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Article

Effect of the Nitrification Inhibitor DMPP on Blueberry Planted in Neutral Soil

by
Yiru Yang
,
Qilong Zeng
*,
Hong Yu
,
Jiguang Wei
,
Jiafeng Jiang
and
Liangliang Tian
Jiangsu Key Laboratory for the Research and Utilization of Plant Resources, Institute of Botany, Jiangsu Province and Chinese Academy of Sciences (Nanjing Botanical Garden Mem. Sun Yat-Sen), Nanjing 210014, China
*
Author to whom correspondence should be addressed.
Agronomy 2024, 14(9), 2029; https://doi.org/10.3390/agronomy14092029
Submission received: 12 August 2024 / Revised: 28 August 2024 / Accepted: 3 September 2024 / Published: 5 September 2024
(This article belongs to the Section Horticultural and Floricultural Crops)
Browse Figure
Review Reports Versions Notes

Abstract

:
In order to increase nutrient input and alleviate the poor growth of blueberry (Vaccinium corymbosum L.) in neutral soil with strong nitrification, the application of nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) as an enhanced efficiency fertilizer is a strategy to reduce nitrogen (N) loss and improve N supply. However, few studies have systematically investigated the effect of DMPP application on blueberry and its soil condition in detail so far. In this study, a pot experiment was conducted to elucidate the effect of DMPP at four gradient levels including 0.5% (w/w applied-N) DMPP (DL), 1% DMPP (DM), 2% DMPP (DH), and no DMPP (CK) on the dynamics of soil mineral N (NH4+-N and NO3−-N), soil chemical properties, as well as the agronomic characteristics and physiological indexes of blueberry plants in the neutral soil–blueberry system. The addition of DMPP significantly increased the retention of soil ammonium nitrogen and the content of total mineral nitrogen. qPCR analysis showed that DMPP inhibited the ammoxidation process mainly by reducing the abundance of the ammonia-oxidizing bacteria (AOB) amoA gene rather than the ammonia-oxidizing archaea (AOA) amoA gene. No significant inhibitory effect of DMPP was observed for the nitrite dehydrogenase gene nxrA and nitrite reductase gene nirS. Soil NH4+-N and available phosphorus content were both enhanced with the DMPP application rates both in bulk and rhizosphere soil. Applying 1% DMPP to the neutral soil for blueberry was sufficient to safely inhibit soil nitrification, not only increasing ammonium nitrogen content by 10.42% and 26.79%, but also enhancing available phosphorus content by 9.19% and 22.41% compared with CK in bulk and rhizosphere soil, respectively. Moreover, 1% DMPP addition increased the nitrogen and phosphorus concentration of blueberry leaves by 12.17% and 26.42%, respectively, compared with CK. The total branch length and the dry weight of blueberry plant were also increased by 16.8% and 33.1%, respectively. These results provide valuable agronomic information for the application of DMPP in blueberry cultivation. Fertilization applied with 1% DMPP has great economic potential to improve both nitrogen and phosphorus absorption of blueberry so as to promote the vegetative growth of blueberry.

1. Introduction

Blueberry (Vaccinium spp.), one of the few fruits native to North America [1], was first introduced into China in the 1980s. In recent decades, the blueberry industry in China has been booming rapidly, as the berry fruit has a rich flavor as well as high nutritional value and is also rich in phenols and anthocyanins, which possess a variety of medicinal benefits including antioxidant, anti-inflammatory, and antitumor effects [2]. Cultivated species of the genus Vaccinium are grown commercially, ranging over a very large area of China [3]. However, there is still a big gap between the blueberry production per unit area in China and the global average. As an economically important small pulp shrub, blueberries are well adapted to acidic soils with optimum pH values in the 4.0–5.5 range [4]. Paradoxically, the relatively high pH in many areas of neutral soil rarely meets the needs of blueberry growth, causing blueberry plants to exhibit a number of undesirable states such as nutrient deficiency symptoms, growth retardation, and decreased fruit quality [5,6]. An ecological explanation for blueberry’s limitation to a low pH environment would be the absence of a nitrate-reducing system for the blueberry plant to utilize nitrates [7,8]. Numerous studies have proved that blueberry belongs to ammonium-loving plants. The growth of both highbush and rabbiteye blueberry under the application of ammonium nitrogen fertilizer was better than that under the application of nitrate nitrogen fertilizer [9]. Thus, the fertilizer for blueberry is mainly ammonium nitrogen. After fertilization, the pH in the rhizosphere soil and subsequently in the bulk soil can be altered by proton excretion from the blueberry roots by reason of cation absorption exceeding anion absorption under the ammonium nutrition regime [10].
Nevertheless, ammonium (NH4+) is rapidly converted to nitrite (NO2−) catalyzed by the key enzyme ammonia monooxygenase (AMO) encoded by the amoA gene in the ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA), and then to nitrate (NO3−) via nitrite oxidoreductase (NXR) in most agricultural soils [11]. The strong nitrification in neutral soil after the application of ammonium nitrogen fertilizer results in increased N mobility in the soil matrix. Notably, blueberry is an oligotrophic species with shallow roots and rare root hairs that limit the efficient absorption of water and nutrients. The root system of blueberry is sensitive to fertilization, so that either insufficient or excessive fertilization will have a negative impact on the vegetative growth of blueberry [12]. The nitrogen source required for blueberry is usually ammonium sulfate [(NH4)2SO4], and an optimal application rate is 20–140 kg·ha−1·year−1, which is conducive to the biomass accumulation and allocation [13,14]. Overapplying the N fertilizer is costly and leads to more production of nitrification (NO3−), which can leach into the environment and be transformed to nitrous oxide or di-nitrogen gas through denitrification by nitrate reductase encoded by the narG gene, nitrite reductases encoded by the nirS/nirK gene, and nitrous oxide reductase encoded by the nosZ genes, so as to be lost to the atmosphere [15,16,17].
The application of the nitrification inhibitor (NI) is a method to reduce the nitrification loss of the nitrogen fertilizer [18]. The NI inhibits the first rate-limiting enzymatic step of the nitrification reaction, i.e., the activity of ammonia monooxygenase, and delays the microbial oxidation of NH4+ to NO3− by reducing the availability of copper, the co-factor of AMO, when applied in the soils in combination with the N fertilizer at very low doses [19,20]. Synthetic nitrification inhibitors, including dicyandiamide (DCD), nitrapyrin, and 3,4-dimethylpyrazole phosphate (DMPP), are widely used to delay nitrification [21,22]. DMPP, which has lower phytotoxicity and enhanced efficiency and durability at much lower application doses in comparison with other NIs, can be able to delay nitrification for several weeks according to different climatic environments and soil physicochemical properties [23,24,25]. DMPP has been recognized as a practical method to reduce the potential nitrate loss and promote nitrogen use efficiency (NUE) and primary production [26,27,28].
However, these benefits of DMPP are not always achievable, which may be influenced by crop type, environmental condition (soil type and properties) [29], climate (temperature and moisture), and management practice (fertilizer types and application methods) [30,31]. The variable efficacy of DMPP was manifested across studies from year to year in both agronomic and environmental benefits. For instance, in a three-year field trial in Germany, no significant effect on the NH4+ concentrations of the treatment with DMPP were observed in comparison with the control [32]. The nitrogen loss reduction efficiency of DMPP varied greatly [33]. DMPP increased the rice yield, the nitrogen content in maize grain, and vegetable biomass, but did not significantly impact on nitrogen absorption and the biomass of grasses [34]. Likewise, DMPP had no significant influence on the yields of summer maize, barley, and winter wheat [32]. In addition, DMPP had no significant impact on the spinach yield at the nitrogen rates of 50 and 75 μg·N·g−1 soil [35], which may be attributable to the preference of different crop for ammonium and nitrate nitrogen [36,37]. Blueberries prefer the ammonium nitrogen fertilizer. Theoretically, DMPP can increase the absorption of nitrogen in blueberry, thereby improving physiological indicators such as photosynthesis [38]. However, to date studies on the effects of NI application on plants were mainly focused on cereal crops, grasses, or vegetables. It is well known that the efficiency of DMPP in increasing crop absorption depends largely on the crop species. For winter wheat and cotton crops, DMPP has no effect on the yield and nitrogen uptake [39]. Few studies have been undertaken to investigate DMPP application on the cultivation of small berries and their soil nitrogen content in detail. Data for blueberries are in particular limited.
In this context, a greenhouse pot experiment was carried out to investigate the effect of different DMPP levels on the growth of blueberry plant and soil property in the neutral soil–blueberry (Vaccinium corymbosum L.) system. The aim of this study was as follows: (1) to clarify the impact of DMPP on soil chemical properties and the dynamics of ammonium and nitrate nitrogen concentration; (2) to examine the influence of DMPP on plant nutrients and the agronomic traits of blueberry; (3) to identify the effect of DMPP on the key nitrogen transformation functional genes in rhizosphere soil of blueberry. We hypothesized that the application of DMPP combined with chemical fertilizer would be beneficial to increase nitrogen content both in soil and plants, promoting the vegetative growth of blueberry.

