontinued at 9 DAG and allowed to gradually dry to impose the water stress treatment (NW) (Bertin et al. 2003).At 7 DAG, plants were watered once with half strength Hoagland's nutrient solution (Hoagland and Arnon 1950).At 14 DAG, roots were harvested by separating roots from the adjacent metal screen with a razor blade.The fresh roots from each experimental unit (i.e., replicate of a cultivar × moisture treatment com- bination) were submerged in 25 ml of Milli-Q water at 25 °C separately for 30 min to extract water soluble root exudates (RE) (Gransee and Wittenmayer 2000).The extracts were filtered using Whatman No 42 filter paper and stored at 4 °C until needed.After recording fresh weights, roots were oven-dried at 50 °C for 48 h.Dry weights of roots were recorded, roots were ground to a powder, and the water-soluble compounds were extracted with 30 ml Milli-Q water by placing containers on a shaker overnight at 120 rpm at room temperature followed by centrifuging and filtering the extracts through Whatman No 42 filter paper.This extract is referred to as root tissue extract (RT), hereafter.Solutions were stored at 4 °C until needed to prepare stock solutions.Both RE and RT extracts were concentrated using a rotary evaporator, evaporated under vacuum to obtain the dry matter, and reconstituted in water to obtain 10 mg ml −1 stock solutions, which were stored at -20 °C until needed.Aseptic conditions were maintained throughout the RE and RT preparation process.

The effect of rice-root derived compounds on nitrification
A pure culture of Nitrosomonas europaea (ATCC 19718) was used to assess the impact of different dilution levels of RE (0.05 and 0.50 mg ml −1 ) and RT (0.05, 0.50, and 1.0 mg ml −1 ) on the potential ammonia oxidation activity (PAOA).Activity of the N. europaea culture with added sterile distilled water to replace the RE/RT volume served as the control.Nitrosomonas europaea cells were cultivated in NH 4 + containing P-buffer medium at pH 7.8 (ammonium sulfate ((NH 4 ) 2 SO 4 ) added at 2.5 g L -1 ) in an incubator shaker (250 rpm; temperature 30 °C) (Subbarao et al. 2006b).After 14 days of incubation the N. europaea culture was used in the BNI bioassay.The Vol.: (0123456789) population level of N. europaea on day 14 was 10 4 cells ml −1 as determined by a most probable number technique (Rowe et al. 1977).
For the BNI bioassay, 400 µl of 14-day old N. europaea culture were mixed with 10, 100, or 200 µl of 10 mg ml −1 stock solution of RE or RT and 1590, 1500, or 1400 µl of fresh NH 4 + containing P-buffer medium to obtain 0.05, 0.50, and 1.0 mg ml −1 concentrations of root-derived compounds, respectively.The mixture was incubated at 30 °C while on a shaker at 250 rpm for 15 h.After 5, 10, and 15 h of incubation, 400 µl subsamples were collected and analyzed for nitrite colorimetrically using the sulfanilamide method (Shinn 1941).The BNI bioassay was performed in two laboratory replicates for each dilution level of the three stock solutions (RE/RT) from the combination of rice cultivar and water treatment.For the colorimetric analysis, a standard curve was prepared using sodium nitrite (Na 2 NO 2 ).The BNI potential of rice was calculated as the percent reduction in PAOA by RE or RT at a given concentration compared with the PAOA of the control.Nitrapyrin, a commercially available nitrification inhibitor (N-serve® with 99% active ingredient) was used with the N. europaea culture in a test series (5, 10, 15, and 20 µg ml −1 ) to compare with the BNI results (Zacherl and Amberger 1990).

Allelopathic potential of rice-root derived compounds
Due to its high sensitivity to allelochemicals, lettuce (Lactuca sativa L.) is commonly used in allelopathy bioassays (Elakovich and Wooten 1991).To assess the allelopathic potential of root-derived compounds, bioassays using lettuce (Harris® seeds, Rochester, NY, USA) were conducted.For each stock solution, a dilution series of RE (0.05 and 0.50 mg ml −1 ) and RT (0.05, 0.50, and 1.0 mg ml −1 ) were prepared in two laboratory replicates and used in bioassays.Ten lettuce seeds were placed on a Petri dish lined with a Whatman No.1 filter paper, which was moistened with 1.0 ml of aqueous extract at each respective dilution level.A control, which received 1.0 ml of Milli-Q water was included in the bioassay.Petri dishes were sealed and kept in a growth chamber with 12 h light/12 h dark cycle at 25 °C for 4 d.Germination percentage and root and shoot lengths of lettuce seedlings were measured at the end of 4 days and percent inhibition was calculated based on seedling growth in the control treatment.
Root-derived substance extractions from rice growing on capillary mats, and subsequent bioassays with ammonia oxidizers and lettuce seeds were repeated once to confirm the repeatability of results.

