*Article* **Efficiency of Reductive Soil Disinfestation Affected by Soil Water Content and Organic Amendment Rate**

**Rui Zhu 1,2, Xinqi Huang 3,4,5,6, Jinbo Zhang 3,4,5,6, Zucong Cai 3,4,5,6, Xun Li 1,2,6 and Teng Wen 3,4,5,6,\***


**Abstract:** Reductive Soil Disinfestation (RSD) is a good method which can restore degraded greenhouse soil and effectively inactivate soil-borne pathogens. However, the approach needs to be optimized in order to facilitate its practical application in various regions. In the present work, we investigated the effect of soil water content (60% water holding capacity (WHC), 100% WHC and continuous flooding) and maize straw application rates (0, 5, 10, and 20 g kg−<sup>1</sup> soil) on the improvement of soil properties and suppression of soil-borne pathogens (*Fusarium oxysporum*, *Pythium* and *Phytophthora*). The results showed that increasing the soil water content and maize straw application rate accelerated the removal of excess sulfate and nitrate in the soil and elevated the soil pH. Elevating the water content and maize straw application rate also produced much more organic acids, which could strongly inhibit soil-borne pathogens. Soil properties were improved significantly after RSD treatment with a maize straw amendment rate of more than 5 g kg<sup>−</sup>1, regardless of the water content. However, RSD treatments with 60% WHC could not effectively inactivate soil-borne pathogens and even stimulated their growth by increasing the maize application rate. RSD treatments of both 100% WHC and continuous flooding could inactivate soil-borne pathogens and increase the pathogens mortality indicated by cultural cells relatively effectively. The inhibited pathogens were significantly increased with the increasing maize application rate from 5 g kg−<sup>1</sup> to 10 g kg<sup>−</sup>1, but were not further increased from 10 g kg−<sup>1</sup> to 20 g kg−1. A further increased mortality of *F. oxysporum*, indicated by gene copies, was also observed when the soil water content and maize straw application rate were increased. Therefore, RSD treatment with 60% WHC could improve soil properties significantly, whereas irrigation with 100% WHC or continuous flooding was a necessity for effective soil-borne pathogens suppression. Holding 100% WHC and applicating maize straw at 10 g kg−<sup>1</sup> soil were optimum conditions for RSD field operation to restore degraded greenhouse soil.

**Keywords:** application rate; nitrate; organic acid; soil-borne pathogens; soil pH; sulfate

#### **1. Introduction**

Greenhouse cultivation plays a very important role in vegetable production all over the world. The health status of greenhouse soil directly influences the yield and the quality of the products from greenhouse cultivation [1]. Yet, driven by the economic benefits, farmers usually apply a large amount of chemical fertilizer for intensive vegetable production in greenhouses in China [2,3]. As a result, acidification and salinization widely occur in greenhouse soil, which not only greatly deteriorates the physical and chemical

**Citation:** Zhu, R.; Huang, X.; Zhang, J.; Cai, Z.; Li, X.; Wen, T. Efficiency of Reductive Soil Disinfestation Affected by Soil Water Content and Organic Amendment Rate. *Horticulturae* **2021**, *7*, 559. https://doi.org/10.3390/ horticulturae7120559

Academic Editor: Giovanni Bubici

Received: 26 October 2021 Accepted: 6 December 2021 Published: 7 December 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

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properties of soil, but also induces serious soil-borne disease [3,4]. This problem has become the bottleneck for sustainable development of greenhouse cultivation all over the world, especially in China [5,6].

Reductive soil disinfestation (RSD), also known as biological soil disinfestation (BSD) or anaerobic soil disinfestation (ASD), is an emerging environmentally friendly method and effective alternative to chemical fumigations [7]. This method was first proposed by Shinmura [8] and Blok [9], and was characterized by applying labile carbon to develop microbial-driven anaerobic soil conditions in moist or flooded soil covered with polyethylene mulch [7]. It was proven to control a wide range of soil-borne pathogens and nematodes, as well as improve soil properties and microbial environment [7,10,11].

RSD needs to apply a large amount of organic material to the soil. Various kinds of organic materials have been used for RSD treatment, including rye-grass, straw stalk, wheat bran, syrup, ethanol, animal waste, and so on [12–16]. However, organic material is not always easily accessible; sometimes this entails expensive purchase and delivery costs. In China, crop residues, such as crop straws, which are usually recognized as agricultural production wastes, can be easily obtained in most areas. Their management and recycling were also long-standing problems for sustainable agriculture. Open-field burning is the most common approach to treat crop residues, but this undoubtedly leads to heavy air pollution. For this reason, applying crop straw as an organic amended material not only helps to recycle waste straw, but also reduces the costs of RSD treatment. Previous studies discovered that maize straw performs well as an organic, amended material for RSD, in which application of 5 g maize straw kg−<sup>1</sup> in saturated soil (100% water holding capacity, WHC) could inactivate more than 90% soil-borne pathogens [17].

RSD also needs large amount of water, but it is not appropriate for flooding in all areas. Soil water holding capacity is affected by soil texture and topography greatly; soil abundant in organic matters can hold more water than sandy soil [18], and it is difficult to implement flooding in hilly areas and uplands. Moreover, water resources are very precious; therefore, it is very necessary to develop a more efficient water-saving method for RSD. Previous studies showed that irrigating soil to 100% WHC and the simultaneous mulching with polyethylene films could create same highly anaerobic environments [18,19] and effectively kill soil-borne pathogens with a maize straw application rate of 5 g kg−<sup>1</sup> soil, as in flooded soil [11]. However, in view of the cost and practicability, an optimum soil water content and amendment rate have not yet been proposed. Accordingly, the aim of this study was to explore the effects of water content and organic amendment rates of RSD treatment on the suppression of soil-borne pathogens and improvement of soil properties, and then to propose an optimum soil water content and maize straw application rate for RSD field operation.

#### **2. Materials and Methods**

#### *2.1. Materials*

The soil for the present laboratory experiment was collected from a greenhouse in Baguazhou, Nanjing, China (118.81 E, 32.19 N). The soil was of a clay loamy texture and used as growing substrate of *Artemisia selengensis* for several years. *Fusarium* wilt disease perniciously spread among the field, and the soil was severely degraded in this area. All soil samples collected from this area were sieved through 2 mm sieve before experiment. The maize straw was collected as the organic material from Hongta county, Lilou towns, Bengbu, Anhui, China (117.44 E, 32.87 N). Before mixed into soil, the straw was air-dried, crushed and sieved through 2 mm sieve. The total organic carbon (TOC), total N (TN), easily oxidized carbon (EOC) and C/N of the soil sample and maize straw are shown in Table 1.


**Table 1.** Total N, total C, easily oxidized carbon (EOC) content and C/N ratio of maize straw and the soil used in this study.

#### *2.2. Experiment Design*

Prepared soil samples (250 g soil on air-dry basis), which were thoroughly mixed with maize straw at designed rates, were placed into self-closure bags. The PE bags were 15 cm long, 9 cm wide, and 0.1 mm thick. The soil water content was set at 60% WHC, 100% WHC, and continuous flooding, respectively. The straw amendment rate was set at 0, 5, 10, and 20 g kg−<sup>1</sup> soil, respectively. Soil samples subjected only to water content modification were referred as CK treatments, while those subjected to both water and maize straw modifications were designed as RSD treatments. Thus, there were 12 treatments which were listed in Table 2. Each treatment set three replicates and all closed bags were incubated randomly at 30 ◦C. Previous studies showed that organic acids were one of the key factors in the sterilization of RSD [20]. For measuring organic acids in the soil solution during the incubation, a soil solution (about 2 mL) was collected by pre-embedded porous-cup soil solution sampler (DIK-8393; Daiki Rika Kogyo, Saitama, Japan) [21] on days 4, 8, 12, and 15 after incubation. The sampler was completely buried into the mixed soil in each bag for the nondestructive collection of soil solution samples through a syringe with a two-way valve, then by adding water to the objective soil water content [22]. At the end of 15 days of incubation, the physical and chemical properties of soil samples and the populations of *Fusarium oxysporum* spp., *Pythium* spp. and *Phytophthora* spp. in soil samples were determined.


