Differential N₂O-Producing Activity of Soil Fungi Across Agricultural Systems: High in Vegetable Fields and Vineyards, Low in Paddies

Abstract

The substrate-induced respiration-inhibition (SIRIN) method has been used to estimate fungi-derived N₂O emissions, but its contribution to soil N₂O emissions remains unclear. There is a need to quantify the fungal fraction of N₂O production more precisely. Here, using isotopocule analysis, we assessed the relative contribution of fungi to soil N₂O production potential under denitrifying conditions, where key limiting factors of denitrification (soil moisture, soil NO₃⁻, and electron donor) were removed. The result showed that the ratio of fungi-derived N₂O emissions (R_F) was 0.83~4.28% in paddy soils, 13.80~23.21% in vineyard soils, and 15.34~65.94% in vegetable field soils, respectively. This indicated that the bacteria were the dominator of soil N₂O production potential in most cases, but fungal pathways could be significant in vegetable field soils. The experiment with bactericide addition showed that inhibitors could affect non-target microorganisms in the SIRIN method. Our further analyses suggest that it is worth to explore the effect of soil organic carbon and microbial synergies on fungi-derived N₂O emissions.

1. Introduction

Nitrous oxide (N₂O) is a potent greenhouse gas and primary ozone-depleting substance [1,2]. Arable soils contribute 60% of the global anthropogenic N₂O emissions [1]. Historically, bacteria were regarded as the dominant denitrifying microorganism. However, the leading role of fungi in N₂O emissions was also observed across diverse ecosystems [3,4,5,6,7]. While denitrifying bacteria commonly possess N₂O reductase, this enzyme appears absent in most denitrifying fungi. Consequently, N₂O reduction to N₂ rarely occurs during fungal denitrification [8]. If the bacterial niches were occupied by fungi under certain conditions, the truncation of fungal denitrification process may lead to a large amount of soil N₂O emissions. This scenario may occur in intensively managed fields [5]. For example, vegetable fields are often characterized by high nitrogen fertilizer inputs and multiple cropping. Notably, most fungi exhibit greater acid tolerance than most bacteria, and therefore fungi-to-bacteria abundance ratios can increase with soil acidity [9]. Continuous cropping of the same or similar vegetable species can lead to the accumulation of phytopathogenic fungi, some of which are recognized as significant N₂O-producing microorganisms [4,10]. Furthermore, due to fungal great ability to utilize complex substances from organic materials [11,12], manure application can increase the relative abundance of soil fungi in fields [13].

In soil incubation experiments, the substrate-induced respiration–inhibition (SIRIN) method is generally used to assess fungal and bacterial contributions to N₂O emissions in real time. In this method, cycloheximide (fungicide) and streptomycin (bactericide) are used to selectively inhibit soil fungal and bacterial activity, respectively. Using the SIRIN method, we previously found fungi contributed more to soil N₂O emissions than bacteria in intensively managed vegetable field soils, where nearly all N₂O emissions (99.09%) were suppressed by fungicide treatment [5]. Fungal dominance in N₂O emission has also been reported in the cropland soil following manure applications [11]. However, the contribution of fungal denitrification to soil N₂O emissions remains controversial because it is unclear whether inhibitors completely suppress the activity of soil target microorganisms, and the inhibition efficacy may wane during incubation [5]. In addition, some studies in liquid growth medium also suggested that the contribution of fungal denitrification may have been overestimated [4]. Given that microbial denitrification contributes approximately 60% to N₂O emissions from agricultural soils [14], elucidating the role of fungal denitrification is essential.