Microcosm construction
Microcosms were constructed using PVC couplings of 5.1 cm diameter and 3.8 cm height (Charlotte® pipe 1½ inch PVC coupling-Model #: PVC 00,100 0800).A Nitex 30 µm nylon mesh (Genesee Scientific, San Diego, CA, USA) was glued (vinyl fabric and plastic flexible adhesive) to the middle wall to divide the coupling into two compartments; a top rhizosphere compartment and a bottom soil compartment (SI Appendix, Study 2).The nylon membrane allowed free nutrient flow but restricted roots to the rhizosphere compartment.A soil sample collected at 0-10 cm depth from a rice paddy in the Cornell Botanic Gardens, Ithaca, NY, USA, was air-dried and sieved through a 2 mm mesh sieve.To increase the efficiency of separating rhizosphere soil from roots and to avoid crack formation and soil crusting during the experiment, soil was mixed with sterilized sand to achieve 70% sand, 12% clay, and 18% silt composition.The pH of the soil was 7.2.The two compartments of the microcosm were filled with soil.The bottom compartment was sealed, and a small hole was created and plugged with cotton to facilitate drainage.Each microcosm was placed in a water reservoir, which helped to regulate the soil moisture level in the rhizosphere compartment.Two soil moisture treatments were imposed, (i) continuously saturated soil (T C ) and (ii) unsaturated soil kept at near field capacity (T U ), starting six days prior to seeding rice.Microcosms were incubated at 25ºC.
Four rice cultivars were used in the microcosm experiment including ApCr, PI312777, and PI338046, and Apo was replaced with Rexmont, a well-known non-allelopathic rice cultivar, due to the availability of seeds.Rice seeds were surface-sterilized with 1% NaClO for 30 min, rinsed with sterile-distilled water and pre-germinated in Petri-dishes lined with wet filter papers for three days.Six germinated seeds with equal radicle lengths were planted in the rhizosphere compartment.Three replicates per cultivar per moisture treatment combination were used.The microcosms were placed in a growth chamber and plants were subjected to 28 °C day and 25 °C night temperature and a photoperiod of 14/10 h light/dark.Plants were fertilized with half strength Hoagland's nutrient solution at 7 and 10 DAG.Soil and plant sampling were performed at 14 DAG.Soil was separated from roots and placed in plastic sampling bags and immediately analyzed for potential nitrification rate (PNR) and plant available N content.A subsample of rhizosphere soil was stored at -20˚C to extract soil DNA for microbial community profiling.Shoots were separated from roots and oven-dried at 65 °C for 48 h.

Soil and plant analyses
The potential nitrification assay was conducted using the shaken-slurry method as described by Hart et al. (1994) modified for sample size.In brief, for each sample, 3 g of soil (oven dry weight equivalent) was placed into a 125 ml Erlenmeyer flask and a 25 ml aliquot of phosphorus buffer solution containing 1.5 mM (NH 4 ) 2 SO 4 was added.Flasks were shaken at 180 rpm on an orbital shaker and aliquots were removed at 1.5, 3, and 5 h after incubation, and analyzed colorimetrically for NO 3 − -N as described by Cataldo et al. (1975).
Available inorganic nitrogen in soil from the microcosm experiment was extracted with 2 M potassium chloride (KCl) and analyzed colorimetrically for NH 4 + and NO 3 − using an auto-analyzer (Seal Analytical Inc., Mequon, WI, USA).Shoots were oven-dried, weighed, and analyzed for total C and N using the dry combustion method (automatic carbon-nitrogen analyzer NC2100, EA/NA 1110, ThermoQuest Italia S.p.A., Milan, Italy).The physiological NUE (PNUE) of the plants was calculated as dry biomass accumulated per gram of nitrogen acquired by the plant.