**Table 2.** Treatments list.

#### *2.3. Soil Analyses*

Soil pH was measured in slurry (soil: water = 1:2.5) by a pH electrode (Mettler S220K, Greifensee, Switzerland) [23,24]. Nitrate and ammonium concentrations in soil were extracted by 2 mol L−<sup>1</sup> KCl solution (soil: water = 1:5) after 250 rpm shaking for 1 h, and then measured by a continuous-flow analyzer (Skalar San++, Breda, Holland) [17]. Soil EC was measured in filtrate (soil: water = 1:5) by a conductivity meter (KangYi Corp., Shanghai, China) after 180 rpm shaking for 20 min [23]. SO4 <sup>2</sup><sup>−</sup> concentration in the soil was measured in filtrate (soil: water = 1:5) by ion chromatography (Thermo Dionex ICS 1100, Waltham, MA, USA) [25]. Soil TOC was measured by wet digestion [26]. Soil TN was measured by Macro Kjeldahl method [27]. Soil EOC was measured by the potassium permanganate oxidation method [28]. The maximum water holding capacity of soil was measured as follows: First, the soil was thoroughly mixed with the corresponding amount

of maize straw. Second, the soil was thoroughly flooded in a funnel equipped with filter paper for 2 h. Then, free water was leached out for 6–8 h. Until this point, the soil water content was defined as 100% WHC. The amended water holding capacity of soil without maize straw was calculated at the same time. The soil water content was determined by drying the soil at 105 ◦C to a constant mass.

Organic acids produced during RSD in the soil solution were measured by HPLC (Agilent 1260, Santa Clara, CA, USA) with a modification according to the literature [29]. The column was XDB-C18 (4.6 × 250 mm, Agilen, USA), and the mobile phase was consisted of different gradient dilution of 2.5 mM H2SO4 (A) and methanol (B) at a flow rate of 1 mL min<sup>−</sup>1. The composition of gradients was set as follows: 0–5 min, 95% A plus 5% B; 5–13 min, 95% A plus 5% B; 13–53 min, 85% A plus 15% B; and 53 min stop, 85% A plus 15% B. The wavelength of UV detector was set at 210 nm. The quantification of organic acid was calculated by comparing the time of the peak appearance and the peak area with the standard samples.

#### *2.4. The Population of the Main Soil-Borne Pathogens: Fusarium oxysporum spp., Pythium spp. and Phytophthora spp.*

Plate count method was used to count the cultural cells in different soil samples. Ten-times-step dilution was applied when performing the dilution plating procedure. K2 medium [30] was used for the growth of *Fusarium oxysporum* spp. (K2HPO4 1 g, MgSO4·7H2O 0.5 g, KCl 0.5 g, Fe-Na-EDTA 0.01 g, L-Asparagine 2 g, D-galactose 2 g, agar 24 g, distilled water 1000 mL. PCNB 1 g, Na2B4O7·10H2O 0.5 g, Streptomycin 0.12 g, 10% H3PO4 for adjusting pH to 3.8–4.0 after sterilization). Maize meal agar was used for the growth of *Pythium* spp. (maize meal agar 20 g, distilled water 1000 mL; Pimarincin 50 mg and Ampicillin 250 mg after sterilization). Oatmeal agar was used for the growth of *Phytophthora* spp. First, by adding 20 g of oatmeal to 900 mL of water, and then boiling the water lightly for 45 min, filtering the solution, adding distilled water and 24 g agar to 1000 mL. Ampicillin (200 mg) and rifampicin (20 mg) was amended after sterilization.

#### *2.5. Extraction of Soil DNA and Quantification of Fusarium oxysporum*

Soil DNA was extracted through the Power Soil TM DNA Isolation Kit (MOBIO Laboratories Inc., Carlsbad, CA, USA) according to the kit instructions and [31]. Real-time PCR was performed on the CFX-96 thermocycler (Bio-Rad Laboratories Inc., Hercules, CA, USA). The 20 μL reaction system was designed as follows: 2 μL DNA template, 10 μL SYBR GREEN premix EX Taq (2×, TaKaRa, Dalian, China), 6 μL sterile deionized water, 1 μL primer of F and 1 μL primer of R (ITS1-F (F): 5- -CTTGGTCATTTAGAGGAAGTAA-3- [32] and AFP308 (R): 5- -CGAATTAACGCGAGTCCCAAC-3- (10 μmol L−1) [33]). Reaction conditions were designed as follows: Pre-denaturation step at 95 ◦C for 2 min followed by 40 cycles, including denaturation at 95 ◦C for 10 s, annealing at 58 ◦C for 15 s, extended at 75 ◦C for 20 s. Melt curve profiles were obtained at annealing step in each cycle.

#### *2.6. Statistical Analyses*

Differences among treatments were assessed with ANOVA analysis. LSD test and SNK test was applied in SPSS 22.0 (SPSS Inc., Chicago, IL, USA) when ANOVA analysis revealed significant differences at *p* < 0.05. Origin 2016 was used to create diagrams.

#### **3. Results**

#### *3.1. Soil pH and EC*

The soil pH in all RSD treatments was significantly increased at the end of treatments (Figure 1 and Table 3). The initial soil pH was 4.42. After the RSD treatments, soil pH was slightly increased in CK, and was further increased in RSD treatments. Among treatments with the same maize straw application rates, flooded soil and 100% WHC soil always had higher pH values than 60% WHC soil. Adding more maize straw helped to achieve higher pH values in 100% WHC and flooded treatments. The highest pH (5.34) was observed in the flooded plus 20 g maize straw kg−<sup>1</sup> soil treatment. In 60% WHC treatments, however, soil pH was not significantly increased with the increasing maize straw application rates from 5 g kg−<sup>1</sup> to 20 g kg<sup>−</sup>1. Generally, soil EC had a decreased trend with increasing water content (Figure 1). However, the effects of the water content and straw amendment rate, and their interaction on soil EC, were not significant (Table 3).

**Figure 1.** The effects of water content and maize amendment rate on soil EC and pH after RSD treatment. Error bars indicate standard deviations. Different uppercase letters in EC implicate the a significant difference among the same maize straw amended rate under different soil water contents, and lowercase letters within the same water content show significant differences among the various maize straw amended rates (SNK tests, *p* < 0.05). Different lowercase letters show a significant difference among the various treatment of pH (SNK tests, *p* < 0.05). The dashed lines represent the initial value of the original soil.


**Table 3.** ANOVA analyses of the RSD effects of maize application rates and soil water contents on soil properties and populations of soil-borne pathogens.

*3.2. Soil SO4 <sup>2</sup>*−*, NH4 <sup>+</sup> and NO3* − *Contents*

Across all RSD treatments, SO4 <sup>2</sup><sup>−</sup> and NO3 − concentrations in soil were significantly decreased. On the contrary, soil NH4 <sup>+</sup> concentration was increased distinctly in 60% WHC and 100% WHC treatments when compared to CK (Figure 2). The effects of straw amendment on the SO4 <sup>2</sup>−, NO3 − and NH4 <sup>+</sup> concentrations were significant, while the significant effect of water content only appeared in soil NH4 <sup>+</sup> (Table 3). In CK treatments, soil SO4 <sup>2</sup><sup>−</sup> and NO3 − concentrations were effectively reduced (*p* < 0.05) and soil NH4 +

increased when compared with their initial contents. This trend was much more clear after maize straw was amended. The contents of NH4 <sup>+</sup> were double or triple their initial values, whereas NO3 - was decreased from 3.36 mg kg−<sup>1</sup> to almost zero (*p* < 0.001), and SO4 <sup>2</sup><sup>−</sup> was reduced from 302 mg kg−<sup>1</sup> to 79-128 mg kg−<sup>1</sup> (*p* < 0.001) in all RSD treatments. Although SO4 <sup>2</sup><sup>−</sup> content shows the lowest in 100% WHC and flood–RSD treatment with the straw amendment rate of 20 g kg−1, no significant differences in NO3 - and NH4 <sup>+</sup> contents were observed among different straw amendment rates in the same soil water content treatments. On the other hand, despite the fact that NO3 - and SO4 <sup>2</sup><sup>−</sup> were significantly decreased in RSD treatment, there were no significant differences among different soil water content under the same straw amendment rate. RSD treatment with 60% WHC and 100% WHC showed the same efficiency in lifting soil NH4 <sup>+</sup> content and removing soil NO3 − content as the continuous flooding treatments.