Recently, the isotopocule analysis was used to discern N₂O producing processes. In the N₂O molecule, the central and terminal N atoms have been referred to as α and β. The difference of δ¹⁵N between the central and terminal N atoms is expressed as site presence (SP = δ¹⁵N_α–δ¹⁵N_β). Although some N₂O production pathways share similar SP values, the ability of isotopocule analysis to precisely partition N₂O sources at the ecosystem scale is limited. For example, the SP value of fungal denitrification (including autotrophic nitrification and chemodenitrification) is 32.8 ± 4.0‰, and bacterial denitrification (including nitrifier denitrification) exhibits a significantly lower SP value (−1.6 ± 3.8‰) [15]. Nevertheless, the SP values of fungal denitrification and bacterial denitrification are distinctly different. Therefore, isotopocule analysis can be used to quantitatively evaluate the potential of fungal and bacterial denitrification under denitrifying conditions. In this study, using the N₂O isotopocule analysis, we assessed the contribution of denitrifying fungi to soil N₂O production potential in four agricultural ecosystems: intensively managed vegetable fields, vineyards, paddy fields, and a bare field. Specifically, we hypothesized that fungi are important contributors to N₂O emissions in fertilized soils, but the role has been overestimated in previous studies [3,5,6].

2. Materials and Methods

2.1. Soil Collection

Soil samples were collected (at 0–20 cm depth) from nine fields in Suzhou, Jiangsu Province, China, including four intensively managed vegetable fields (VF1~VF4), two vineyards (VY1~VY2), two paddy fields (P1~P2), and one bare field (CK). Paddy field and bare field soils were sampled from the Changshu Agroecosystem Experimental Station, the China Academy of Sciences (120°42′ E, 31°33′ N). The bare field, historically untilled for ≥10 years and never fertilized, served as the control. Soil samples from intensively managed vegetable fields and vineyards were collected 2 km south and 1.5 km north of the station, respectively. At each field, according to “S” shape, five soil cores were collected in an “S” shape and then mixed into one sample. The samples were sieved through a 2 mm mesh, and stored at 4 °C for the soil incubation experiment and −20 °C for DNA extraction. Detailed field management practices and soil physicochemical properties were described previously [13].

2.2. N₂O Production Potential Measurement Based on Isotopomers Analysis

N₂O production potential measurement was modified from previous study [16]. Briefly, twenty-seven soil microcosms (9 soils × 3 replicates) were established. For each microcosm, fresh soil (20 g dry weight) was added to a 50 mL flask with a solution capable of supplying 100 mg kg⁻¹ KNO₃-N dry weight soil (analytical reagent, Sinopharm, Beijing, China) and 6 mg g⁻¹ glucose-C dry weight soil (analytical reagent, Sinopharm). Soil moisture was adjusted to 100% water hold capacity (WHC). Flasks and KNO₃-N/glucose solutions were sterilized prior to use. Then, the flask was evacuated for 2 min, flushed with N₂O-free air, and incubated under 25 °C. Gas samples were collected with syringes after 12 h, and determined with an Agilent 7890A gas chromatograph. Isotopic data (δ¹⁵N_α, δ¹⁵N_β, and SP) was determined on the off-axis integrated cavity output spectroscopy (Los Gatos Research Company, 914-0027, San Jose, CA, USA). Isotopic data could remain stable when N₂O concentration was more than 2.5 ppm, requiring a 2 L gas sample volume (Figure S1). Gas samples were diluted to 2.5~3 ppm in 2 L gas bags prior to isotopic analysis. A schematic diagram of sampling and microcosm design are shown in Figure S2. The fraction of N₂O produced by fungi (f_FD) and bacteria (f_BD) were calculated according to the following Equation [17]

SP = f_FD × SP_FD + f_BD × SP_BD

(1)

f_FD + f_BD = 1

(2)

where SP_FD and SP_BD represent process-specific SP values for denitrifying fungi and bacteria (32.8‰ and −1.6‰, respectively) [15].

Although SP can be affected by isotopic fractionation when there is a N₂O reduction [17], significant SP increases occur only when >80% of N₂O is reduced to N₂ [15]. Our previous studies showed that such substantial N₂O reduction (>80%) occurs in the CK soil [13]; therefore, N₂O reduction was not considered in this study.