Terminal restriction fragment length polymorphism (T-RFLP) analysis
DNA was extracted from sampled soils using the PowerSoil™ DNA extraction kit (MoBio Laboratories, Carlsbad, CA) and used for PCR to perform T-RFLP analysis.Ammonia-oxidizing bacterial amoA genes were amplified using primers amoA-1F (5'-GGG GTT TCT ACT GGT GGT -3') and amoA-2R (5'-CCC CTC KGSAAA GCC TTC TTC -3') (Rotthauwe et al. 1997).From the primer pair amoA-2R was fluorescently labeled to generate T-RFLP 'fingerprint' profiles.Reaction mixture consisted of 10 mM PCR buffer, 4 mM MgCl 2 , 200 μM dNTPs, 0.1 μM Primer, 1 mg ml −1 BSA, and 0.025 U μl −1 Taq polymerase.PCR cycles were as follows: an initial denaturation for 5 min at 94 ˚C; then 35 cycles of 1 min denaturation at 94 ˚C, 1 min annealing at 57 ˚C, 90 s extension at 72 ˚C; finished by a 10 min final extension at 72 ˚C.Reactions were performed in a 50 μl reaction volume with approximately 100 ng DNA per reaction.For each soil DNA extract, a PCR reaction was performed in triplicate and products were pooled, vacuum-dried, and reconstituted in sterile molecular grade water to obtain 20 ng DNA μl −1 prior to use in restriction digestion (Singh et al. 2006).Therefore, each pooled PCR products per treatment contained amplicons from nine reactions (3 replicates of DNA extracts × 3 PCR).PCR products were restricted using the TaqI enzyme.Restriction digest products were column-purified using an EdgeBio purification plate (Applied Biosystems, Foster City, CA) and were lyophilized for a final time.DNA was resuspended in a 10 μl mix containing 9.85 μl of formamide and 0.15 μl of Liz 500 size standard (Applied Biosystems) and terminal restriction fragments (T-RFs) size analysis was performed using an ABI 3730 electrophoretic capillary sequencer (Applied Biosystems) located at the Cornell Biotechnology Core Laboratory, Ithaca, NY.
To predict which species the T-RFs represented we performed a simulation experiment using online tools.We retrieved a set of nucleotide sequences of amoA genes of Nitrosospira and Nitrosomonas species from NCBI database used in NEBcutter (https:// nc3.neb.com/ NEBcu tter/) a restriction analysis tool, to determine the DNA restriction pattern corresponding to TaqI enzyme.The results were compared with the information reported in literature.

Statistical analysis
Data generated from the allelopathy, BNI, and microcosm experiments were tested for normality and used in analysis of variance (ANOVA) with the generalized linear model (GLM) procedure in a factorial design to determine significance of treatment effects using JMP 8.0® software (SAS Institute Inc., Cary, NC, USA).
Rice cultivar, moisture treatment, and dilution level of root-derived compounds were used as the grouping factors in the allelopathy and BNI studies.Rice cultivar and moisture treatment were used as the grouping factors in the microcosm study.When treatment effects were significant at p < 0.05, means were compared using the LSD mean separation technique (p < 0.05).
T-RFLP profiles were analyzed using GeneMapper Software v 3.0 (Applied Biosystems).T-RFLP profiles were further analyzed using T-REX, a web-based tool (http:// trex.biohpc.org/), as described previously (Culman et al. 2009).To align T-RFLP profiles, we used a clustering threshold of five for amoA based on positive controls used for correction of shifts in peaks due to limitations in the T-RFLP method related to the fluorescent signals (Culman et al. 2009).To avoid detection of primers and uncertainties of size determination, terminal fragments (T-RFs) smaller than 40 bp and larger than 500 bp were excluded from further analysis (Wang et al. 2012).The relative abundance of different T-RFs within the sections was determined by calculating the ratio between the area of each peak and the total area of all peaks within one sample.The T-RFs with relative abundance below 3% were regarded as background noise and excluded from the analysis (Wang et al. 2012).

Results
The average osmolality of 10 mg ml −1 stock solutions of RT and RE was 53 ± 11 mOsmols kg −1 .The pH of RE and RT at 1.0 mg ml −1 in water ranged from 4.5 to 6.5 but adding RE or RT did not change the pH of the phosphate buffer medium (pH 7.8) used in the BNI assay.The osmotic potential and pH of RT and RE were not significantly different (p > 0.05) among the three dilution levels used in the bioassay and treatment and cultivar effects were not significant (p > 0.05) on these two variables.

