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Article

Effect of Litter Removal and Addition on Root Exudation and Associated Microbial N Transformation in a Pinus massoniana Plantation

by
Chengfu Zhang
1,
Qingxia Zhao
2,*,
Yinmei Cai
3,
Tao Zhang
2,
Limin Zhang
1 and
Tengbing He
2,3
1
Guizhou Institute of Mountain Resources, Guizhou Academy of Sciences, Guiyang 550001, China
2
Institute of New Rural Development, Guizhou University, Guiyang 550025, China
3
College of Agriculture, Guizhou University, Guiyang 550025, China
*
Author to whom correspondence should be addressed.
Forests 2023, 14(7), 1305; https://doi.org/10.3390/f14071305
Submission received: 18 May 2023 / Revised: 18 June 2023 / Accepted: 21 June 2023 / Published: 25 June 2023
(This article belongs to the Section Forest Ecology and Management)
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Abstract

:
In forest ecosystems, variations in aboveground litter input caused by global changes, substantially alter soil N cycling. In field-grown plants, few studies have directly measured root exudation rates and quantified their effects on N transformations under litter manipulation. We quantified soil N transformation rate responses to litter manipulation in a Pinus massoniana plantation, and unravelled the effect of root exudation on soil N transformations. We measured in situ P. massoniana root exudation rates as well as soil microbial biomass, soil C and N concentrations, the activities of four soil enzymes involved in soil N transformations, and net N mineralization and net nitrification rates after experimental litter removal and litter addition treatments. Litter removal and litter addition treatments had little impact on soil C and N concentrations, microbial biomass, soil enzyme (urease, hydroxylamine reductase, nitrate reductase, and nitrite reductase) activity, and net N mineralization rates. However, both litter removal and addition increased net N nitrification rates. Additionally, litter removal significantly decreased root C exudation rates (in April 2021 and annually), whereas litter addition had no significant effects on root C exudation rates across all seasons. Furthermore, root C exudation rates were positively associated with urease and nitrate reductase activities, but negatively associated with hydroxylamine reductase and nitrite reductase activities, as well as net N nitrification rate. Overall, we demonstrated that root exudates may be an important physiological adjustment by which trees respond to changes in litter input caused by global environmental changes, regulating underground N biochemical processes. Furthermore, we provide new evidence from root exudates for understanding the potential influence of litter inputs on soil N cycling. A strong correlation exists between root exudates and N transformation, shedding new light on the dynamics of rhizosphere nutrient cycling crucial for maintaining forest ecosystem stability and productivity under changing environmental conditions.