2. Materials and Methods

2.1. Experimental Site Description

A pot experiment was carried out in a greenhouse located in Nanjing Botanical Garden Mem. Sun Yat-Sen, Jiangsu Province, in eastern China (32°3′ N, 118°49′ E). The region has a humid subtropical monsoon climate with an average annual temperature and precipitation of 16.2 °C and 1013 mm, respectively. The soil used in this study was a neutral loam soil (mountain yellow-brown soil) collected from the surface horizon (0–20 cm) of an arboretum land in the botanical garden. Roots and stones were removed from the soil. The soil was then air-dried and sifted at 4 mm until used. The soil had a pH value (1:2.5 H2O) of 6.3, 7.77 and 5.96 μg·N·g−1 dry soil of NH4-N and NO3-N, respectively. Additionally, 5 kg of soil was filled into a plastic pot with a diameter of 15 cm and a height of 18 cm mixed thoroughly at the ratio of 5% with perlite, which was helpful to aerate soil. The blueberry cultivar used in this experiment was ‘Lanmei 1’, an annual highbush blueberry seedling (Vaccinium corymbosum L.), which was selected and bred by Zhejiang Lanmei Technology Co., Ltd. (Zhuji, China). One blueberry plant was planted into each pot on 1 June 2020 and lasted 180 days.

2.2. Experimental Design and Management

The chemical fertilizer was applied uniformly in each pot according to optimal agronomic recommendations of 10 g (NH4)2SO4 pot−1 and 10 g KH2PO4 pot−1. The treatment design included a different nitrification inhibitor DMPP (purity 97.00%, Shanghai yuanye Bio-Technology Co., Ltd., Shanghai, China) rates factor at four levels: unfertilized as a control; fertilized with 0.5% (w/w applied-N) DMPP (equivalent to 10.5 mg DMPP pot−1); fertilized with 1% DMPP (equivalent to 21 mg DMPP pot−1); and fertilized with 2% DMPP (equivalent to 42 mg DMPP pot−1). Four treatments were conducted following a completely randomized design with six pots per level. The samples of two pots from the same level were pooled together into one composite sample, so that three replicates of the data for each treatment were obtained. In order to enable homogenous handling, the fertilizer and DMPP were dissolved in 800 mL of deionized water and the solutions were evenly distributed onto the corresponding soil surface manually. The first application was on 1 June 2020. As the nitrification-inhibiting effect of DMPP was presumed to last for several weeks, top dressings of the same were carried out after 71 and 154 days (11 August and 2 November) of the experiment, respectively. Blueberries were grown under conventional and consistent management practices. The plants were irrigated with deionized water so that the moisture lost was replenished. A tray was placed at the bottom of each pot. Little leaching water occurred during the experiment. If there was by accident, it would be poured back into the pot immediately. To warrant uniformity of the conditions, the place of each treatment and replication was replaced in rotation.