Allelopathic and BNI potentials of rice-root derived compounds
Lettuce root growth and ammonia oxidation were not significantly (p > 0.05) affected by rice cultivar, RE concentration, or the moisture stress experienced by rice plants.There was no relationship between BNI activity and lettuce root growth inhibition by RE (Fig. 1a), but a significant correlation between these two variables (r = 0.83; p < 0.05) was observed (Fig. 1b) when RT was applied.In some instances, RE enhanced lettuce root growth and N. europaea activity (Fig. 1a).Lettuce root growth was significantly inhibited by RT at all three concentrations (Fig. 2), but RT from all four rice cultivars inhibited the activity of N. europaea only when applied at the 1.0 mg ml −1 concentration (Fig. 3).Varietal differences, moisture stress, and their interaction were significant only at RT concentrations of 0.5 and 1.0 mg ml −1 .At 1.0 mg ml −1 concentration of RT, the two allelopathic rice cultivars, PI312777 and PI338046, had higher BNI potential (Fig. 3) when the plants were grown under water stress than when the plants were continuously watered.However, this effect was not observed for lettuce growth (Fig. 2).The BNI potential of RT at 1.0 mg ml −1 ranged from 12 to 52% (Fig. 3), which was equivalent to the nitrification inhibition by 3 to 10 µg ml −1 of nitrapyrin, respectively (Fig. 4).

Nitrification inhibition in rice-rhizosphere soil
The rhizosphere compartment of the microcosms was filled with roots providing maximum exposure of soil to plant roots.The PNR in the rhizosphere soils of all four rice cultivars (Rexmont, ApCr, PI312777, and PI338046) was significantly lower than that of the control (bulk soil) (Table 1).The effects of rice cultivar, soil moisture treatment, and their interactions on PNR were significant (p < 0.05).Cultivars PI312777 and PI338046, the Fig. 2 Inhibition of lettuce root growth by water-soluble root tissue extract (RT) from 14-day old seedlings of four rice cultivars at three concentrations.RT was extracted from rice grown at two moisture levels: continuously watered (CW) and water stressed (NW).Percent inhibition was calculated as the reduced activity under treatment compared with the activity when inoculated with water.Vertical bars, within one RT concentration level having a different letter are significantly different (p < 0.05).Error bars represent the standard deviation Fig. 3 Biological nitrification inhibition of N. europaea in a laboratory bioassay by root tissue extract (RT) from 14-day old seedlings of four rice cultivars applied at three concentrations.Rice was grown at two moisture levels: continuously watered (CW) and water stressed (NW).Percent inhibition was calculated as the reduced activity under treatment in comparison to the activity when inoculated with water.Vertical bars within one RT concentration level having a different letter are significantly different (p < 0.05).Error bars represent the standard deviation two cultivars known for their allelopathic effects on weeds, and Rexmont (the non-allelopathic cultivar) had comparable PNR in their respective rhizospheres (Table 1).Physiological NUE (PNUE) was significantly different among the four rice cultivars only under saturated soil conditions (T C ) (Table 1).Potential nitrification rates in the rhizosphere were not correlated with plant PNUE, plant available inorganic N in soil, or the shoot biomass.But, soil PNRs were strongly correlated with the NO 3 − :NH 4 + ratio therein (r = 0.8 at p < 0.05), suggesting the effect of nitrification on speciation of available N in soil.In the analysis of bacterial amoA gene T-RFLP profiles, five T-RFs remained for analysis after excluding T-RFs with relative abundance below 3%.Each T-RF was considered as an operational taxonomic unit (OTU).The OTU 1 to OTU 5 corresponds to the reverse T-RFs; R49, R68, R86, R106 and R205 respectively.The total relative abundance of the excluded T-RFs (those with relative abundance below 3%) in the 40 bp to 500 bp category was less than 5%.The relative abundance of OTU1 and OTU5 varied greatly among the studied treatments with OTU5 being the most abundant type (Fig. 5).
The simulation experiment conducted using online molecular biology tools indicated that the amoA gene of Nitrosospira spp.and Nitrosomonas europaea amplified using the amoA1F primer and the fluorescently labeled amoA2R primer and digested with TaqI enzyme would result in T-RFs of approximately 206 bp and 272 bp (R206 and R272), respectively.As summarized in Table 2, the studies conducted with double labeled T-RFLP to identify group-specific T-RFs representing several groups of AOB indicates the complexity in assigning phylogenetic identities to T-RFs.For example, OTU5 could represent Nitrosospira-like and/or Nitrosomonaslike sequence types.We did not detect revers T-RFs of 270-272 bp length that could directly designate to Nitrosomonas europaea in this study (Fig. 5 and SI Appendix, S1).The relative abundance of OTU5 was higher in soils that received T C compared to  T U moisture treatment in all tested cultivars except non-allelopathic Rexmont (Fig. 5).