1. Introduction

In forests, nitrogen (N) is a primary limiting element for plant growth and productivity. Of the forest ecosystem N pool, soil N accounts for more than 90%, but most of it is organic N; inorganic N accounts for only 1%~5% [1]. Thus, soil N transformations play a crucial role in N cycling in forest ecosystems. Numerous investigations have examined global environmental changes such as elevated temperatures, altered precipitation patterns, and severe climatic phenomena, all of which may affect litterfall by altering tree net primary productivity [2,3]. Moreover, changes in aboveground litter production will affect underground N-cycle processes [4]. Therefore, assessing the effects of litterfall changes on soil N transformations and its driving factors would aid in gaining a more comprehensive knowledge of the forest ecosystem N cycling mechanism.
Changed litter inputs could affect N input, loss, and retention in soils [5]. Litter is the main source of soil N, but its changes have inconsistent influences on the soil N transformation rate [6]. Generally, litter removal reduces total and available soil N concentrations [3,7] and N mineralization rates due to the direct reduction in N input to the soil [8]. Conversely, litter addition has positive, negative, or neutral effects on soil N concentration and transformation rates. There was an increase in soil NO3−-N [9] and N-acetyl-β-D-glucosaminidase activities [10] after litter addition in lowland tropical forest. Increasing organic matter input can result in an increment in N fixation inputs [11], while also promoting N retention due to greater biological N demand [12]. However, some studies have demonstrated that the amounts of inorganic and total soil N are not affected, or even decrease with litter addition [13,14]. Deng et al. [15] also reported a decline in the net N mineralization rate after litter addition. Therefore, more fieldwork is required to understand the impact of litter manipulation on soil N biochemical processes.
Soil N transformation response to litter manipulation is readily influenced by abiotic and biotic factors. Abiotic factors such as soil nutrient status, soil water content, soil temperature, bulk density, soil permeability, and oxygen content could all affect N transformation rates [4,16]. Whereas biotic factors including soil microorganisms and plants, play more critical roles than abiotic factors in forest soil N transformations. For example, changes in the litter amount greatly influenced soil N transformation processes through changes in soil microbial communities [17] and soil enzyme activities [10]. Plant root-induced acceleration of N mineralization has been reported in temperate and boreal forest soils [18,19,20], and attributed to labile carbon (C) inputs via root exudates into the rhizosphere through priming effects [21]. Thus, knowledge of how litter experimental manipulation affects N transformations via root exudation could provide new insights into understanding biogeochemical process feedback to litter manipulation.
Root exudates are soluble organic substances released from roots into the surrounding soil, mostly as soluble sugars, amino acids, or organic acids [22]. Root exudates can be used by microorganisms to increase microbial activity and community growth, thus enhancing soil organic matter (SOM) decomposition and N transformation [22,23,24,25]. In temperate forest soils, root-accelerated mineralization and priming explain up to 33% of total C and N mineralized [26,27]. Many studies have reported an increase in underground C allocation in trees exposed to elevated CO2, elevated temperature, and low nutrient content [19,20,28,29]. Root C allocation can increase soil urease and hydroxylamine reductase activities and accelerate soil N transformations [30]. Moreover, there is now sufficient evidence that increased crop root exudates can inhibit nitrification [31,32]. Root exudates affect soil nitrification by inhibiting ammonia monooxygenase and hydroxylamine oxidoreductase, which is beneficial for reducing agroecosystem nitrate leaching and N2O emissions [31,32,33,34]. Although the root exudation role on N transformation is increasingly recognized in agroecosystems, related studies in forests are still lacking. Much less is known about how root-derived C inputs affect microbial and soil enzyme activities and biogeochemical cycles under litter manipulation.
There is abundant proof for the N limitation of various ecosystem processes in forest plantations [35]. Here, we conducted an experimental manipulation of aboveground litter in a P. massoniana plantation (litter removal and litter addition) to examine the impacts of variable litter inputs on soil N transformation rates. Moreover, we measured root exudation rates in situ, as well as soil microbial biomass, and enzyme activities involved in soil N transformations. We hypothesize that (1) litter removal reduces soil N concentrations and N mineralization and nitrification rates, while litter addition increases them because of the higher N inputs from litter; and (2) litter removal increases root exudation rates, but litter addition lowers them.

2. Materials and Methods

2.1. Site Description

This study was carried out in a P. massoniana experimental plantation located in southern Guiyang, Guizhou Province, China (106°398″ E, 26°2815″ N, altitude 1145 m). The study area annually receives 1178.3 mm precipitation, has an average temperature of 14.9 °C, and sunshine duration of 1354 h. The study area is characterized by a subtropical monsoon climate, and the main landforms are mountainous and hilly. At the beginning of the study, the P. massoniana plantation was approximately 35 years old and had a tree density of 853 stem hm−2 with a canopy density of 0.8. Tree average diameter and height were 32.2 cm and 28.32 m, respectively. The United States Department of Agriculture (USDA) categorizes the soil at this site as an Oxisol developed from red clay during the Quaternary period. A detailed description of soil properties and dominant understory species can be found in our previous study [36].

2.2. Experimental Design

The litter manipulation experiment comprised nine 10 × 10 m plots in a randomized block design and was initiated in October 2018. Three similar blocks were created, and within each block, litter treatments were randomly assigned to three plots. These treatments consisted of litter removal (LR), no intervention (CT), and litter addition (LA). Litter in the 3 LR plots was removed monthly and added to the 3 LA plots, nominally doubling litter inputs, while leaving three plots as undisturbed controls.