**Figure 2.** The contents of NO3 −, SO4 <sup>2</sup><sup>−</sup> and NH4 <sup>+</sup> after RSD treatment. Error bars indicate standard deviations. Different uppercase letters implicate the significant difference among the same maize straw amended rate under the different soil water contents of NO3 - and SO4 <sup>2</sup>−, and lowercase letters within the same water content show a significant difference among the various maize straw amended rates (SNK tests, *p* < 0.05). Different lowercase letters show a significant difference among the various treatments of NH4 <sup>+</sup> (SNK tests, *p* < 0.05). The dashed lines represent the initial value of the original soil.

#### *3.3. Dynamics of Organic Acids during the RSD Treatment*

A great quantity of organic acids, which were toxic to soil-borne pathogens [34], were produced in all RSD treatments, but none of them were detected in CKs (Figure 3). Acetic acid exhibited the highest concentration among the four detectable acids, followed by propionic acid and butyric acid, while isovaleric acid was the most minor acid and only showed low concentrations in the flooded RSD treatment amended with a high amount of maize straw. The elevating application rate of maize straw helped to produce more organic acids. The concentrations of acetic acid in RSD treatments with maize amendment rate of 20 g kg−<sup>1</sup> were fivefold to tenfold compared to those with 5 g kg−1. Moreover, organic acids concentrations maintained high levels throughout the treating period in RSD applied with high amended rates of maize straw, but declined to almost zero after 15 days' incubation in those with low amended rates of maize straw.

**Figure 3.** Dynamics of organic acids during the RSD treatments. Error bars indicate standard deviations. Different lowercase letters show significant differences among the various maize straw amended rates and soil water contents. (*p* < 0.05). CKs were not displayed in the figure because no organic acid was detected throughout the incubation period.

#### *3.4. Community of Soil-Borne Pathogens*

The populations of soil-borne pathogens (*F. oxysporum* spp., *Phytophthora* spp. and *Phyrium* spp.) were counted through selective cultural mediums. Compared with CK, in which the populations of pathogens were only slightly reduced or even increased from the initial values, RSD treatments by applying maize straw into 100% WHC or flooded soil dramatically killed pathogens (*p* < 0.001, Figure 4). Both water content and straw amendment rate and their interaction significantly affected the populations of studied soil-borne pathogens (Table 3). However, the water content in the soil has a smaller effect on the content of pathogenic microorganisms than the addition of straw to 100% WHC and flooded soil. The highest mortalities of all three pathogens were found in treatments applying with maize straw at 20 g kg−1, which inactivated more than 96% of pathogens (*F. oxysporum* spp., *Phytophthora* spp. and *Phyrium* spp.) in both flooded and 100% WHC

soil. Applying maize straw at 5 g kg−<sup>1</sup> into flooded soils also effectively reduced up to 90% of *F. oxysporum* spp. and *Pythium*, but no statistically significant differences were observed between this treatment and CK in the 100% WHC condition. Increasing the straw applying rate to 10 and 20 g kg−<sup>1</sup> in both flooded and 100% WHC soil significantly decreased the populations of studied soil-borne pathogens by more than 95%, and the differences between the straw applying rate of 10 and 20 g kg−<sup>1</sup> were not significant. In 60% WHC soil; however, the highest pathogens mortality was less than 60% even when the highest rate of maize straw (20 g kg<sup>−</sup>1) was applied. Instead, it seemed that the more maize straw that was used, the less the pathogens were suppressed. In the treatments of 60% WHC + 20 g maize straw kg−<sup>1</sup> soil, the populations of three pathogens increased twofold, fivefold and onefold, respectively, compared with the CK (*p* < 0.05).

**Figure 4.** The populations of *F. oxysporum*, *Phytophthora* and *Pythium* in soils after RSD treatment. Error bars indicate standard deviation. Different lowercase letters show significant differences among the various treatment (SNK tests, *p* < 0.05). The dashed lines represent the initial value of the original soil.

Real-time PCR results of *F. oxysporum* were consistent with the plate-count results mentioned above (Figure 5). Applying maize straw at a rate of 5 g kg−<sup>1</sup> into 100% WHC or flooded soil could reduce the populations of the pathogen beyond 95%, and over 98% of pathogens were killed by enhancing the maize straw application rate to 10 g kg−<sup>1</sup> or 20 g kg<sup>−</sup>1. The lowest density of *F. oxysporum* was found in the treatment of flooding coupled with a 20 g maize straw kg−<sup>1</sup> application rate (5.55 log copies g−<sup>1</sup> dry soil) while they remained at high levels throughout the experiment (about 7.55~7.82 log copies g−<sup>1</sup> dry soil) in CKs and 60% WHC RSD treatment, as well as in variants with 100% WHC and flooded soil.

**Figure 5.** The population of *F. oxysporum* in soils after RSD treatments through real-time PCR. Error bars indicate standard deviations. Different lowercase letters show significant differences among the various treatments (SNK tests, *p* < 0.05). The dashed lines represent the initial value of the original soil.

#### **4. Discussion**

Reductive Soil Disinfestation is a highly efficient and environmentally friendly method of remediating the degraded greenhouse soil over a short period [12]. So far, it has been applied in the field by amending organic material into flooded or water-saturated soil (irrigating soil to 100% WHC), and then mulching plastic film to achieve the desired effect [9,19,35]. Maize straw, alfalfa, wheat bran and other easily decomposable agricultural waste could effectively suppress soil-borne pathogens in RSD treatments [23,36,37]. In this study, aiming to promote RSD application in fields where water and straw are difficult and costly to access, we optimized the RSD-treating conditions from different soil water contents (60% WHC, 100% WHC and continuous flooding, respectively) and maize straw application rates (0, 5, 10, and 20 g kg<sup>−</sup>1, respectively).

Our results showed that all the RSD treatments with different soil water contents and maize straw application rates could effectively remove excessive soil SO4 2- and NO3 - , but increase NH4 <sup>+</sup> in degraded soil (Table 3). A relatively low original soil EC could be the reason that RSD treatments did not reduce soil EC significantly. The results showed significant interactions between water content and the maize straw application rate on soil pH and NH4 <sup>+</sup> (Table 3). Applying a low amount of maize straw (5 g kg−1) and irrigating to 60% WHC significantly reduced soil SO4 2- and NO3 - contents and increased NH4 + content, but no further changes in these variables were observed when the maize straw

application rate was increased from 5 g kg−<sup>1</sup> to 20 g kg−<sup>1</sup> (Figures 1 and 2). As we know, RSD creates a strong anaerobic environment in soil where nitrate can transform into N2O or N2 mainly through denitrification, and sulfate can be reduced to H2S or assimilated to organic sulfur by soil microbes [10]. Previous research suggested that inputting a large amount of organic matter into anaerobic soil by flooding or irrigating to 100% WHC could dramatically decrease soil Eh over several days [17]. Here, although 60% WHC could not fulfill the requirement of completely anaerobic soil, we still found that soil SO4 <sup>2</sup><sup>−</sup> and NO3 − experienced a sharp decline after amending maize straw. Moreover, our previous work revealed that increasing the application rate of maize straw might accelerate the speed of Eh decline, but the final Eh were not significantly different [17]. In other words, the low amendment rate of maize straw in 60% WHC could induce a similar strong reductive environment to reduce nitrate and sulfate contents in the end.