Discussion
Isolating the effects of biologically active compounds on the activity of nitrifiers from other possible nitrification-suppressing mechanisms is challenging (Verhagen et al. 1994;Nardi et al. 2013Nardi et al. , 2020;;Subbarao et al. 2015;Illarze et al. 2021).In addition to BNI, nitrification in the rhizosphere can be suppressed by plants and other microorganisms via competition for NH 4 + , and by favoring fast-growing heterotrophs in the rhizosphere that outcompete slow-growing nitrifiers (Verhagen et al. 1994;Bottomley et al. 2012;Nardi et al. 2013).Therefore, we experimented at first on BNI of rice in the absence of a soil matrix.It should be noted that the method of extraction, choice of extractant, presence or absence of stress signals to plants and plant growth conditions affect the allelochemical composition of root exudates (Gransee and Wittenmayer 2000;Bi et al. 2007;Subbarao et al. 2007;Al-Sherif et al. 2013;Tesfamariam et al. 2014).Osmotic potential and pH of the extractant can also influence the outcome of allelopathy bioassays by modifying the growth medium (Elakovich and Wooten 1991).Lettuce seedling growth is not inhibited at osmolality below 70 mOsmols kg −1 and is not affected by pH ranging from 4 to 8 (Chou and Young 1974;Elakovich and Wooten 1991).Based on a previous study, the growth rate of N. europaea in liquid culture medium is not affected by 0.10 M NaCl, which is equivalent to 200 mOsmoles kg −1 (Wood and Sørensen 1998).We observed that pH of the phosphate buffer growth medium used in the BNI assay was not significantly affected by RE or RT addition.Therefore, we can rule out the effect of osmotic potential and pH of RE and RT concentration we used on lettuce seedling growth and PAOA of N. europaea in this study.
The expression of BNI traits in Brachiaria is sensitive to the NH 4 + concentration in the growth medium; thus, immersing intact roots in 1 mM NH 4 Cl solution has been used as an approach to extract exudates from plant roots in previous BNI studies (Subbarao et al. 2007).In the present study, we used water as the extractant, which had been used for extracting exudates from plant roots including allelochemicals (Gransee and Wittenmayer 2000;Zhang and Fu 2010;Al-Sherif et al. 2013).Some BNI compounds are known to be hydrophobic (Subbarao et al. 2007) and such compounds may not have been extracted by cold water (at 25 °C).This may be one reason for not observing the BNI activity with RE.The composition of the trap solution used for root exudate extraction from rice affected BNI potential of the extract (Tanaka et al. 2010).We observed that water-soluble, root-derived compounds of all the rice cultivars at 0.05 mg ml −1 concentration enhanced the activity of N. europaea and activity did not correlate with allelopathic effects on lettuce seedling growth.Dandeniya (2014) reported water-soluble root exudates (derived from 0.4 mg roots ml −1 ) of 10 rice cultivars augmented the activity of nitrifiers in a laboratory culture medium but had variable effects on nitrifiers in soil.However, the same study reported that water-soluble root tissue extracts (at 0.4 mg ml −1 ) suppressed the activity of nitrifiers in culture and in soil.Although N. europaea is a well-known chemolithoautotroph that uses CO 2 as the cellular C source, its ability to grow as a chemolithoorganotroph, using fructose and other organic compounds, such as pyruvate and amino acids, has been shown in several studies (Clark and Schmidt 1967;Hommes et al. 2003).Water-extracted rice root exudates contain amino acids, whose concentration is highest during the first two weeks after seeding relative to other growth stages (Bacilio-Jimenez et al. 2003).Also, there may be other organic compounds that promote N. europaea growth in RE.Gransee and Wittenmayer (2000) reported that cold water extracts of root exudates from maize and pea plants at early vegetative stage contained sugars, amino acids and carboxylic acids.Our observations indicate that water-soluble RT of all four rice cultivars had a high potential to inhibit the activity of N.
Table 2 Phylogenetic information of the dominant T-RFs generated from T-RFLP profiles of bacterial amoA genes based on literature review # Only the studies that used T-RFs generated from PCR amplification using the primers amoA-1F (5'-GGG GTT TCT ACT GGT GGT -3') and amoA-2R (5'-CCC CTC KGSAAA GCC TTC TTC -3') (Rotthauwe et al. 1997)  Vol:.( 1234567890) europaea at 1.0 mg ml −1 concentration.Rhizodeposition of 1 mg ml −1 is a high concentration that is likely to occur in the vicinity of a mass of decaying roots (Uren 2007).It should be noted that a BNI enabled plant will be continuously releasing inhibitory compounds to the rhizosphere at low rates in contrast to one-time application of commercial nitrification inhibitors like nitrapyrin and DCD at high dose.
Apo and ApCr are two cultivars bred for use in unsaturated soil conditions; whereas PI312777 and PI338046 are well-known allelopathic cultivars bred for paddy systems, where soil is mostly water-saturated.Given the strong correlation between suppression of lettuce root growth and BNI activity by root extracts, rice cultivars known for their allelopathic effects on weeds and their lineages can be considered as candidates in the search for BNI potential.Useful information to regulate nitrification and thereby enhance NUE could be derived from analyzing the composition of RT of the four rice cultivars identifying active compounds responsible for BNI.The BNI potential is a trait that has been shown to be genetically transferred via traditional plant breeding approaches in the Tribe Triticeae (Subbarao et al. 2015(Subbarao et al. , 2021)).Hence, identifying those compounds responsible for BNI and knowing their genetic level regulation in plants will be useful in breeding rice with high NUE, especially for upland/aerobic rice cropping systems where nitrification is a major contributing factor of N losses (Haefele et al. 2008;Farooq et al. 2022).