2.3. Root Exudation Measurements

An in situ collection device [37] was utilized to collect root exudates in July, October, and December 2020, and April 2021. In each plot, two target trees with similar growth conditions were selected for root exudate collection. From each target tree, three terminal fine roots (with laterals and an average diameter of 2 mm) were carefully extracted from the topsoil layer (0–10 cm). The excavated roots were triple washed with deionized water followed by a double rinse with a nutrient solution containing 0.2 mM K2SO4, 0.3 mM CaCl2·2H2O, 0.1 mM KH2PO4, and 0.2 mM MgSO4·7H2O to remove any extraneous materials. Next, the cleaned roots were placed in a 30 mL sterile syringe prefilled with 1 mm diameter glass beads to maintain their structure, and loaded into the syringe barrel with glass wool at the bottom to prevent bead clogging. Before vacuum pump-assisted root exudate extraction, 15 mL of nutrient solution was injected into each syringe to fulfill the fine roots’ metabolic needs for growth and activity. The syringe was then treated with aluminum foil and reintroduced into the original plot soil environment, allowing a 24 h equilibration period. The nutrient solutions in the syringe were collected thrice via the same method over three days cultivation, during which an additional 15 mL of nutrient solution was injected into the system each day. Additionally, each plot included six blank syringes without roots. The daily collected nutrient solution was combined and transferred to brown bottles for further analysis. After collection, exudates were passed through a filter (0.22 μm) and stored in a refrigerator (−20 °C). Root C exudates were analyzed using a TOC-TN analyzer (Vario, Hana, Germany). Roots in each syringe were harvested, oven-dried at 65 °C, and weighed. The rates at which C was exuded from Root C were determined by computing the mass of C (μg) that was washed out from each root system during a 24 h incubation period (with the average C concentration in control cuvettes subtracted). To obtain the exudation rates (μg C g−1 root biomass h−1), the total C flushed out from the root system was divided by the entire fine root biomass.

2.4. Soil Sampling

Soils were sampled in July, October, and December 2020 and April 2021, immediately after the exudation collections. Three soil samples were collected along the same three directions from each tree where root exudates were collected, and combined into one sample. Fine roots plus attached soil were meticulously separated from non-adhering soil with precise tweezers to obtain the rhizosphere soil [18]. Soil samples were taken to the laboratory and split into two parts. One part was passed through a 2 mm diameter sieve and then stored in a refrigerator at 4 °C for NH4+-N, NO3−-N, urease, hydroxylamine reductase, nitrate reductase, nitrite reductase, microbial biomass C (MBC), and microbial biomass N (MBN) analyses, while the other part was air-dried for several days and then filtered through a 1 mm sieve for soil pH measurement. The sample was additionally filtered using a 0.149 mm sieve to determine soil organic C (SOC) and total N (TN) concentrations.

2.5. N Mineralization and Nitrification Experiments

We conducted measurements of net N mineralization and nitrification rates in October 2020, although these rates showed a strong seasonal pattern. Numerous litter manipulation experiments have demonstrated that litterfall showed a pronounced seasonal tendency. Increased litterfall was associated with the release of N and P from the litter and had a significant positive correlation with the amount of N and P that was introduced into the litter [38,39,40]. In our previous study, litterfall varied strongly among litter treatments in October, indicating that N release from the litter varied greatly [36]. Soil net N mineralization and nitrification rates were determined using the PVC tube closed-top in situ incubation method. Specifically, 3 PVC tubes (5 cm diameter, 15 cm tall) were hammered vertically into the soil near the roots in each plot. The PVC pipe tubes were buried at a depth of 10 cm, with their top 5 cm above the ground, and sealed with a polyethylene bag (to prevent rainwater from entering and to avoid nitrate leaching). Concurrently, an initial soil sample was extracted with a soil corer (5 cm diameter, 10 cm depth) from 5 to 10 cm away from the PVC tubes. After 30 days culturing, 0–10 cm soil samples in each PVC tube were collected as final samples and immediately analyzed for soil NH4+-N and NO3−-N concentrations using an Auto Discrete Analyzer (CleverChem380, Hamburg, Germany). Simultaneously, soil moisture content was determined by the drying method. Net N mineralization rates were calculated by the difference between initial and final inorganic N concentrations (NH4+-N and NO3−-N), and nitrification rates were calculated by the difference between the initial and final NO3−-N concentrations.