2.3. Soil Sampling and Analysis

The destructive soil sampling was performed at each time point with an interval of several days during the planting process after fertilizer and DMPP application in the three replicates per treatment until the third fertilizer application. Three individual soil cores (10 mm diameter, 0–5 cm depth) were collected randomly from each sampling pot and mixed together. About 5.0 g of fresh bulk soil was sampled from each pot and quickly transferred from the greenhouse to a refrigerator at 4 °C in the laboratory for the concentrations of mineral N (NH4+-N and NO3–-N) and soil moisture. The net nitrification rate (n) was calculated according to the following equation within the first fertilization period [40]: n(μg·N·g−1 soil day−1) = [(NO3−-N)t2 − (NO3−-N)t1]/t, where (NO3−-N)t2 is the NO3−-N concentration in the soil at time 2 (10 August), (NO3−-N)t1 is the NO3−-N concentration in the soil at time 1 (2 June), and t is the number of days between two sampling dates (69 day).
When the experiment was terminated on 29 November, the blueberry rhizosphere and corresponding bulk soil samples were collected uniformly. Soils away from growing plants were collected as bulk soil samples. The blueberry plant in each pot was uprooted along with its surrounding soil. The roots were shaken vigorously to remove the loose soil. The soil that was tightly attached to the roots was considered to be the rhizosphere soil and collected using sterile brushes. Three samples of rhizosphere and bulk soil, respectively, from each treatment were used for subsequent analyses. Each replicate sample was well homogenized and divided into three subsamples. One portion was immediately frozen in liquid nitrogen and stored at −80 °C for DNA extraction, another portion was analyzed for mineral N (NH4+-N and NO3–-N) and soil moisture content using fresh soil, and the rest was air-dried and sieved for EC, pH, available P, and exchangeable K assays.
Soil ammonium and nitrate nitrogen were extracted from 1 g of fresh soil by shaking on a reciprocal shaker with 10 mL of 1 M KCl for 30 min. The samples were centrifuged at 3000 rpm (revolutions per minute) for 5 min. The supernatant was then filtered and NH4+-N and NO3–-N were analyzed. Soil ammonium nitrogen content was quantified colorimetrically using the indophenol blue method [41]. The content of nitrate nitrogen was determined by ultraviolet spectrophotometry, using 220 and 275 nm wavelengths for nitrate and organic matter, respectively. The soil water content was gravimetrically determined by drying at 105 °C for 8 h to a constant weight.
The air-dried soil samples were sifted through a 2 mm sieve to remove impurities such as roots and crop residues. The soil EC was measured in a saturated solution extract and soil pH was measured in deionized water using a standard pH meter (METTLER TOLEDO S200, Shanghai, China) with a soil–water ratio of 1:2.5 (w/v) [42,43]. Soil available phosphorus (AP) was extracted with 0.5 mol L−1 NaHCO3 and analyzed by the molybdenum blue method [44]. Soil exchangeable potassium was extracted with 1 mol·L−1 ammonium acetate (CH3COONH4) and determined by the flame photometry method. Soil urease and acid phosphatase activities were determined by the following method. Fresh soil samples were pre-treated with the toluene, and then cultured at 37 °C for 24 h in the presence of urea (100 g·L−1) and citrate buffer (pH = 6.7). The generated NH4+-N was quantified by the indophenol blue colorimetry method, and the urease activity was expressed as μg NH4+-N g−1 dry soil d−1. Hydrolyses of disodium phenyl phosphate were performed at pH 6.5 (citrate—borate buffer) for 24 h at 37 °C to determine the activities of acid phosphatases. The resulting p-nitrophenol was measured at a 400 nm wavelength, and its phosphate activity was expressed as μg p-nitrophenol g−1 dry soil h−1.

2.4. Soil DNA Extraction and Quantitative PCR

Total soil DNA was extracted from the subsamples mentioned above of 0.3 g of soil using the PowerSoil DNA Isolation Kit (MP Biomedicals, Santa Ana, CA, USA), following the manufacturer’s instructions. Soil DNA concentration and quality were determined spectrophotometrically using a NanoDrop™ ND-1000UV-Vis Spectrophotometer (NanoDrop Technologies, Rockwood, TN, USA).
The abundances of microbial nitrogen-cycling functional genes including the amoA genes for ammonia oxidation, the nitrite oxidoreductase alpha subunit (nxrA) gene, and the nirS gene encoding the nitrite reductase for heterotrophic denitrification in the rhizosphere soil across the treatments with 2% DMPP (DH) and without DMPP (CK) were detected by real-time quantitative PCR using SybrGreen as a fluorescence dye to examine DMPP effects on microorganisms. We attempted to amplify the hzsB, narG, and nrfA genes from the tested soil using the universal primers, but failed to obtain any positive amplification products. qPCR was performed on the AOA and AOB amoA genes for ammonia-oxidizing archaea and bacteria to estimate their abundance. The primers AmoA-1F_AmoA-2R were used for targeting AOB [45], and Arch-amoAF_Arch-amoAR were used for AOA [46]. For targeting nxrA, primers nxrA-1F_nxrA-2R were used, whereas for targeting nirS, primers cd3AF_R3cd were applied. The real-time PCR was carried out on an ABI7300 RealTime PCR System (Applied Biosystems, Foster City, CA, USA). Each gene in the sample was quantified in triplicate with a standard curve and negative control. The reaction volumes of 20 μL consisted of 10 μL 2X ChamQ SYBR Color qPCR Master Mix (P211-02, Vazyme Biotech Co., Ltd., Nanjing, China), 0.8 μL of each primer (5 μM, DSL purification, Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China), 0.4 μL 50 X ROX Reference Dye 1, 2 μL template (DNA), and 6 μL ddH2O. The thermal cycling steps for qPCR amplification were as follows: an initial cycle of 95 °C for 3 min and continued with 40 cycles of 95 °C for 5 s, 58 °C for 30 s, and 72 °C for 60 s. The constructed plasmid (2692bp pMD18-T) was identified by sequencing and then analyzed by an ultraviolet spectrophotometer (Thermo Scientific NanoDrop 2000/2000c Spectrophotometer, Waltham, MA, USA). The value of plasmid OD260 was determined and converted into copy number (copies/μL) by formula. The standard plasmids were diluted to yield a series of tenfold concentrations and subsequently used to establish qPCR standard curves. Finally, data were analyzed using 7500 software (version 1.0.6) to obtain the parameter CT (cycle threshold) and calculate the copy numbers of each target gene per gram of dry soil in each sample.

2.5. Determination of Leaf Chlorophyll Content in Blueberry

Before harvesting, chlorophyll including chlorophyll-a and chlorophyll-b in fully expanded leaves of blueberry from each treatment was extracted using acetone and then absorption at 663 nm and 645 nm was measured by spectrophotometry to calculate the chlorophyll concentration. For this purpose, the leaves in three positions, i.e., on the branches which were 6 cm from the tip of the branches (outer leaves), 6 cm from the base of the branches (inner leaves), and in the middle of the branches (middle leaves), were selected. The chlorophyll content of three leaves was randomly recorded in each position, and its average value was taken as the leaf chlorophyll content of each treatment.