The activity of organotrophs and heterotrophs, chelation, adsorption to clay and organic matter, dilution by soil moisture, and root exudation as affected by plant stress and plant age affect plantderived secondary metabolite concentrations in soil (Gopalakrishnan et al. 2009;Kaur et al. 2009).Illarze et al. (2021) observed that the expression of BNI by rice in soil varied depending on rice cultivar and soil type.Zhang et al. (2019) reported that release of 1,9-decanediol, a BNI compound from rice roots is enhanced by low to moderate concentrations of NH 4 + (≤ 1.0 mM), low pH, and aeration of the rhizosphere.Hence, results from laboratory bioassays on allelopathic effects of root-derived compounds on biological processes such as growth of weeds and nitrification are relative and provide information about potential activities only (Tanaka et al. 2010;Subbarao et al. 2015).Furthermore, in the bioassays we used only N. europaea standard culture, which is not representative enough of the diverse ammonia oxidizers in soil (Kaur-Bhambra et al. 2021;Nardi et al. 2020).Therefore, In-situ experiments are required to evaluate the effect of BNI compounds on nitrification in the presence of complicated interactions in soil-plant system (Nardi et al. 2020).To determine the direct effect of BNI on nitrifiers it is important to take a polyphasic approach and study the changes in gross nitrification and the expression of amoA genes (Nardi et al. 2020).In order to determine gross nitrification, 15 N tracer technique will have to be used and gene expression work involve RNA transcriptome analysis.
In soil-plant system, the differences in rates of net nitrification and gross nitrification would be high because of the co-occurrence of mineralization and denitrification along with nitrification (Nardi et al. 2020).The PNR assay with shaken slurry method is a better technique than assessing net-nitrification to understand the treatment effects on the potential activity of nitrifiers (Hart et al. 1994).A main assumption in PNR is that the rate depends on the initial population size of the nitrifiers.Unlike in netnitrification rate assay, a high NH 4 + concentration like 1.5 mM is used in the P-buffer medium during PNR assay, which is much higher than the general NH 4 + levels reported in soil solution.Thus, in PNR assay, the interference from mineralization is minimum and substrate limitation would not be a factor contributing to the differences observe among treatments.Continuous shaking at 150 rpm oxygenates the slurry discouraging the denitrifiers (Hart et al. 1994).However, the method would discourage the growth of AOA, oligotrophic type slow growing AOB and comammox, who are important players in gross nitrification in soil.Because these groups would get discouraged by the high NH 4 + concentration and neutral pH used in the P-buffer medium, and the short duration (less than 48 h) used for the assay.Hazard et al. (2021) reported that PNR is bias towards AOB.Thus, predicting nitrifiers' abundance with PNR results becomes challenging when the complexity of nitrifying communities increases (Hazard et al. 2021).In rice-paddy soils with neutral pH, AOB was found to be more responsive than AOA to N-fertilizer application (Wang et al. 2009).
We established a microcosm study using soil as the growth medium to elucidate the effects of rice cultivars on nitrifiers in the rhizosphere.We expected Vol.: (0123456789) a higher PNR in rhizosphere soils than in bulk soil, especially in those collected from the microcosms that were subjected to T C treatment because rice plants aerate the root zone; thus, facilitating the activity of nitrifiers (Briones et al. 2002).But PNR in bulk soil was greater than that in the rice rhizosphere in both the T U and T C treatments (Table 1).Competition with plants for NH 4 + can reduce the activity of nitrifiers in the rhizosphere (Verhagen et al. 1994).Increase in labile organic carbon availability and higher affinity of microorganisms for NH 4 + /NH 3 would determine the success of heterotrophs in outcompeting the AOB (Bottomley et al. 2012).Therefore, BNI and/ or enhanced growth of heterotrophic microorganisms may have outcompeted slow-growing nitrifiers in the rice rhizosphere which led to the lower rates of PNR observed in rhizosphere soils.However, considering the strong positive correlation between PNR and NO 3 − :NH 4 + ratio (r = 0.8 at p < 0.05), we suggest that nitrification contributed to the speciation of available N in soil.
Since we had a limited supply of seeds for the rice cultivar 'Apo', this cultivar was not included in the microcosm study.Instead, we used the nonallelopathic cultivar, Rexmont, which has been bred for paddy rice systems.The effects of the Rexmont cultivar on the various parameters assessed were similar to the two allelopathic cultivars, but less so for the ApCr cultivar (Table 1).One plausible explanation for the similar impact of the two allelopathic rice cultivars and the Rexmont cultivar is that all three cultivars were bred for paddy systems.The rhizosphere of ApCr, the cultivar bred for aerobic rice systems, had significantly higher PNR compared with the other rice cultivars under saturated moisture conditions.In contrast, root tissue extracts of all four cultivars suppressed nitrification in the laboratory bioassays.It would be interesting to investigate whether the ability of rice cultivars to suppress nitrification in aerobic rice cropping systems can be selected for during the breeding process.None of the cultivars augmented PNR in the rhizospheres compared with PNR in bulk soil, although activity of N. europaea was enhanced by root exudates of these same cultivars in the laboratory bioassays.Previous studies have reported on genetically regulated differences in the release of inhibitory compounds from seedlings under hydroponic and soil cultures resulting in differences in BNI capacity that may persist in the rhizosphere (Subbarao et al. 2007;Tesfamariam et al. 2014;Zhang et al. 2019).