2.3. The Effect of Streptomycin and Formate on Fungi Denitrification

To evaluate SIRIN reliability, a N₂O production potential assay with bactericide (streptomycin, Chemically Pure, Aladdin, Riverside, CA, USA) addition was conducted for P1 and VF1. Briefly, besides KNO₃-N and glucose, streptomycin (50 mg kg⁻¹ dry weight soil) was added to soils. Six soil microcosms (2 soils × 3 replicates) were incubated under 25 °C and N₂O-free air, and the soil moisture was adjusted to 100% WHC. N₂O concentration and isotopic information of gas samples were determined after 12 h.

Additionally, because exogenous formate (analytical reagent, Sinopharm) can stimulate the denitrification of Fusarium oxysporum (a denitrifying fungus) [18], the effect of formate addition on fungal pathway was also evaluated in P1 and VF1 by another incubation experiment. In this experiment, besides KNO₃-N, formate (6 mg g⁻¹ C dry weight soil) was added to soils. Six soil microcosms were incubated under 25 °C, 100%WHC and N₂O-free air. N₂O concentration and isotopic information were determined after 12 h. Total soil DNA (P1 and VF1) was extracted using the Fast DNA SPIN Kit (MP Biomedical, Santa Ana, CA, USA) according to the manufacturer’s protocol and stored at −80 °C. Subsequent high-throughput sequencing of bacterial and fungal communities was performed on an Illumina HiSeq 2500 platform (Novogene, Beijing, China). Details regarding the target regions, amplification primers (including barcodes), and data processing followed established methods [19,20].

2.4. Statistical Analyses

Least Significant Difference (LSD) tests were performed to compare the contribution of fungi (or bacteria) to N₂O emissions in soils. For VF1 and P1, one-way analysis of variance (ANOVA) was used to analyze (1) the effect of streptomycin on N₂O emissions from fungal and bacterial denitrification, (2) the effect of glucose and formate on fungal N₂O emissions, and (3) the relative abundance ratios of denitrifying fungal genera (>0.1%). Statistical analyses were performed on IBM SPSS Statistics 16.0.

3. Results and Discussion

3.1. The Fungal Role in N₂O Emission Based on Isotopomers Analysis

Previous reviews showed that the process-specific SP value of N₂O fungal denitrification is 35.2 ± 4.3% and that of bacterial denitrification is −2.2 ± 4.2% [15]. In this study, a sufficient carbon source (glucose) and nitrogen source (KNO₃) were provided, and the incubation was short-term. Thus, it is reasonable to assume that N₂O primarily originated from denitrification. The SP values of gas samples ranged from −1.89% to 22.46%, indicating concurrent fungal denitrification and bacterial denitrification in the soils.

As shown in Figure 1, the ratios of fungi-derived N₂O emissions (R_F) were 18.73% in the CK soil, 2.55% (range: 0.83~4.28%) in the paddy soils, 18.5% (13.80~23.21%) in vineyards, and 39% (15.34~65.94%) in vegetable field soils. The medium R_F across all soils was 17.84%, similar to the CK soil. The R_F in CK soil can be regarded as background value in the study region. Thus, fungi were important contributors to soil N₂O production potential in vegetable field soils but less important in paddy soils, consistent with our previous findings [5,13].

Notably, intensive management practices favor soil fungi-derived N₂O emissions. Resultant soil acidification and nutrient accumulation may impact bacteria more severely than fungi [13]. Furthermore, fungal growth could be stimulated by manure application [21], and substantial manure (ca. 400 kg N ha⁻¹ year⁻¹) was applied in these vineyards. However, the R_F in vineyard soils was not significantly higher than in CK soil, which was likely that the role of fungi may be offset by high N₂O reduction capacity in vineyards [13].