RSD treatment can also remediate severe soil acidification [7]. In this study, soil pH after RSD treatments was markedly increased and displayed a rising trend with the increasing soil water content (Figure 1). Furthermore, there was a significant interaction between the maize application rate and water content regarding soil pH (*p* < 0.05, Table 2). In 60% WHC condition, there were no significant differences of soil pH among the RSD treatments with maize straw application rates of 5, 10 and 20 g kg−1, whereas in flooded RSD treatments, soil pH was increased with the increasing maize straw application rate from 5 g kg−<sup>1</sup> to 20 g kg−<sup>1</sup> (Figure 1). Organic amendment decomposition yielded energy and soluble organic carbon, which could promote soil microbe proliferation and accelerate the consumption of soil oxygen simultaneously. Then, the anaerobic soil environment was established. Anaerobic microbes transported electrons to oxidized substances (SO4 <sup>2</sup>−, NO3 <sup>−</sup>, Fe2+, Mn2+) in soil through anaerobic respiration. The reduction process of these oxidized substances was concomitant with the consumption of H+, which eventually increased soil pH [38]. The results suggested that if soil pH needed to be elevated further, a higher soil water content and larger organic material application rate were required for RSD treatment. However, our previous research compared the 100% WHC and flooded RSD treatments with equal organic material input, and clearly pointed out that their different effects on soil pH were entirely diminished after 25 days of treatment [17]. The implication was that the RSD treatment with 60% WHC could achieve the same high pH levels as 100% WHC or flooded treatment when the incubation time was extended.

RSD needs to apply a large amount of organic materials to soil, where microorganisms are the major agents used to degrade fresh organic matter [39]. Previous studies showed that a huge amount of organic acid was produced by anaerobes in an anaerobic soil environment, especially acetic acid [40], as well as the antagonistic activity of soil microorganisms in the mechanisms of RSD [20]. Our experiment showed the same trend that RSD treatments produced a large amount of organic acid while the proliferation of soil-borne pathogens was suppressed simultaneously after 15 days of incubation. Treatment with organic matter produced acetic acid, propionic acid, butyric acid and isovaleric acid throughout the culture period. Acetic acid was the major organic acid produced by RSD treatment, and was increased with the increasing amended amount of maize straw (from 5 g kg−<sup>1</sup> soil to 20 g kg−<sup>1</sup> soil). Continuous flooding treatments with maize straw produced much higher acetic acid and propionic acid in soil solutions than those in 60% WHC and 100% WHC conditions, especially in 20 g maize straw kg−<sup>1</sup> treatment. Previous studies showed that organic acids in soil produced during RSD incubation could effectively suppress soil-borne pathogens [34,41]. This may be the reason for the better sterilizing effect of flooding coupled with 20 g maize straw kg−<sup>1</sup> soil treatment.

However, unlike RSD treatments of continuous flooding or 100% WHC, in which more than 90% soil-borne pathogens were suppressed, only 30~58% pathogens were inactivated in RSD treatments with 60% WHC, even lower than that of CK (flooding alone). Applying more maize straw did not increase the mortality of pathogens, but promoted their propagation instead when the water content was set at 60% WHC (Figure 4). This result is not consistent with existing studies on RSD. A previous study showed that the

incorporation of organic matter in soil may be utilized by strains of the saprophytic fungus, which led to a growth in soil pathogen population density [42].

All three fungal species tested in this research were mostly saprophytic. This must be why these populations increased at a 60% WHC condition at high organic amendment rates. Generally, RSD sterilize soil-borne pathogens via three methods: (1) creating a strong reductive and completely anaerobic environment in soil to prevent the aerobe reproduction [7]; (2) producing toxic and volatile fatty acids to inactivate soil-borne pathogens during the process of organic materials anaerobic fermentation [43]; (3) inducing compositional shifts of microbes by diverse *Clostridium* sp. becoming the dominant species and improving soil biodiversity [44]; (4) inducing proliferation and dynamic compositional changes in the Firmicutes community, including methyl sulfide compounds, through a metabolic method [45]. The three ways work together to achieve the suppression of soil-borne pathogens in soil [9]. At 60% WHC amended with maize straw, the anaerobic environment was less sufficient than that in 100% WHC or flooded soil. What is more, organic acids contents were kept at very low levels during the whole RSD treatment period and were not dependent on maize straw application rates from 5 g kg−<sup>1</sup> to 20 g kg−<sup>1</sup> (Figure 3). This could be one of the most important reasons why RSD treatment with soil water content of 60% WHC could not effectively inactivate soil-borne pathogens. Conversely, when soil water content was set at 60% WHC for RSD treatment, the populations of soil-borne pathogens were even increased when the maize straw application reached 20 g kg−<sup>1</sup> (Figure 4). This result implied that the trapped soil air at a soil water content of 60% WHC could temporarily support soil-borne pathogens, as aerobes [44], to grow and reproduce, provided that they were rich in carbon and nitrogen [34].

#### **5. Conclusions**

RSD is an economic and effective method to restore degraded greenhouse soil. Although applying maize straw into 60% WHC soil is adequate to alleviate soil acidification and salinization, we found that it could not effectively inactivate soil-borne pathogens. Developing a complete anaerobic soil condition by irrigating soil to at least 100% WHC is necessary in order to suppress soil-borne pathogens; therefore, 100% WHC must be held and 10 g kg−<sup>1</sup> of maize straw must be applied (18 Mg per ha per 15 cm depth in the field).

Soil was recommended for the RSD field operation to both improve soil properties and suppress soil-borne pathogens.

**Author Contributions:** Conceptualization, Z.C. and J.Z.; methodology, Z.C., T.W. and R.Z.; software, R.Z.; validation, T.W.; formal analysis, R.Z.; resources, Z.C, X.H. and T.W.; data curation, T.W., X.H. and X.L.; writing—original draft preparation, R.Z.; writing—review and editing, T.W. and R.Z.; visualiza-tion, R.Z.; supervision, T.W. and X.H.; project administration, Z.C. and X.H.; funding acquisition, Z.C. and X.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was financially supported by the Key-Area Research and Development Program of Guangdong Province (2020B0202010006), the National Natural Science Foundation of China (42090065), and the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


#### *Article* **High NH4 +/NO3** *−* **Ratio Inhibits the Growth and Nitrogen Uptake of Chinese Kale at the Late Growth Stage by Ammonia Toxicity**

**Yudan Wang, Xiaoyun Zhang, Houcheng Liu, Guangwen Sun, Shiwei Song \* and Riyuan Chen \***

College of Horticulture, South China Agricultural University, Guangzhou 510642, China; ydwang@stu.scau.edu.cn (Y.W.); safiyazhang@163.com (X.Z.); liuhch@scau.edu.cn (H.L.); sungw1968@scau.edu.cn (G.S.)

**\*** Correspondence: swsong@scau.edu.cn (S.S.); rychen@scau.edu.cn (R.C.); Tel.: +86-20-85280228 (S.S.); +86-20-85280228 (R.C.)

**Abstract:** The aim of this study was to determine the effects of various NH4 +/NO3 − ratios in a nutrient solution on the growth and nitrogen uptake of Chinese kale under hydroponic conditions. The four NH4 +/NO3 − ratios in the nutrient solution were CK (0/100), T1 (10/90), T2 (25/75), and T3 (50/50). An appropriate NH4 +/NO3 − ratio (10/90, 25/75) promoted the growth of Chinese kale. T2 produced the highest fresh and dry weight among treatments, and all indices of seedling root growth were the highest under T2. A high NH4 +/NO3 − ratio (50/50) promoted the growth of Chinese kale seedlings at the early stage but inhibited growth at the late growth stage. At harvest, the nutrient solution showed acidity. The pH value was the lowest in T3, whereas NH4 <sup>+</sup> and NH4 +/NO3 − ratios were the highest, which caused ammonium toxicity. Total N accumulation and N use efficiency were the highest in T2, and total N accumulation was the lowest in T3. Principal component analysis showed that T2 considerably promoted growth and N absorption of Chinese kale, whereas T3 had a remarkable effect on the pH value. These findings suggest that an appropriate increase in NH4 <sup>+</sup> promotes the growth and nutrient uptake of Chinese kale by maintaining the pH value and NH4 +/NO3 − ratios of the nutrient solution, whereas excessive addition of NH4 <sup>+</sup> may induce rhizosphere acidification and ammonia toxicity, inhibiting plant growth.