The best method to assess the impact of BNI on ammonia oxidizers would be to conduct a transcriptome analysis targeting the mRNA of the functional gene amoA (Nardi et al. 2020).However, the reliability of data generated will depend on the coverage of the primers used in the in PCR (Pjevac et al. 2017).Information on the composition of targeted community is required to design primer pairs for the efficient quantification of specific members (Pjevac et al. 2017).In our study, we considered T-RFLP community fingerprinting technique to gather preliminary information on community composition of ammonia oxidizers, the group involved in the rate limiting step of nitrification (Horz et al. 2000;Bae et al 2011;Wang et al. 2012).We observed that the rhizosphere AOB community was responsive to moisture management and cultivar.The dominant T-RF of the soil used in the microcosm study collected from a rice crop in the Cornell Botanical Gardens was R205 (OTU5), which could be Nitrosospira spp.and/or AOB distantly related to Nitrosomonas communis/ europaea lineage (Table 2).In this soil, the less abundant OTU1 could be Nitrosomonas oligrotropha like species.However, a high throughput DNA sequence analysis is required to confirm the exact taxonomic assignment of the T-RFs.Considering the complexity in AOB community, quantitative-PCR will have to be performed targeting different AOB species to generate more information on species level differences in responsiveness of AOB to BNI (Nardi et al. 2020).
Although both Nitrosomonas spp.and Nitrosospira spp.have been detected in paddy soils the dominance of one group over the other in paddy soils has mixed reports (Wang et al. 2009;Lu et al. 2019;Farooq et al. 2022).Nitrosomonas spp. is reported to be a dominant AOB contributing to nitrification at fluxes of NH 4 + in soils with neutral pH conditions (Wang et al. 2009;Zhang et al. 2017;Farooq et al. 2022).Thus, this group of species has been widely used in nitrification inhibition research (Matsuba et al. 2003;Subbarao et al. 2006b;Gopalakrishnan et al. 2009;Tanaka et al. 2010;Papadopoulou et al. 2020).Both Nitrosomonas spp.and Nitrosospira spp.are suppressed by synthetic nitrification inhibitors such as DCD, nitrapyrin, 2-amino-4-methyl-6-trichloromethyl-1,3,5-triazine (MAST), and 3,4-dimethylpyrazole-phosphate (DMPP) to different degrees (Matsuba et al. 2003;Papadopoulou et al. 2020).Bacterial strain level differences in the degree of activity suppression have been observed for Nitrosomonas spp.and Nitrosospira spp.(Matsuba et al. 2003;Papadopoulou et al. 2020).Differences in sensitivity of AOA and AOB to biological nitrification inhibitors found in plants showing BNI have been reported (Kaur-Bhambra et al. 2021;Illarze et al. 2021).We observed changes in AOB community compositions in soil depending on rice cultivar.The relative abundance of OTU5 in the rhizosphere of the PI338046 cultivar subjected to the moisture stress treatment (T U ) was greatly reduced compared with other cultivars (Fig. 5) indicating that this allelopathic cultivar might have BNI.This observation is in agreement with the results obtained from laboratory bioassay on BNI for the same cultivar.Based on this observation we suggest that the BNI of the rice cultivars tested as expressed on N. europaea in the laboratory bioassay may have influenced the dominant AOB in the paddy soil used in our study.Kaur-Bhambra et al. (2021) highlighted the importance of not limiting the BNI bioassays to N. europaea and using a range of bioassay strains including, AOA and other AOB.We propose that it would be worth considering a wide group of ammonia oxidizers including AOA and AOB in future BNI research.
Moreover, root turnover rate and exudation rates may be different among cultivars when grown in soil compared with hydroponic settings.Thus RT:RE in the rhizosphere environment of plants grown in soil would differ from that in hydroponically grown rice.This difference may have contributed partly to the inconsistency in results between the microcosm experiment and the laboratory bioassays in this research.The effects of BNI on rhizosphere nitrifiers cannot be isolated from the effects of the soil matrix, competition for NH 4 + , and other microorganisms in soil in the microcosm study.This highlights the difficulty of extrapolating findings from hydroponic experiments to soil-based experiments (Dandeniya 2014;Nardi et al. 2020).The BNI potentials could be overestimated or underestimated depending on the characteristics of the soil used in a study because the interactions between biotic and abiotic components vary among soils (Illarze et al. 2021).Therefore, hydroponic culturing and laboratory bioassays are useful tools for initial screening of nitrification inhibitors as seen in our study and many others reported in literature (Matsuba et al. 2003;Subbarao et al. 2006b;Gopalakrishnan et al. 2009;Tanaka et al. 2010;Tesfamariam et al. 2014;Papadopoulou et al. 2020;Kaur-Bhambra et al. 2021;Subbarao et al. 2021).