2.6. Laboratory Analyses

Soil pH was determined with a pH electrode (Orion) while suspended in water at a 1:2.5 (m/v) ratio. Additionally, the Walkley–Black method, which relies on dichromate digestion, was used to determine soil organic C content. After sulfuric digestion, soil TN concentration was analyzed using an Auto Discrete Analyzer (CleverChem380, Hamburg, Germany). Soil sample NH4+-N and NO3−-N concentrations were measured with an Auto Discrete Analyzer (CleverChem380, Hamburg, Germany). Specifically, 20 g fresh soil samples were extracted and shaken horizontally at 200 rpm for an hour at 25 °C, using 100 milliliters of 1 M KCl solution. NH4+-N and NO3-N in the extracts were analyzed with an Auto Discrete Analyzer (CleverChem380, Hamburg, Germany). Mineral N was calculated as the sum of NH4+-N and NO3−-N. Soil organic N (SON) was calculated as TN minus mineral N. MBC and MBN concentrations were determined by the chloroform fumigation extraction method [41]. Soil was extracted with 0.5 mol·L−1 K2SO4 and agitated at 300 rpm for 30 min on a flat shaker. Subsequently, extracted dissolved organic C and N were strained using Whatman No. 1 filter paper and directly assessed using a TOC-TN analyzer (Vario, Hana, Germany). MBC and MBN conversion factors (Kec and Ken) were 0.45 and 0.54, respectively [42].
We measured the activities of four enzymes involved in N transformations. Urease is an enzyme that hydrolyzes substrates of the urea type. Hydroxylamine reductase, nitrate reductase, and nitrite reductase are key enzymes in soil nitrification and denitrification. Hydroxylamine reductase catalyzes the NH2OH to NH4OH reduction, while nitrate reductase catalyzes NO3−-N to NO2−-N reduction in soil. Soil nitrite reductase catalyzes the reduction of NO2−-N to NO or NH3. Soil urease activity was assessed by employing the sodium phenate-sodium hypochlorite colorimetric technique. Briefly, 5 g of fresh soil sample was placed in a 50 mL Erlenmeyer flask, and 1 mL of toluene was added. After 15 min, 10 mL of a 10% urea solution and 20 mL of potassium citrate-citric acid buffer (pH = 6.7) were added to the sample. Then, flasks were stoppered, shaken, and incubated at 37 °C for 24 h. The amount of ammonia released by urea hydrolysis was determined from the filtrate using the sodium phenate-sodium hypochlorite colorimetric method. Urease activity was expressed as mg of NH4+-N released per kg dry soil per 24 h. Briefly, 1 g of fresh soil sample was placed in a 10 mL test tube, and 2 mL of 0.5% hydroxylamine hydrochloride and 1 mL of 1% glucose were added. The test tubes were stoppered and incubated at 30 °C for 5 h. Hydroxylamine reductase activity was determined from hydroxylamine hydrochloride content changes in the soil solution. Briefly, 1 g of fresh soil sample was placed in a 10 mL test tube and 1 mL of 1% potassium nitrate and 1 mL of 1% glucose were added. Test tubes were stoppered and incubated at 37 °C for 48 h. Nitrate reductase activity was determined from nitrate content changes in the soil solution. Briefly, 1 g of fresh soil sample was placed in a 10 mL test tube and 1 mL of 0.25% sodium nitrite and 1 mL of 1% glucose were added. Test tubes were stoppered and incubated at 37 °C for 48 h. Nitrite reductase activity was determined from nitrate content changes in the soil solution.

2.7. Statistical Analyses

We subjected the data to normality and variance homogeneity tests prior to any further analysis. Then, a two-way ANOVA was used to evaluate the impact of litter treatments, season, and their interactions on soil properties, root exudation rates, and soil enzyme activity involved in N transformation. We utilized Tukey’s test to evaluate significant treatment differences when variances were equal, while Dunnett’s test was employed for unequal variances. A p-value of less than 0.05 was deemed to indicate statistically significant differences. All statistical analyses were conducted using SPSS version 22.0.