**Keywords:** growth rate; root morphology; ammonium to nitrate ratio; nutrient solution composition; nitrogen efficiency

#### **1. Introduction**

Nitrogen is an essential nutrient in plant growth. It is the main component of many important organic compounds and participates in many physiological and biochemical processes in plants [1]. The forms of nitrogen that plants can absorb and utilize include ammonium (NH4 +), nitrate (NO3 −), nitrite (NO2 −), soluble protein, and free amino acids; higher plants mainly absorb NH4 <sup>+</sup> and NO3 − [2]. However, the processes of absorption, storage, transportation, and assimilation of the two types of N in plants are very different. The different forms of N affect the growth and development of plants and ultimately affect their yield and quality.

Most crops prefer to absorb NH4 <sup>+</sup> rather than NO3 − under the condition of hydroponics because a single NO3 − nutrient consumes a large amount of energy during the reduction process. The absorption of NO3 − increases the pH value of the solution, which leads to an insufficient supply of iron and other trace elements and decreases the content of chlorophyll, thus affecting the yield and quality [3]. The energy cost of NH4 <sup>+</sup> absorption and assimilation is lower than that of NO3 − [4]. However, single NH4 <sup>+</sup> nutrition causes many problems, such as ammonium toxicity, blocked leaf expansion, reduced organic acid synthesis, and decreased osmotic regulation [5].

**Citation:** Wang, Y.; Zhang, X.; Liu, H.; Sun, G.; Song, S.; Chen, R. High NH4 +/NO3 − Ratio Inhibits the Growth and Nitrogen Uptake of Chinese Kale at the Late Growth Stage by Ammonia Toxicity. *Horticulturae* **2022**, *8*, 8. https://doi.org/10.3390/ horticulturae8010008

Academic Editor: Pietro Santamaria

Received: 24 November 2021 Accepted: 20 December 2021 Published: 22 December 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

An increasing number of studies have shown that adding a mixture of NH4 <sup>+</sup> and NO3 − in appropriate proportions is more advantageous for crop growth and development and that NH4 <sup>+</sup> and NO3 − can interact when both N forms are provided together [4,6]. Compared with the addition of NH4 <sup>+</sup> or NO3 − alone, appropriate NH4 +/NO3 − ratio nutrition can significantly promote plant growth, increase plant biomass, soluble sugar, soluble protein, and vitamin C content, and reduce nitrate content in strawberry [7], mini-Chinese cabbage [8], flowering Chinese cabbage [9], and Chinese kale [10]. In addition, it is well-known that the absorption of NH4 <sup>+</sup> can induce net release of H<sup>+</sup> and acidify the rhizosphere, whereas the absorption of NO3 <sup>−</sup> can increase H+ uptake through the H<sup>+</sup> cotransport system in PM and alkalize the rhizosphere [11,12]. A plant's uptake of different forms of N and its transport and assimilation mechanisms depend on its NH4 +/NO3 − ratios. Our recent research further showed that an appropriate NH4 +/NO3 − ratio triggers plant growth and nutrient uptake of flowering Chinese cabbage by optimizing pH value in nutrient solution [13].

Chinese kale (*Brassica alboglabra* L. H. Bailey) is an important vegetable in south China. The flower stalk and leaves of Chinese kale are rich in anticarcinogenic compounds and antioxidants, including glucosinolates, carotenoids, vitamin C, and total phenolics [14,15]. Chinese kale best absorbs NO3 − under hydroponic conditions and easily accumulates nitrate. In our previous study, an appropriate increase of NH4 <sup>+</sup> in nutrient solution enhancement improved the yield and quality of Chinese kale and significantly reduced the nitrate content in its product organs [16]; however, the physiological mechanism of this regulation is still unclear.

Based on previous research, this study further investigates the effects of different NH4 +/NO3 − ratios on plant growth, seedling root morphology, nutrient solution composition, and plant nutrient absorption to reveal the physiological mechanism by which different NH4 +/NO3 − ratios regulate the growth and N uptake of Chinese kale.

#### **2. Materials and Methods**

#### *2.1. Plant Material*

The experiment was conducted in a greenhouse at the College of Horticulture, South China Agricultural University from March 2013 to January 2014. The average temperature from colonization to the end of the experiment was 24–30/20–24 ◦C (day/night). Chinese kale seeds 'lvbao' were provided by Guangzhou Academy of Agriculture Science. Chinese kale seeds were sown in a perlite medium. Seedlings with one developed true leaf and one core were watered with a 1/4 dose of Hoagland nutrient solution every 4 days. After 1 month, three consistent seedlings with three developed leaves and one core were selected and transplanted into hydroponic containers with 5.5 L of nutrient solution. There were 10 replications in each treatment arranged in a randomized complete block design.

#### *2.2. Treatments*

In this experiment, four different NH4 +/NO3 − ratios were set based on 1/2 dose of Hoagland sloe NO3 − nutrient solution formula (Table 1): CK, 0/100; T1, 10/90; T2, 25/75; and T3, 50/50. General formula for mineral elements: B, 0.5 mg L−1; Mn, 0.5 mg L−1; Zn, 0.05 mg L−1; Cu, 0.02 mg L−1; Mo, 0.01 mg L−1. Fe was supplied by EDTANaFe at a concentration of 50 mg L−1. Additionally, 0.2 g L−<sup>1</sup> ampicillin (excellent grade pure) was added to inhibit microbial activity. Chinese kale seedlings were transplanted and watered with a 1/4 dose of Hoagland nutrient solution (adjusted according to Table 1), adding 3/4 dose of mother liquor after 12 days, and adding pure water to the original volume of 5.5 L. Pure water was added to the original volume every 3 days. Electrical conductivity and pH were measured during the experiment. In nutrient solutions with different NH4 +/NO3 − ratios, NH4 <sup>+</sup> was supplied by NH4Cl. KCl or CaCl2 was added to maintain a constant concentration of K+ and Ca2+ among the treatments. All nutrient solutions were aerated for 15 min per hour using a controlled pump.


**Table 1.** Nutrient solution with different NH4 +/NO3 − ratios (Unit: mM).

The seeds of Chinese kale were sown in a medium containing 0.5% agar. After 5 days, seedlings with a radicle length of 1.5 cm were transplanted into hydroponic containers with 1.1 L of nutrient solution for different NH4 +/NO3 − ratio treatments.

#### *2.3. Parameter Measurements*

Chinese kale seedlings were harvested when they reached marketable maturity, plant height, and stem diameter, and their fresh and dry weight (determined after 1 h at 120 ◦C and 48 h at 75 ◦C in a drying oven) were measured (3 biological replicates per treatment, 12 plants per replicate). The fresh weight of the product organ (flower stalk above the 4th node) is called the economic yield. The fresh and dry weight were measured at 9, 15, 21, and 27 days after treatment to calculate the growth rate. The growth rate was measured by dividing the difference in fresh weight before and after sampling by the number of days.

The seeds of Chinese kale were accelerated to bud on a medium containing 0.5% agar. After 5 days, seedlings with a radicle length of 1.5 cm were selected and treated with different NH4 +/NO3 − ratios. After 2 weeks, the root morphological indices were analyzed (3 biological replicates per treatment, 12 plants per replicate). The root samples were stained with 0.16% neutral red solution, scanned using an applied digital scanner (LA2400), and quantitatively analyzed using the WinRHIZO Pro LA2400 software (Regent Instruments, Quebec City, QC, Canada) for total root length, root surface area, root volume, and average root diameter.

Total N content was determined according to the method suggested by Avery and Rhodes [17], and total N content was multiplied by the dry weight of the whole plant to calculate N accumulation. N loss (NL), N loss rate (NLR), mean residence time of N (MRT), N productivity (NP), and N use efficiency (NUE) were calculated as described by Eckstein and Karlsson [18] using the following formulae: NL = [(NSapplied − NSremain) − (Nharvest − Ntransplant) × n]/n; NLR = NL/(NSapplied − NSremain); MRT = (Nharvest − Ntransplant)/[(lnNharvest − lnNtransplant) × (NL/t)]; NP = [(Wharvest − Wtransplant)/(Tharvest − Ttransplant)] × [(lnNharvest − lnNtransplant)/(Nharvest − Ntransplant)]; NUE = NP ×MRT. In the above formulae, W represents the dry weight of the plant, T represents the sampling time, N represents the amount of N absorbed by seedlings, NS represents the amount of N in nutrient solution, n represents the number of seedlings in each hydroponic bucket, and t represents the number of days in the whole growth period. Total P and K were determined at 660 nm using a spectrophotometer and atomic absorption spectrophotometer [19].