2.6. Agronomic Index and Plant Nutrient Analysis

The whole plants were photographed and harvested after 180 days of growth on 29 November. The number of primary, secondary, and tertiary branches under each treatment was counted and recorded. The total branch length, plant height, and basal diameter was measured with a ruler or a vernier caliper. The plants were divided into the roots, stems, and leaves. The aboveground vegetative organs were then dried at 70 °C to the constant weight of the plant dry biomass. Next, the leaves were ground using a mixing grinder to pass through a 0.25 mm sieve for total nitrogen (TN), total phosphorus (TP), and total potassium (TK) analysis. Briefly, the leaf sample was digested with concentrated H2SO4 and H2O2. The NH4+-N produced was quantified by the indophenol blue colorimetry method to obtain the TN content of leaves. The content of TP was determined by the molybdenum blue method, and TK content was determined by the flame emission spectrometry [41].

2.7. Statistical Analysis

The means ± the standard error (SE) were calculated using Excel version 16.16.4. One-way analysis of variance (ANOVA) based on the Duncan test was conducted to analyze the difference between the means of more than two groups with SPSS version 24 (IBM, Armonk, NY, USA). The independent two sample t-test was used to compare two population means. The influences of DMPP application on the relationships between the nitrogen and phosphorus content of soil and blueberry leaves, soil enzyme activities, and plant agricultural traits were evaluated by Pearson correlation. Any difference between the mean values was considered significant at p ≤ 0.05.

3. Results

3.1. Dynamic Change of Soil Mineral Nitrogen Content

The dynamic changes of soil NH4+-N and NO3–-N content reflected the effect of fertilization and DMPP addition. The addition of DMPP significantly increased the mineral N content in soils as the blueberry plants grew during the 180-day trial. At just three days after the first application, the total mineral N content in all soils treated with DMPP was higher than that without DMPP (Figure 1). DMPP increased the average N content, respectively, by 6.19%, 11.24%, and 16.72% in the DL, DM, and DH treatments than the CK treatment from 2 June to 19 October on the whole. Soil NH4+-N content increased with the DMPP application rate, the average value of which was 16.3% and 23.74% higher in the DM and DH treatments than that in the CK treatment. By contrast, there was no significant differences in the soil NO3–-N content of all the treatments by and large. The dynamic changes of NH4+-N and NO3–-N content were different. As the blueberry plants grew, soil NH4+-N content increased rapidly after each fertilization, and then gradually decreased week by week, whereas the NO3–-N content showed a slowly growing trend during the first two months. The addition of DMPP significantly slowed down the decline of NH4+-N levels in the first two fertilization periods. According to the calculation, the average net nitrification rate was 1.63 μg NO3−-N g−1 soil day−1 in the DM treatment, significantly lower by 12% than that in the CK treatment (1.86 μg NO3−-N g−1 soil day−1).

3.2. Soil Chemical Properties Analysis

The results of the determination of soil chemical properties when the blueberry plants were harvested showed that soil NH4+-N content increased with the DMPP application rate both in the bulk and rhizosphere soil (Table 1). The NH4+-N content in the DL, DM, and DH treatments was 6.33%, 10.42%, and 10.64% higher than that in the CK treatment in the bulk soils, respectively, whereas a greater growth rate of 16.3%, 26.79%, and 32.15% was shown in corresponding rhizosphere soil, respectively. No significant difference was found among the three levels of DMPP in the bulk soil, while the difference was obvious in medium or high DMPP levels compared to the low level in the rhizosphere soil. In contrast, the addition of DMPP significantly decreased the NO3–-N content. The NO3–-N content in the DL, DM, and DH treatment was 21.02–23.31%, 43.28–46.57%, and 40.41–40.29% lower than that in the CK treatment in the bulk and rhizosphere soils, and the difference was statistically significant.
No significant difference was found in soil pH among all treatments either in the bulk soil or the rhizosphere soil. The pH was almost the same as the initial value, ranging from 6.19 to 6.31. The EC value of soil tended to increase with the DMPP rate, but there was no significant difference among all the treatments except for the soil EC in the DM treatment in the rhizosphere soil, which was 12.55% higher than that in the CK treatment.
Soil available phosphorus content increased significantly with the increase in the DMPP rate not only in the bulk soil but also in the rhizosphere soil. Treatment means comparison showed that there was no significant difference in AP content between the DM and DH treatments, but the AP content in them was obviously higher than that in the CK treatment by 9.19–9.21% and 22.41–27.93%, respectively, in the bulk and rhizosphere soil. For exchangeable potassium, there was no significant difference in all the treatments in bulk as well as rhizosphere soil.
The urease and acid phosphatase activity was significantly increased with the DMPP rate both in the bulk and rhizosphere soil, and the lowest activity was observed obviously in the CK treatment. The highest values of urease and acid phosphatase activity were obtained in the DH treatment, which increased, respectively, by 25.29% and 32.88% in the bulk soil and increased, respectively, by 18.15% and 23.69% in rhizosphere soil compared with CK.

3.3. Leaf Chlorophyll Content of Blueberry

The chlorophyll content of blueberry leaves firstly increased and then decreased with the increase in the DMPP rate (Table 2). The chlorophyll-a and chlorophyll-b content of the inner leaves in the DL treatment was 43% and 25.23% higher than that in the CK treatment, respectively. The increase in the chlorophyll-a content of the inner, middle, and outer leaves in the DM treatment was more obvious, which was 41.98%, 35.57%, and 64.92% higher than that in the CK treatment, respectively. The increase in the chlorophyll-b content of the inner and outer leaves in the DM treatment was 24.59% and 39.91% higher than that in the CK treatment, respectively. The chlorophyll-a and chlorophyll-b content in the DH treatment was also higher than that in the CK treatment, respectively, but had no significant difference of the content in both the inner and middle leaves with CK.

3.4. Agronomic Characteristics and Biomass of Blueberry Plants

The total branch length and the number of blueberry branches treated without DMPP were the lowest, and the highest were observed in the DH treatment (Table 3). Treatment means comparison showed that there was significant difference of the above indexes between the DM and DH treatment with the CK and DL treatments. Blueberry total branch length in the DM and DH treatments was 16.8% and 34.93% higher than that in the CK treatment, respectively. No significant difference was observed in the plant height and basal diameter of blueberry after DMPP application in all the treatments. The highest value of biomass was found in the DM treatment, which increased the dry weight of blueberry plant by 33.1% compared with the treatment without DMPP, and resulted in significant difference compared with CK and all the other treatments indicated by treatment means comparison.