3.2. The Fungal Role in N₂O Emission Based on the SIRIN Method

The SIRIN method has been widely used to distinguish bacterial and fungal contributions to N₂O emission in previous studies, These results showed the fungi contributed to 30~99% of N₂O emission in vegetable field soils [5,21], 28~56% in paddy soils [6,22], and 17.4~89% in other ecosystems [3,23,24], often suggesting fungal dominance. In contrast, our results based on isotopocule analysis generally showed a lower fungal contribution compared to bacterial contribution (Figure 1). Only in VF4, fungal contribution (65.94%) exceeded the bacterial contribution, while in P1, it was only 3.8%. This indicated that the contribution rate of fungi may be overestimated by the inhibitor method in the past. To further investigate and evaluate SIRIN reliability, a laboratory incubation experiment amended with bactericide (streptomycin) was conducted in VF1 and P1 soils. The result showed that streptomycin expectedly restrained bacteria-derived N₂O emissions; however, the bactericide also affected fungal N₂O-producing activity (Figure 2). A similar study also suggested a small or missing fungal contribution in three arable soils under anoxic conditions [19]. These suggested that inhibitors can affect non-target microorganisms, especially in the paddy soil leading to substantial overestimation of the fungal contribution.

3.3. The Actual N₂O-Producing Activity of Fungi in Soils

In pure culture studies, N₂O production rates of fungal isolates in liquid growth medium are typically 1 to 5 orders of magnitude lower than those of bacteria [4]. Given the comparable biomass of fungi and bacteria, R_F should not exceed 10% or should be even lower in soil microcosm experiments. In this study, however, R_F was more than 10% in vineyard and vegetable field soils. The discrepancy between culture-incubation experiments and soil-incubation experiments indicated that some factors in favor of fungal N₂O-producing activities are not fully understood. Firstly, the cultured microorganisms represent a small fraction of all denitrifying fungi; however, N₂O production capacity of unidentified soil fungi may be equal to or even greater than that of bacteria [4]. Secondly, culture media in previous research may not provide optimal conditions for fungal N₂O production. For example, in fungal denitrification, the NO₃⁻ reductase (narG) and formate dehydrogenase (UQFdh) coupled electron transport system allow NO₃⁻ to efficiently accept electrons and be reduced to N₂O [18,25]. Therefore, exogenous formate can stimulate fungal denitrification in soil. In this study, compared to glucose addition, exogenous formate increased R_F by about 34% in vegetable field soil and only 4% in paddy soil (Figure 3a), which may be related to higher fungal abundance in vegetable soil [5]. Additionally, microbial synergies can significantly promote N₂O emission, such as in liquid growth medium, when the fungus (Penicillium citrinum) and bacterium (Citrobacter freundii) are cultured separately. The content of N₂O in the culture medium was 0.29 mg L⁻¹ and 0.05 mg L⁻¹, respectively [26]. However, when the fungus and bacterium were co-cultured, the content of N₂O in the culture medium reached 2.51 mg L⁻¹. Interestingly, the Penicillium genus was also detected in the vegetable field, and the effect of synergies between microorganisms should be evaluated in the future (Figure 3b). Together, these underlying mechanisms indicate that fungi can be important contributors to N₂O production in fertilized soils, especially for vegetable fields and vineyards in present study.

4. Conclusions

Effective N₂O mitigation requires a deeper understanding of microbial N₂O pathways and associated microbial communities. Elucidating the significance of soil fungi as contributors to N₂O emissions across ecosystems is crucial. Our findings expand this work. In summary, the presented results showed that inhibitors used in the SIRIN method can affect non-target microorganisms, potentially leading to an overestimation of the fungal contribution to soil N₂O production potential in previous studies employing this method. Incubation experiments based on isotopomer analysis indicated that the bacteria dominated soil N₂O production potential in most cases, but fungal pathways could also be significant in some scenarios such as soil acidification, cropping obstacles, and manure application. However, our results reflect the potential for fungal denitrification rather than actual emission rates in situ. To more accurately assess the fungal role in N₂O production, further studies are imperative to validate the reliability and applicability of current methodologies for distinguishing fungal-derived and bacterial-derived N₂O emissions.