#### *2.4. Data Analysis*

The data were analyzed by one-way analysis of variance (ANOVA) using SPSS 19.0. The differences between treatments were compared using the least significant difference (LSD) with a significance level of *p <* 0.05. The tables and figures were created using Excel 2013 and SigmaPlot 11.0, respectively. Multivariate principal component analysis (PCA) was performed using OriginPro 9.0.

#### **3. Results**

#### *3.1. Growth and Biomass*

The growth of Chinese kale was significantly affected by the different NH4 +/NO3 − ratios (Figure 1 and Figure S1). Compared with CK, plant height increased by 18.89% and 24.70% in T1 and T2, respectively (Figure 1A). Similarly, the stem diameter was the highest in T2, increasing by 13.89%, whereas there was no significant difference between CK and T3 (Figure 1B). The economic yield was the highest in T2 and lowest in T3 at the harvest stage of Chinese kale (Figure 1C). To further study the dynamic changes in Chinese kale growth under different NH4 +/NO3 − ratios, fresh and dry weights were measured at 9, 15, 21, and 27 days after treatment. T1, T2, and T3 significantly increased the biomass of Chinese kale during the growth stage (0–21 d) compared to the control. However, in contrast to T1 and T2, the fresh weight and dry weight of T3 at harvest time decreased by 17.57% and 12.60%, respectively, compared with CK (Figure 1D,E). This result is consistent with economic yield. At the early stage of Chinese kale growth (0–15 d), the root/shoot ratio was the highest in CK, followed by T1, and the difference between T2 and T3 was not significant, indicating that the treatment of increasing NH4 <sup>+</sup> in the nutrient solution would reduce the dry matter distribution of the roots. At the harvest stage of Chinese kale (27d), the root/shoot ratio of T3 was 1.13, 1.24, and 1.12 times that of CK, T1, and T2, respectively, indicating that the effect of high NH4 +/NO3 − ratio treatment on the shoot was greater than that on the root at the late growth stage of Chinese kale (Figure 1F).

**Figure 1.** Effect of different NH4 +/NO3 − ratios on the growth and biomass of Chinese kale. (**A**) plant height at the harvest stage. (**B**) stem diameter at the harvest stage. (**C**) economical plant yield at the harvest stage. (**D**) dynamic changes in fresh weight. (**E**) dynamic changes in dry weight. (**F**) dynamic changes in the ratio of root to shoot fresh weight. CK = 0/100, T1 = 10/90, T2 = 25/75, T3 = 50/50. DAT, days after treatment. The data represent mean ± SE (n = 3). Different letters in Figure 1C–F indicate significant differences at *p* < 0.05.

Furthermore, we analyzed the effects of different NH4 +/NO3 − ratios on the growth rate of Chinese kale seedlings and found that the growth of Chinese kale was slow in the early growth stage and fast in the late growth stage (Table 2). During the period of 0–9 d after treatment, seedling growth rates were highest in T2 and T3, with no significant difference between them, followed by T1, and were the lowest in the control. During the period of 9–15 d after treatment, the change trend of the four treatments was consistent with that of 0–9 days. During the period of 15–21 d after treating, the growth rates of seedlings reached the maximum in T2 and T3, which were 519.6 mg d−<sup>1</sup> plant−<sup>1</sup> and 388.4 mg d−<sup>1</sup> plant−1, respectively. During the period of 21–27 d after treatment, the seedling growth rates reached the maximum in CK and T1, which were 656.1 mg d−<sup>1</sup> plant−<sup>1</sup> and mg d−<sup>1</sup> plant−1, respectively. In conclusion, at 0–21 d, the growth rate of seedlings was the highest in T2, followed by T3. However, the difference between them was not significant: at 0–27 d, the growth rate of seedlings was the lowest in T3, which was only 87.45%, 79.47%, and 73.08% that of the control, T1, and T2, respectively, indicating that the growth was significantly inhibited.

**Table 2.** Effects of different NH4 +/NO3 <sup>−</sup> ratios on the growth rate of Chinese kale (Unit: mg d−<sup>1</sup> plant<sup>−</sup>1).


Data are presented as mean ± SE (n = 3). Different letters indicate significant differences at *p* < 0.05.

To study the effects of different NH4 +/NO3 − ratios on the root growth of Chinese kale seedlings, we measured the main root length, root total length, root surface area, root volume, and average root diameter (Table 3, Figure S2). The main root length of Chinese kale seedlings in T1 and T2 was slightly higher than that in CK, and that of T3 was slightly lower than that of CK. Compared with CK, the total root lengths of Chinese kale seedlings in T1 and T2 were 1.38 and 1.91 times than those of CK, respectively. Root volumes of Chinese kale seedlings in T1 and T2 were 1.74 and 2.87 times that of CK, respectively. The root surface area and average root diameter of Chinese kale seedlings were maximum in T2. We concluded that the optimal root growth and root indices of Chinese kale seedlings occurred under T2 (NH4 +/NO3 − = 25/75).. However, a high NH4 +/NO3 − ratio (50/50) may inhibit the main root elongation of Chinese kale seedlings to a certain extent. In addition, root activity showed no significant difference between the control, T1, and T2; however, it decreased by 20.85% in T3 compared with the control. (Figure S3).

**Table 3.** Effects of different NH4 +/NO3 − ratios on root morphological indices of Chinese kale seedlings.


Data are presented as mean ± SE (*n* = 3). Different letters indicate significant differences at *p* < 0.05.

*3.2. Dynamic Changes in Nutrient Solution Composition*

To elucidate the mechanism by which different NH4 +/NO3 − ratios affect the growth of Chinese kale, we conducted dynamic monitoring of the physicochemical properties of the nutrient solution and N content (Figure 2). The initial electrical conductivity (EC) value of the nutrient solution increased as the NH4 <sup>+</sup> proportion increased. The EC values were 629, 673, 732, and 879 μs cm−<sup>1</sup> in the control, T1, T2, and T3, respectively (Figure 2A). With the growth of Chinese kale, both the EC value and ion concentration of the nutrient solution gradually decreased. During the entire growth period of the plant, the decrease in EC value in T2 was the largest, and the decrease in T1 and T3 was also greater than that of the control, but the decrease in EC value in T3 was the lowest during the harvest period. This indicates that an appropriate NH4 +/NO3 − ratio could promote the ion absorption of Chinese kale plants, and the NH4 +/NO3 − ratio (50/50) could promote ion absorption of plants at the early and middle stages but significantly inhibit ion absorption of plants at the late growth stage.

**Figure 2.** Changes in electrical conductivity value (**A**), pH value (**B**), NO3 − content (**C**), NH4 <sup>+</sup> content (**D**), total N content (**E**), and NH4 +/NO3 − ratios (**F**) in nutrient solution under different NH4 +/NO3 − ratios. CK = 0/100, T1 = 10/90, T2 = 25/75, T3 = 50/50. DAT, days after treatment. The data represent mean ± SE (*n* = 3).

The initial pH value of the nutrient solution decreased as the NH4 <sup>+</sup> proportion increased; however, the difference was not significant, both were at approximately 6.75 (Figure 2B). During the entire growth period, the pH value in the CK was mostly weakly alkaline. Before and after the replenishment of the nutrient solution, the average pH of

the nutrient solution showed a gradual increase and reached 8.15 at harvest time. The nutrient solution in T1 was weakly acidic at the early stages, and the pH value was stable at approximately 6.5. At 21 d, the pH value was greater than 7.0, the nutrient solution became weakly alkaline, and the pH of the nutrient solution further increased to 8.20 at harvest time. The nutrient solution in T2 was always weakly acidic, and the pH value decreased gradually at the early stage and reached the lowest value at 5.24 at 18 d. The pH value gradually increased and reached 6.73 at harvest time. The nutrient solution in T3 was acidic during the entire growth period, the pH of the nutrient solution decreased gradually, and the pH of the nutrient solution was 3.63 at the harvest stage. In combination with plant biomass, Chinese kale was found to be insensitive to environmental pH and could grow normally in a nutrient solution pH range of 4.73–8.15. An appropriate acidic environment was more conducive to the growth of Chinese kale; however, an over-acidic environment (pH < 4) could significantly inhibit the growth of Chinese kale and reduce the growth rate of plants.