3.5. Nutrient Content of Blueberry Leaves

The total nitrogen and phosphorus concentration of blueberry leaves gradually increased with the increase in the DMPP rate (Table 4). The highest N and P concentration of leaves was observed in the DH treatment. Leaf N concentration was enhanced by 4.73%, 12.17%, and 21.22% in the DL, DM, and DH treatments compared with CK, respectively. Similarly, leaf P concentration was enhanced by 10.23%, 26.42%, and 28.31%. No significant difference was found in the DM and DH treatments in leaf N or P concentration. It was also showed by treatment means comparison that there was no significant difference in potassium concentration in blueberry leaves among all treatments.

3.6. Nitrifier and Denitrifier Gene Abundance

The difference in the abundance of the key nitrifier (amoA and nxrA) and denitrifier gene (nirS) in the rhizosphere soil at harvest time was quantified to investigate the impact of DMPP addition on soil nitrogen transformation by real-time qPCR analysis (Table 5). The result showed that the population size of AOB was almost equivalent to that of AOA in the treatment without DMPP in the neutral soil–blueberry system. Comparing the AOB amoA gene abundance between the two treatments with and without DMPP, it was detected that the addition of DMPP significantly inhibited the mean abundance of the AOB amoA gene. The gene abundance in the DH treatment (2.72 × 104 copies g−1) was only about 2.5‰ of that in the CK treatment (1.09 × 107 copies g−1). The mean copy number of AOA amoA in the DH treatment was slightly higher than that in the CK treatment though without significant difference, resulting in a much larger AOA population size than AOB under DMPP application. The competition between nitrifying bacteria and archaea populations was significantly reduced, resulting in a high AOA/AOB ratio of 634 in the DMPP-treated samples. For the nitrite dehydrogenase gene nxrA and nitrite reductase gene nirS, no significant inhibitory effect of DMPP was observed. A slight decrease in the number of nxrA genes and a minor increase in the population size of nirS were found in the DH treatment.

3.7. Pearson Correlation Analysis among Investigated Parameters

The Pearson correlation coefficient between variables showed that there were significant positive correlations between the number of branches, total branch length, dry weight of blueberry plant, NH4+-N and AP content of the bulk and rhizosphere soil, and the total nitrogen and phosphorus concentration of blueberry leaves (Table 6). Urease activities of the rhizosphere soil and phosphatase activities of the bulk soil had significant positive correlations with the number of branches, total branch length, and the leaf nitrogen concentration. The NO3–-N content of the bulk and rhizosphere soil had significant negative correlations with all the above parameters.

4. Discussion

Many experiments have estimated the effect of DMPP, but the present study is the first attempt to simultaneously monitor the temporal dynamics of soil NH4+-N and NO3−-N content as well as the properties of near-neutral soil under blueberry growth conditions as effected by DMPP. Regarding the effect of DMPP on soil NH4+-N content, previous studies had mixed results. A meta-analysis showed that the combined application of DMPP with ammonium sulfate can effectively increase the content of NH4+-N content in soil [47]. The result was different from Tufail’s research [34], in which soil NH4+-N content was, respectively, reduced by the addition of DMPP in vegetable fields and grasslands by 27% and 32%. In the present experiment, all the treatments with different DMPP levels significantly increased soil NH4+-N content compared with the treatment without DMPP, which indicated that DMPP showed good nitrogen retention effects in just half a year. There was almost no lag time before DMPP took effect in the case of blueberry cultivation, similar to the other data which demonstrated an obvious inhibition of NH4+-N oxidation during the first several weeks after DMPP application [48]. Although currently unknown, it is plausible that DMPP is not easy to lose in clay loam soil. The inhibitory effect of DMPP can prevail for a month at suitable soil moisture and temperature [30]. Other scholars also believe that DMPP was easily adsorbed onto soil particles with high clay content [49,50], resulting in low availability and a weak inhibition effect [51]. Perhaps the uneven distribution of DMPP may hinder its short-term effect of reducing nitrification in specific micro-areas. In this study, however, DMPP may be neither easily absorbed on soil particles nor easily lost, showing a good nitrification inhibition effect. In addition, the results might also depend on soil physicochemical properties, especially soil pH [23]. Soil pH has been considered one of the main factors affecting soil nitrification and the effect of nitrification inhibitors. It may affect the migration and degradation rate of NIs in soil [52]. Nitrification generally occurs more in neutral soil than in acidic soil [53]. The effect of DMPP application on near-neutral soil may be better than that on acidic soil. Thus, the inhibitory effect of the phased application of DMPP on the transformation from NH4+-N to NO3−-N in the soil planted with blueberry was observed throughout the whole period in the current research.
Ammonium–nitrate transformation in soil is mainly driven by key contributors such as ammoxidation (AOA and AOB) and denitrification genes [54,55]. We investigated the influence of DMPP on the AOB and AOA amoA genes for ammonia oxidation in rhizosphere soil of blueberry plants, and the nitrite oxidoreductase alpha subunit nxrA as well as nirS encoding the nitrite reductase for heterotrophic denitrification. The abundance of functional genes quantified by qPCR indicated that DMPP addition had little impact on ammonia-oxidizing archaea and nitrite oxidoreductase in treatments. The main mechanism of nitrification inhibition by DMPP was inhibiting ammonia oxidation rather than nitrite oxidation, and no adverse effects of DMPP on the growth or activity of denitrifiers were found in this study, which is consistent with previous research [15,56]. They believe that DMPP inhibits nitrification mainly by reducing AOB abundance and metabolic activity, reinforcing the view that there are different metabolic pathways (fundamental metabolic and cellular differences) of ammonia oxidation in AOA [57]. In the current study, the populations of ammonia oxidizers significantly plunged in AOB amoA gene copies in the presence of DMPP relative to the treatment without DMPP, which indicated that DMPP inhibits the ammoxidation process primarily by reducing the abundance of AOB amoA rather than AOA amoA genes. Previous studies reported that ammonia-oxidizing archaea were little affected by DMPP [58,59]. Some believe that archaea are at a disadvantage when they compete with bacteria in an ammonia-rich environment [60]. Li et al. believed that the AOB community played more important functional roles than the AOA community and the nitrogen accumulation of maize was closely related to AOB, especially Nitrosospira but not AOA [61]. Segal et al. found that the responses of bacterial and archaeal ammonia oxidizers to long-term tillage and fertilizer management in a continuous maize field were different [62]. It was shown in the present study that ammonia-oxidizing archaea remained relatively unresponsive compared with ammonia-oxidizing bacteria when the nitrification substrate was provided to promote the nitrification reaction, and when the nitrification reaction was inhibited as well. In the present study, AOA were even increased in population size, indicating that AOA counteracted the inhibition effect of DMPP on nitrification to some extent. The microbial community AOB can be divided into five genera (Nitrosomonas, Nitrosospira, Nitrosococcus, Nitrosolobus, and Nitrosovibrio), and the common ammonia-oxidizing bacteria in blueberry rhizosphere and the species changed by DMPP application have not been studied so far. The change of the ammonia-oxidizing bacteria community may indirectly affect the microbial community structure and even the nitrogen use efficiency of blueberry roots, because plant species may affect the composition of nitrifying communities [63]. Therefore, the effects of DMPP on the bacterial and fungal communities in rhizosphere soil of blueberry and their contribution to the nutrient uptake of blueberry need further study.
In the current study, DMPP increased the content of ammonium nitrogen in soil, which is conducive to enhancing the NH4+-N absorption of blueberry plants. The benefits of DMPP on plants were mainly reflected in the nutrient and chlorophyll content of blueberry leaves. Compared with the treatment without DMPP, medium and high levels of DMPP application significantly improved the nitrogen absorption of blueberries. Due to the close relationship between leaf chlorophyll and nitrogen content [64], the chlorophyll content of leaves increased with the increase in the DMPP application rate, and reached the peak value when 1% DMPP was added. The results indicated that the higher retention of NH4+-N in soil under the DMPP application plays an important role in improving the availability of soil nitrogen [65], and the increase in plant nitrogen uptake also had a positive effect on the total branch length, branch number, and dry weight. Ammonium accumulation in blueberry shoots was generally correlated with increased plant growth [66]. It was shown in our research that a significant positive correlation between related agronomic indexes and the nitrogen and phosphorus content of blueberry plants was manifested.
In addition, it is worth noting that the nitrogen content in blueberry leaves increased simultaneously with the phosphorus content, which may be due to the close nitrogen and phosphorus coordination mechanism in plants. Nitrogen addition can promote plant phosphorus absorption [67]. Studies on the molecular mechanism of the efficient absorption and utilization of rice nutrition showed that when the nitrogen transporter and receptor NRT1.1B gene mediated the degradation of cytoplasmic inhibitory protein SPX4 to release nitrogen signal NLP3, the phosphorus signal PHR2 core transcription factor was released at the same time, thereby activating the expression of the nitrogen and phosphorus response gene, promoting phosphorus absorption and realizing the cooperative utilization of nitrogen and phosphorus [68,69]. This was for calcareous soils with relatively high pH values, and the mechanism in near-neutral mountain yellow-brown soil needs further study. In addition, the use of NH4+-N can improve the absorption of insoluble phosphorus by plants, because rhizosphere acidification in ammonium-dominated nitrogen nutrition improves phosphorus solubility, which has been proved in wheat, kidney beans, and other crops [70].