Conclusions
This study demonstrated the potential effects of varietal differences in root-derived compounds of rice on a nitrifying culture, N. europaea, and also the relationship between the allelopathic effects of the extracts studied and the BNI potential.Moreover, this study indicated the differences in response between rice genotypes in their allelopathic effects and BNI potential depending on whether the plants have been subjected to water stress or not.
More specifically, the results indicate that the BNI potential of root tissue extracts of the two allelopathic cultivars (PI312777 and PI338046) were enhanced when rice seedlings were subjected to moisture stress.Water-soluble compounds in crushed rice roots and not the root wash containing exudates inhibited ammonia oxidation activity of N. europaea when applied at rates above 0.5 mg ml −1 .The rate of application of RE and RT largely determined whether the activity of N. europaea was enhanced or suppressed.Moreover, growing rice under moisture stress led to varietal differences in the AOB communities colonizing the rhizosphere environments.Our results indicate that rice cultivars with known allelopathic potential and lineages of these cultivars would be promising candidates in the search for BNI potential given the strong correlation between suppression of lettuce root growth and BNI activity by rice root extracts.
Improving the potential of rice to reduce nitrification in the rhizosphere has positive environmental and agronomic implications; especially, when N input to the system exceeds N uptake by plants, as occurs during the seedling growth stage.Further, if rice plants can inhibit nitrification, there would be less need for amending soils with synthetic nitrification inhibitors, and this would be more economical and advantageous for most rice farmers who do not have access to such inputs.

Fig. 1
Fig. 1 Inhibition of ammonia oxidation activity of Nitrosomonas europaea in relation to inhibition of lettuce root growth by (a) rice root exudates (RE) and (b) rice root tissue extracts (RT) of four rice cultivars (Apo, ApCr, PI312777, and PI334086)

Fig. 4
Fig. 4 Inhibition of ammonia oxidation activity of Nitrosomonas europaea by Nitrapyrin (N-Serve® with 99% active ingredient) in a laboratory bioassay.Percent inhibition was calculated as the reduced activity under treatment in comparison to the activity when inoculated with water

Fig. 5
Fig. 5 Histograms of T-RF relative abundances for TaqI T-RFLP profiles of bacterial amoA.Relative abundance is the ration of the peak area of a given T-RF to the sum of peak area under all T-RFs in that sample expressed as a percentage

Table 1
Potential nitrification rates in soil and rice plant PNUE at 14 days after germination for two different soil moisture treatments (T U -unsaturated and T C -continuously saturated) in a microcosm experiment Means followed by the same letter in a given column are not significantly different (p > 0.05) * According to LSD mean separation, T U and T C treatments for a given soil are significantly different (p < 0.05).NA = Not analyzed and the restriction digestion with TaqI enzyme were considered