During the plant growth period, the NO3 − content in the four treatments decreased gradually as NH4 <sup>+</sup> proportion increased (Figure 2C). After 12 d of treatment, the NO3 − content in the control, T1, T2, and T3 decreased by 45.74, 43.99, 37.38, and 25.12 mg L−1, respectively, compared with that before treatment. After the replenishment of nutrient solution, the NO3 − content in the control, T1, T2, and T3 was 164.26, 145.01, 120.12 and 79.88 mg L−1, respectively. At harvest, the NO3 − content in the control, T1, T2, and T3 decreased by 154.00, 145.00, 120.05, and 79.77 mg L−1, respectively, compared with that after the replenishment of nutrient solution. The proportions of NO3 − absorbed by the four treatments (CK, T1, T2, and T3) to the total NO3 − were 95.11%, 99.99%, 99.96%, and 99.90%, respectively, indicating that the three treatments (T1, T2, and T3) all promoted NO3 − uptake by Chinese kale plants, and there was no significant difference between the different treatments. Similarly, with an increase in NH4 <sup>+</sup> proportion, the NH4 <sup>+</sup> content in the three treatments also decreased gradually (Figure 2D). After 12 d of treatment, the NH4 + content in T1, T2, and T3 decreased by 4.94, 12.26, and 14.74 mg L<sup>−</sup>1, respectively, compared with that before treatment. After nutrient solutions were added, the NH4 <sup>+</sup> content in T1, T2, and T3 were 16.06, 40.24, and 90.26 mg L−1, respectively. After 3 d, they decreased by 9.53, 9.52, and 37.55 mg L−1, respectively. Thereafter, they slowly decreased to 0.03, 0.01, and 34.77 mg L<sup>−</sup>1, respectively, at harvest. The proportions of NH4 <sup>+</sup> absorbed by the three treatments to the total NH4 <sup>+</sup> were 99.86%, 99.98%, and 66.89%, respectively, indicating that low and medium NH4 +/NO3 − ratios could significantly promote the absorption of NH4 + by Chinese kale compared with the high NH4 +/NO3 − ratio.

With the growth of the plants, the total N content in the four treatments decreased gradually with an increase in NH4 <sup>+</sup> proportion (Figure 2E). At harvest, the total N content in the control, T1, T2, and T3 was decreased by 154.00, 161.03, 160.28, and 135.26 mg L−1, respectively, compared with that after the replenishment of nutrient solution. The greatest reduction in total N content was observed in T1 and T2, and the final content at harvest was 0.04 and 0.08 mg L−, respectively. This indicates that a low NH4 +/NO3 − ratio (10/90) significantly promoted the absorption of total N by Chinese kale, whereas a high NH4 +/NO3 − ratio inhibited the absorption of total N. In addition, the three treatments (T1, T2, and T3) promoted the absorption of *p* by Chinese kale, whereas the high NH4 +/NO3 − ratio significantly inhibited the absorption of K by Chinese kale at the late growth stage (Figure S4).

We further analyzed the NH4 +/NO3 − ratio in the four treatments (Figure 2F). Before and after the replenishment of the nutrient solution, the NH4 +/NO3 − ratio in T1 showed a decreasing and then an increasing trend. It decreased to a minimum of 0.001 at 21 d and increased to a maximum of 0.445 at the harvest stage. The NH4 +/NO3 − ratio in T2 remained between 0.243 and 0.435, which decreased significantly to 0.003 at 24 d of treatment and then increased to 0.214 at the harvest stage. The NH4 +/NO3 − ratio in T3 gradually increased during plant growth, and it increased rapidly after the replenishment of nutrient solution and reached a maximum of 95.144 at the harvest stage.

#### *3.3. N Content and N Use Efficiency*

The total N content of Chinese kale first increased and then decreased during the growth period (Figure 3A). The N content of Chinese kale increased rapidly from 0 d to 9 d of growth, and T2 and T3 were significantly higher than those of the control and T1. The N content of Chinese kale increased slowly from 9 d to 15 d of growth, and T3 was higher than that of the other three treatments. Except for the control, the N content of Chinese kale plants decreased significantly from 15 to 21 d of growth. The N content in T3 increased by 5.40%, 5.40%, and 6.93% compared with CK, T1, and T2, respectively, at 27 d. In addition to total N content, seedling dry weight also had a significant effect on total N accumulation. Total N accumulation in Chinese kale increased continuously during the growth period (Figure 3B). In the middle and late periods of plant growth, the total N accumulation was the highest in T2, and the total N accumulation of control plants was the lowest in the period from 0 d to 25 d of growth, whereas the total N accumulation of T3 plants was the lowest at 27 d, which was reduced by 7.88%, 16.13%, and 21.62% compared with CK, T1, and T2, respectively.

**Figure 3.** The content (**A**) and accumulation (**B**) of total N affected by different NH4+/NO3 − ratios during the growth period of Chinese kale. CK = 0/100, T1 = 10/90, T2 = 25/75, T3 = 50/50. DW, dry weight; DAT, days after treatment. The data represent mean ± SE (*n* = 3).

To further study the N utilization of Chinese kale plants in response to different NH4 +/NO3 − ratios, we analyzed the N loss, N productivity, N residence time, and N use efficiency (Table 4). We found that there was a certain amount of N loss in Chinese kale production under hydroponic conditions. Compared with the control, N loss in T1, T2, and T3 was reduced by 7.37%, 27.40%, and 23.71%, respectively, and the N loss rate was reduced by 11.83%, 30.90%, and 12.97%, respectively, with significant differences between treatments. N retention time was prolonged in all three treatments; however, it was most significant in T2. N productivity was the highest in T2, followed by T1, and lowest in T3; however, there was no significant difference between the different treatments. Owing to the difference in N loss rate, N productivity, and N retention time, N use efficiency was significantly different between the four treatments. N use efficiency was the highest in T2, followed by T1; however, there was no significant difference between T1 and T3. The results showed that an appropriate NH4 +/NO3 − ratio (25/75) significantly reduced N loss, prolonged N retention time, and improved N productivity and N use efficiency compared with the control.


**Table 4.** Rate of N loss, N residence time, N productivity, and N use efficiency in nutrient solutions with different NH4 +/NO3 − ratios during the growth period of Chinese kale.

Data are presented as mean ± SE (*n* = 3). Different letters indicate significant differences at *p* < 0.05. Significant differences among the treatments were determined using SPSS 17.0.

#### *3.4. Principal Component Analysis*

Principal component analysis (PCA) was used to visualize the effects of different NH4 +/NO3 − ratios on the growth and N uptake of Chinese kale (Figure 4). The computed model captured 84.5% of the total observed variance with the first two principal components (PCs). Four treatments (CK, T1, T2, and T3) were distributed in distinct quadrants in the PCA scatter plot. All indexes of plant growth and N uptake were the best in the T2 treatment, and biomass and root morphology were strongly correlated with N use efficiency. In addition, the NH4 +/NO3 − ratio in the nutrient solution and root/shoot ratio were the highest in T3 and were significantly negatively correlated with the pH value.

**Figure 4.** Principal component analysis showing differences and correlations in the investigated parameters of Chinese kale under different NH4 +/NO3 − ratio.