5. Conclusions

The current findings have mainly provided an insight into how DMPP addition affected soil and blueberry plant in the neutral soil–blueberry system. The addition of 1% DMPP in neutral loam was sufficient to safely inhibit nitrification in soil, minimizing nitrate leaching and chemical fertilizer cost. DMPP mediated the availability of fertilizer by decreasing AOB amoA gene abundance and enhancing ammonium nitrogen content to further improve nutrient absorption in blueberry plants. Macroscopically speaking, DMPP is beneficial for maintaining the soil nitrogen pool and sustainable nitrogen supply capacity, and has implications for improving N uptake and utilization in the neutral soil–blueberry system. After this observation under short-term pot experiment conditions, further studies under long-term studies including the measurements of microbial community compositions are important to explore the potential of DMPP to the biomass and yield of blueberry during the postharvest season.

Author Contributions

Conceptualization: Y.Y. and Q.Z.; Investigation: Y.Y.; Resources: J.W. and J.J.; Formal analysis: Y.Y.; Visualization: Y.Y. and L.T.; writing—original draft preparation: Y.Y.; writing—review and editing: Y.Y., Q.Z. and H.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

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Agronomy 14 02029 g001
Figure 1. Dynamic changes of ammonium nitrogen, nitrate nitrogen, and total mineral nitrogen content in the soil planted with blueberry as affected by different 3,4-dimethylpyrazole phosphate (DMPP) levels through time. The value represents the mean of three samples, and the error bars indicate the standard deviation of the mean (n = 3). Down arrow ↓ indicates the time of fertilization and DMPP addition. CK = control with no DMPP; DL, DM, and DH = treatments applied with 0.5%, 1%, and 2% DMPP, respectively.
Figure 1. Dynamic changes of ammonium nitrogen, nitrate nitrogen, and total mineral nitrogen content in the soil planted with blueberry as affected by different 3,4-dimethylpyrazole phosphate (DMPP) levels through time. The value represents the mean of three samples, and the error bars indicate the standard deviation of the mean (n = 3). Down arrow ↓ indicates the time of fertilization and DMPP addition. CK = control with no DMPP; DL, DM, and DH = treatments applied with 0.5%, 1%, and 2% DMPP, respectively.
Agronomy 14 02029 g001
Table 1. Soil chemical property including soil NH4+-N, NO3–-N, AP, AK content, pH, EC, urease and acid phosphatase activity as affected by different 3,4-dimethylpyrazole phosphate (DMPP) levels when the blueberries were harvested. CK = control with no DMPP; DL, DM, and DH = treatments applied with 0.5%, 1%, and 2% DMPP, respectively. Data are means ± standard deviations (n = 3). Means followed by different letters within a row are significantly different at the 0.05 probability level.
Table 1. Soil chemical property including soil NH4+-N, NO3–-N, AP, AK content, pH, EC, urease and acid phosphatase activity as affected by different 3,4-dimethylpyrazole phosphate (DMPP) levels when the blueberries were harvested. CK = control with no DMPP; DL, DM, and DH = treatments applied with 0.5%, 1%, and 2% DMPP, respectively. Data are means ± standard deviations (n = 3). Means followed by different letters within a row are significantly different at the 0.05 probability level.
Soil Chemical Properties Treatment
CKDLDMDH
NH4+-N (μg g−1)bulk soil803 ± 18 a854 ± 32 b887 ± 25 b888 ± 14 b
rhizosphere soil406 ± 13 a472 ± 13 b515 ± 8 c537 ± 22 c
NO3–-N (μg g−1)bulk soil131 ± 11 c104 ± 7 b74 ± 10 a78 ± 10 a
rhizosphere soil125 ± 4 d96 ± 4 c67 ± 2 a75 ± 1 b
pHbulk soil6.23 ± 0.04 a6.19 ± 0.03 a6.24 ± 0.06 a6.23 ± 0.06 a
rhizosphere soil6.31 ± 0.10 a6.30 ± 0.10 a6.30 ± 0.04 a6.31 ± 0.04 a
ECbulk soil1.42 ± 0.16 a1.55 ± 0.23 a1.76 ± 0.19 a1.68 ± 0.33 a
rhizosphere soil1.33 ± 0.04 a1.46 ± 0.03 ab1.50 ± 0.10 b1.44 ± 0.07 ab
AP (μg g−1)bulk soil374 ± 4 a392 ± 19 ab408 ± 13 b408 ± 16 b
rhizosphere soil246 ± 4 a282 ± 2 b301 ± 14 c315 ± 13 c
AK (mg g−1)bulk soil2.02 ± 0.03 a2.03 ± 0.06 a2.05 ± 0.04 a2.08 ± 0.05 a
rhizosphere soil1.23 ± 0.05 a1.27 ± 0.03 a1.28 ± 0.04 a1.29 ± 0.01 a
Urease (NH4+-N μg g−1 24h−1)bulk soil503 ± 49 a589 ± 52 ab605 ± 69 ab630 ± 61 b
rhizosphere soil581 ± 46 a608 ± 22 a632 ± 20 ab687 ± 48 b
Phosphatase (p-nitrophenol μg g−1 h−1)bulk soil1.21 ± 0.19 a1.43 ± 0.10 ab1.52 ± 0.13 ab1.61 ± 0.19 b
rhizosphere soil1.90 ± 0.19 a2.14 ± 0.25 ab2.27 ± 2.27 ab2.35 ± 0.07 b
Table 2. The chlorophyll content including chlorophyll-a and chlorophyll-b of the inner, middle, and outer leaves of blueberry as affected by different DMPP levels. CK = control with no DMPP; DL, DM, and DH = treatments applied with 0.5%, 1%, and 2% DMPP, respectively. Data are means ± standard deviations (n = 3). Means followed by different letters within a column are significantly different at the 0.05 probability level.
Table 2. The chlorophyll content including chlorophyll-a and chlorophyll-b of the inner, middle, and outer leaves of blueberry as affected by different DMPP levels. CK = control with no DMPP; DL, DM, and DH = treatments applied with 0.5%, 1%, and 2% DMPP, respectively. Data are means ± standard deviations (n = 3). Means followed by different letters within a column are significantly different at the 0.05 probability level.
TreatmentChlorophyll-a (mg g−1 FW)Chlorophyll-b (mg g−1 FW)
InnerMiddleOuterInnerMiddleOuter
CK0.77 ± 0.06 a0.67 ± 0.05 ab0.34 ± 0.03 a0.40 ± 0.02 a0.39 ± 0.06 a0.25 ± 0.00 a
DL1.10 ± 0.08 b0.64 ± 0.05 a0.35 ± 0.06 a0.51 ± 0.02 b0.37 ± 0.06 a0.26 ± 0.03 a
DM1.09 ± 0.16 b0.90 ± 0.09 c0.56 ± 0.05 b0.50 ± 0.06 b0.45 ± 0.04 a0.35 ± 0.04 b
DH0.86 ± 0.04 a0.77 ± 0.07 b0.57 ± 0.06 b0.44 ± 0.02 a0.42 ± 0.02 a0.34 ± 0.04 b
Table 3. Agronomic characteristics including total branch length, the number of blueberry branches, height, basal diameter, and the dry weight of blueberry plant as affected by different DMPP levels. CK = control with no DMPP; DL, DM, and DH = treatments applied with 0.5%, 1%, and 2% DMPP, respectively. Data are means ± standard deviations (n = 3). Means followed by different letters within a row are significantly different at the 0.05 probability level.
Table 3. Agronomic characteristics including total branch length, the number of blueberry branches, height, basal diameter, and the dry weight of blueberry plant as affected by different DMPP levels. CK = control with no DMPP; DL, DM, and DH = treatments applied with 0.5%, 1%, and 2% DMPP, respectively. Data are means ± standard deviations (n = 3). Means followed by different letters within a row are significantly different at the 0.05 probability level.
Agronomic Characteristics Treatment
CKDLDMDH
Total branch length (cm) 450 ± 4 a457 ± 18 a525 ± 26 b607 ± 16 c
Number of branchesPrimary order8 ± 1 a8 ± 1 ab9 ± 1 ab9 ± 0 b
Secondary order16 ± 1 a21 ± 3 b18 ± 1 ab22 ± 2 b
Tertiary order9 ± 1 b5 ± 2 a10 ± 1 b7 ± 2 ab
Total branching number33 ± 1 a34 ± 2 a37 ± 1 b37 ± 1 b
Dry weight (g) 23 ± 2 a24 ± 5 a31 ± 1 b28 ± 2 ab
Height (cm) 41.7 ± 1.2 a42.3 ± 2.5 a45 ± 4.4 a42.7 ± 4.2 a
Basal diameter (mm) 7.5 ± 0.4 a7.6 ± 0.9 a7.6 ± 0.2 a7.7 ± 0.6 a
Table 4. Nitrogen, phosphorus, and potassium concentration of blueberry leaves as affected by different DMPP levels at harvest. CK = control with no DMPP; DL, DM, and DH = treatments applied with 0.5%, 1%, and 2% DMPP, respectively. Data are means ± standard deviations (n = 3). Means followed by different letters within a column are significantly different at the 0.05 probability level.
Table 4. Nitrogen, phosphorus, and potassium concentration of blueberry leaves as affected by different DMPP levels at harvest. CK = control with no DMPP; DL, DM, and DH = treatments applied with 0.5%, 1%, and 2% DMPP, respectively. Data are means ± standard deviations (n = 3). Means followed by different letters within a column are significantly different at the 0.05 probability level.
TreatmentN (mg g−1)P (mg g−1)K (mg g−1)
CK25.54 ± 1.11 a1.11 ± 0.04 a13.26 ± 0.81 a
DL26.75 ± 1.88 ab1.22 ± 0.05 a12.65 ± 0.61 a
DM28.65 ± 1.98 bc1.40 ± 0.13 b12.65 ± 0.71 a
DH30.96 ± 0.34 c1.42 ± 0.12 b13.03 ± 0.96 a
Table 5. Abundances of nitrifier (ammonia-oxidizing archaeal and bacterial amoA, nxrA), denitrifier (nirS) gene copies per gram dry soil related to microbial nitrogen cycle in rhizosphere soil across the DH and CK treatments. CK = control with no DMPP; DH = treatments applied with 2% DMPP. Data are means ± standard deviations (n = 3).