#### **4. Discussion**

The main forms of inorganic N, NH4 <sup>+</sup> and NO3 − can be absorbed and utilized by plants [20,21]. To date, there have been many reports on the effects of different N forms and ratios on crop biomass [22]. Our previous study showed that the biomass of Chinese kale was largest when the nutrient solution increased NH4 <sup>+</sup> by 25% to 30%; when the nutrient solution increased NH4 <sup>+</sup> by 45% to 50%, the biomass of kale was significantly lower than that of the control treatment with only NO3 − [9]. In this study, the high NH4 +/NO3 − ratio (50/50) significantly increased the biomass of Chinese kale and promoted plant growth at the early stage; however, it showed a strong inhibitory effect at the late growth stage (21–27 d). During this period, the plant growth rate was 304.9 mg d−<sup>1</sup> plant<sup>−</sup>1, which was only 46.5% of plant growth in the control with only NO3 − (Figure 1, Table 2). This showed that the effects of different NH4 +/NO3 − ratios on plant growth could also be closely related to the duration of treatment and growth stage of plants, and that a high concentration of NH4 <sup>+</sup> applied for a short time would not cause ammonium toxicity [23,24]. In this study, 2.25, 5.60, and 11.23 mmol L-1 Cl− was added to the nutrient solution of T1, T2, and T3, respectively, being less than the salt-osmotic stress threshold level of approximately 40 mmol L−<sup>1</sup> NaCl for most plants [25]. Therefore, the amount of Cl<sup>−</sup> in the nutrient solution used in this study was insufficient for salt stress.

Plants mainly absorb nutrients through their roots. Of all mineral nutrients, nitrogen has the greatest influence on root morphology, growth, and distribution in the medium [26]. The absorption of NO3 − by plants results in rhizosphere alkalization, while the absorption of NH4 <sup>+</sup> results in rhizosphere acidification [27]. In the present study, the nutrient solution was weakly acidic under a medium NH4 +/NO3 − ratio (25/75), and the root morphology index was the highest (Table 3). Root morphology and biomass were significantly positively correlated with pH under a medium NH4 +/NO3 − ratio (Figure 4). Appropriate ratios of NH4 +/NO3 − promote root growth and increase root dry weight [28,29]. Pepper treated with a 25:75 ratio increased root length, surface area, and root volume and tips [30]. Therefore, an appropriate NH4 +/NO3 − ratio (25/75) may promote root growth by maintaining the pH value, thereby increasing biomass. The pH of the rhizosphere also affects the absorption of NH4 <sup>+</sup> and NO3 − by plants [31]. With the growth and development of plants, the pH value of the nutrient solution gradually decreased under a medium NH4 +/NO3 − ratio (25/75). The NO3 − was quickly and completely absorbed, and only NH4 <sup>+</sup> remained in the nutrient solution at the late growth stage of Chinese kale (Figure 2). When the proportion of NH4 <sup>+</sup> in the nutrient solution was more than 75%, cabbage growth was reduced by 87% due to the accumulation of a large amount of free ammonia in the leaves [32]. The pH value and NH4 <sup>+</sup> proportion showed a significant negative correlation under a high NH4 +/NO3 − ratio (50/50) (Figure 4). However, the pH value was acidic, which was not conducive to the root development of Chinese kale seedlings. In addition, the accumulation of large amounts of NH4 <sup>+</sup> in Chinese kale leaves resulted in ammonium toxicity, which inhibits plant growth.

The change in pH value in the rhizosphere results in different absorption of NH4 <sup>+</sup> and NO3 −, which affects the content and accumulation of N in plants [9]. In this study, the total N content of the three treatments (T1, T2, and T3) was significantly increased at 0–15 d after treatment compared to that of the control treatment with NO3 − alone (Figure 3A). This is consistent with previous studies on Chinese flowering cabbage [13]. Total N accumulation increased continuously throughout the growth period of the Chinese kale. At the late plant growth stage, the total N accumulation of the high NH4 +/NO3 − ratio (50/50) was the lowest (Figure 3B). This is similar to the results of previous studies [33]. There was a negative correlation between N accumulation and biomass under high NH4 +/NO3 − ratios (Figure 4). NH4 <sup>+</sup> at higher concentrations caused toxicity in bamboo, as it inhibits root growth and N accumulation [34]. Therefore, a high NH4 +/NO3 − ratio reduced the uptake and accumulation of N in plants at the late growth stage of Chinese kale, thus inhibiting plant growth. Different NH4 +/NO3 − ratios also significantly affect the absorption of other nutrients [35]. In this study, a high concentration of NH4 <sup>+</sup> promoted the absorption of P but inhibited the absorption of K by Chinese kale at the late stage (Figure S4). There is a strong correlation between pH change in a solution and net cation or anion uptake in geranium and petunia [36]. The elevation of the NH4 <sup>+</sup> ratio in the fertilizer solution decreased the soil solution pH at 35 days after sowing, resulting in an increase in tissue P and a decrease in K content in French marigold "Orange Boy" [37]. This may be caused by the interaction between ions and the pH change in the rhizosphere.

This study adopts the definition of nutrient use efficiency by Berendse and Aerts [38], and divides nitrogen use efficiency into N productivity and average retention time of N. N productivity reflects a plant's rapid growth strategy. The average retention time

of nitrogen reflects a plant's nutrient retention strategy [39]. In this study, N loss in the nutrient solution increased by NH4 <sup>+</sup> treatment was significantly lower than that of the control with NO3 − alone (Table 4). In our recent research, the contents of NH4 +, NO3 −, and total N in the nutrient solution without plant culture were almost constant during the growth period [13]. Therefore, in a closed hydroponic system, the loss of N may be related to the N metabolism of plants or caused by changes in the nutrient solution composition during plant cultivation. Combined with the experimental conditions, this part of the N loss in this experiment should be attributed to gaseous loss. The volatile forms of gaseous nitrides include NH3, N2O, and NO. Previous studies have found that, compared with NH4 +, when NO3 − is used as the nitrogen source, the volatilization of N2O in wheat and of NO in rice is enhanced [40–42]. However, the main loss forms of nitrides during kale production are still unclear, and further experiments are needed. In this study, the medium NH4 +/NO3 − ratio (25/75) had the highest N use efficiency, unlike *Brassica napus*, which had the highest N use efficiency with a high NH4 +/NO3 − ratio (75/25) [43]. We observed that the NH4 +/NO3 − ratio in T2 was maintained at approximately 0.15 (Figure 2D), which may have contributed to balanced N absorption. The NH4 +/NO3 − ratio in T3 increased rapidly at the late stage of plant growth (Figure 2D), which may have caused insufficient assimilation of NH4 <sup>+</sup> after its absorption or protein degradation owing to NH4 <sup>+</sup> toxicity, thereby reducing N productivity. Thus, applying appropriate ratios of NH4 +/NO3 − is an important way to improve plant N use efficiency [44].

#### **5. Conclusions**

An appropriate NH4 +/NO3 − ratio (25/75) increased biomass and promoted the growth of Chinese kale, whereas high NH4 +/NO3 − (50/50) promoted the growth of Chinese kale seedlings at the early growth stage but inhibited growth at the late growth stage. With the prolongation of treatment time, the pH value of T3 nutrient solution decreased continuously, whereas the NH4 +/NO3 − ratio was the highest in T3 at the harvest stage. In addition, T2 had the highest total N accumulation and N use efficiency, whereas total N accumulation was lowest in T3. Thus, an appropriate NH4 +/NO3 − ratio (25/75) promotes the growth and N uptake of Chinese kale by maintaining the pH value of the nutrient solution, whereas excessive addition of NH4 <sup>+</sup> may induce rhizosphere acidification and ammonia toxicity, thereby inhibiting plant growth. This study provides a theoretical basis for the effects of different NH4 +/NO3 − ratios on plant growth and N absorption and utilization.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/horticulturae8010008/s1, Figure S1: Plant morphology of Chinese kale treated with different NH4 +/NO3 − ratios; Figure S2: The root morphology of Chinese kale seedlings treated with different NH4 +/NO3 − ratios; Figure S3: The root activity of Chinese kale under different NH4 +/NO3 − ratios; Figure S4: Effect of different NH4 +/NO3 − ratios on the content and accumulation of total K (A,B) and P (C,D) in the growth period of Chinese kale.

**Author Contributions:** R.C. and S.S. conceived and designed the study X.Z. carried out the experiments. Y.W. analyzed the data and wrote the manuscript. H.L. and G.S. reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Key-Area Research and Development Program of Guangdong Province (2020B0202010006), the Guangdong Provincial Special Fund for Modern Agriculture Industry Technology Innovation Teams (2021KJ131), and the China Agriculture Research System of MOF and MARA.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