Table 5. Abundances of nitrifier (ammonia-oxidizing archaeal and bacterial amoA, nxrA), denitrifier (nirS) gene copies per gram dry soil related to microbial nitrogen cycle in rhizosphere soil across the DH and CK treatments. CK = control with no DMPP; DH = treatments applied with 2% DMPP. Data are means ± standard deviations (n = 3).
TreatmentQuantity Mean (Copies g−1)
AOB amoAAOA amoAnxrAnirS
CK1.09 × 107 ± 3.55 × 1069.24 × 106 ± 3.00 × 1061.86 × 107 ± 4.89 × 1061.65 × 107 ± 2.52 × 106
DH2.72 × 104 ± 1.48 × 1041.73 × 107 ± 5.93 × 1061.19×107 ± 3.77×1062.14 × 107 ± 1.63 × 106
Table 6. Pearson correlation analysis between nitrogen and phosphorus content of soil and blueberry leaves, soil enzyme activities, and plant agricultural traits. The value represents coefficients and significance, * p ≤ 0.05, ** p ≤ 0.01. BS = bulk soil; RS = rhizosphere soil; Pase = phosphatase; BD = basal diameter; TBL = total branch length; NB = number of branches; DW = dry weight.
Table 6. Pearson correlation analysis between nitrogen and phosphorus content of soil and blueberry leaves, soil enzyme activities, and plant agricultural traits. The value represents coefficients and significance, * p ≤ 0.05, ** p ≤ 0.01. BS = bulk soil; RS = rhizosphere soil; Pase = phosphatase; BD = basal diameter; TBL = total branch length; NB = number of branches; DW = dry weight.
ParameterNH4+-N (BS)NH4+-N (RS)NO3−-N (BS)NO3−-N (RS)pH (BS)pH (RS)EC (BS)EC (RS)AP (BS)AP (RS)AK (BS)AK (RS)Urease (BS)Urease (RS)Pase (BS)Pase (RS)HeightBDTBLNBDWLeaf NLeaf P
NH4+-N (RS)0.793 **—
NO3−-N (BS)−0.768 **−0.920 **—
NO3−-N (RS)−0.846 **−0.942 **0.925 **—
pH (BS)0.0080.165−0.177−0.153—
pH (RS)−0.3320.087−0.0800.0030.131—
EC (BS)0.609 *0.521−0.436−0.551−0.145−0.502—
EC (RS)0.4600.606 *−0.670 *−0.661 *0.3790.2390.017—
AP (BS)0.669 *0.705 *−0.694 *−0.733 **0.024−0.1180.639 *0.559—
AP (RS)0.729 **0.966 **−0.930 **−0.902 **0.2280.0880.4730.662 *0.758 **—
AK (BS)0.3450.375−0.336−0.3040.251−0.1780.529−0.0340.5320.436—
AK (RS)0.4430.623 *−0.352−0.5560.0880.3280.2800.4430.4950.5110.204—
Urease (BS)0.848 **0.567−0.564−0.626 *0.094−0.2510.2670.3440.4400.5290.4330.341—
Urease (RS)0.684 *0.667 *−0.642 *−0.597 *−0.295−0.3350.666 *0.0500.694 *0.667 *0.5040.1930.465—
Pase (BS)0.5580.751 **−0.850 **−0.681 *0.0800.0300.4230.3340.5100.818 **0.5420.0610.4540.681 *—
Pase (RS)0.4990.667 *−0.771 **−0.680 *0.1050.1550.0930.3880.4140.720 **0.3960.1160.590 *0.5210.823 **—
Height0.2630.304−0.429−0.3250.719 **−0.2210.1450.4990.1260.3980.127−0.1930.149−0.0400.3900.182—
BD0.1560.122−0.151−0.0240.4550.250−0.0590.3990.2860.1920.3250.2520.217−0.0850.070−0.1290.322—
TBL0.730 **0.821 **−0.703 *−0.720 **0.165−0.0110.5280.2150.654 *0.779 **0.5510.5310.577 *0.791 **0.612 *0.5330.0980.192—
NB0.687 *0.671 *−0.645 *−0.599 *−0.290−0.3320.665 *0.0560.698 *0.671 *0.5080.1950.4691.000 **0.684 *0.523−0.036−0.0760.794 **—
DW0.595 *0.586 *−0.685 *−0.691 *0.2610.1190.4630.4390.737 **0.581 *0.591 *0.3880.5260.4360.5030.5100.1570.3570.605 *0.441—
Leaf N0.678 *0.779 **−0.710 **−0.712 **−0.0500.1160.3920.1520.5150.694 *0.5230.5110.651 *0.736 **0.664 *0.721 **−0.136−0.0080.862 **0.738 **0.626 *—
Leaf P0.872 **0.766 **−0.677 *−0.850 **−0.012−0.2820.615 *0.2730.616 *0.679 *0.3420.4820.731 **0.690 *0.4810.5730.065−0.1990.777 **0.689 *0.577 *0.759 **—
Leaf K0.030−0.1700.3800.2090.165−0.252−0.106−0.117−0.176−0.302−0.2420.3090.032−0.182−0.653 *−0.565−0.1270.1340.045−0.183−0.249−0.1210.021
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Yang, Y.; Zeng, Q.; Yu, H.; Wei, J.; Jiang, J.; Tian, L. Effect of the Nitrification Inhibitor DMPP on Blueberry Planted in Neutral Soil. Agronomy 2024, 14, 2029. https://doi.org/10.3390/agronomy14092029

AMA Style

Yang Y, Zeng Q, Yu H, Wei J, Jiang J, Tian L. Effect of the Nitrification Inhibitor DMPP on Blueberry Planted in Neutral Soil. Agronomy. 2024; 14(9):2029. https://doi.org/10.3390/agronomy14092029

Chicago/Turabian Style

Yang, Yiru, Qilong Zeng, Hong Yu, Jiguang Wei, Jiafeng Jiang, and Liangliang Tian. 2024. "Effect of the Nitrification Inhibitor DMPP on Blueberry Planted in Neutral Soil" Agronomy 14, no. 9: 2029. https://doi.org/10.3390/agronomy14092029

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