3. Results

3.1. Litter Treatment Effects on Soil Properties and Net N Mineralization and Nitrification Rates

Litter treatment had no significant effects on soil pH, SOC, TN, C:N, SON, NH4+-N, NO3−-N, mineral N, MBC, and MBN concentrations (Table 1). SON in LA plots was significantly higher than in LR plots in October 2020 (Table 1; p = 0.036). Mineral N in LA plots was significantly higher than in LR and CT plots in December 2020 (Table 1; p = 0.033 and p = 0.038, respectively), and was significantly higher than in the CT plots in April 2021(Table 1; p = 0.02). There was no significant difference in soil pH, SOC, TN, SON, MBC, and MBN concentrations among seasons. C:N, NH4+-N, NO3−-N, and mineral N were significantly affected by seasonal variation (Table 1; p = 0.001, p = 0.018, p < 0.001, and p < 0.001, respectively). Generally, the results showed that the impact of litter treatment on soil properties was weak. Litter treatment had no significant effects on net N mineralization rates, although both litter removal and litter addition significantly increased net N nitrification rates (Table 2; p = 0.003 and p = 0.012, respectively).

3.2. Litter Treatment Effects on Soil Enzyme Activities

Litter treatment had no significant effects on urease, hydroxylamine reductase, nitrate reductase, and nitrite reductase activities (Figure 1). Urease, nitrate reductase, and nitrite reductase activity significantly differed among seasons (Figure 1; p < 0.001), but hydroxylamine reductase activity did not (Figure 1).

3.3. Litter Treatment Effects on Root C Exudation Rates

Root C exudation rates in LR plots were significantly lower by 44.1% than in CT plots in April 2021 (Figure 2; p = 0.035). Annual root C exudation rates in LR plots were significantly lower by 22.4% than in CT plots (Figure 2; p = 0.048). There was no significant difference in root C exudation rates among litter treatments in the other seasons (Figure 2). Litter removal slightly decreased root C exudation rates and litter addition had little effect on root C exudation rates. Root C exudation rates showed strong seasonal patterns (Figure 2; p < 0.001) and there were litter × season interactions (Figure 2; p = 0.047).

3.4. Correlations between Root C Exudation Rates and Soil Enzyme Activities and the Rates of Net N Mineralization and Nitrification

Root C exudation rates were positively correlated with urease and nitrate reductase activities, but negatively associated with hydroxylamine reductase and nitrite reductase activities (Figure 3; p < 0.05). Conversely, root C exudation rates showed little relationship with the net N mineralization rate, but were negatively associated with the net N nitrification rate (Figure 4; p < 0.05). Root C exudation rates were closely associated with soil N transformations.

4. Discussion

4.1. Changes in Soil C and N after Litter Removal and Addition

Litter removal and addition had no significant influence on SOC concentration (Table 1), which indicates that SOC is not very sensitive to litter input variations. In contrast, in a meta-analysis of litter manipulation studies, soil C content was lower when litter was removed, whereas it was higher when litter was added as compared to controls [13,43]. Our results are in line with some previous studies that indicated no effect on soil C from changes in litter inputs [14,44]. Although tropical and subtropical forest soils are generally not regarded as C-saturated [45,46,47], C loss by priming effects was compensated by C inputs from added litter inputs; i.e., the additional labile C increased older SOM mineralization [2,48]. It is possible that we did not observe changes in soil C content due to an increase in soil respiration after adding litter [14]. It is commonly believed that adding organic matter to the soil will enhance microbial respiration, as it provides a readily decomposable substrate for microbes [49]. However, the enhanced soil respiration that offset the additional C input from the organic layer resulted in no increase in SOC.
Contrary to our hypothesis, we found little change in soil TN, NH4+-N, NO3−-N, and mineral N following litter inputs (Table 1). This is consistent with previous studies, which found that soil TN concentrations did not vary with litter treatment [17]. In contrast, in a long-term litter manipulation experiment in mature lowland tropical forest after 13 years of treatments, there was a reduction in soil TN after litter removal and a slight increase in TN in litter addition plots [7]. In evergreen lowland tropical forest in Panama, Sayer et al. [9] also observed a substantial increase in extractable soil inorganic N in double-litter plots, and NO3−-N was threefold higher after 7 years litter addition, and NO3−-N also decreased after litter removal. As N in forest ecosystems is mainly cycled in organic form through litter as substrate, its retention and availability are largely dependent on N cycling in organic matter [9]. We did not observe differences in SOC and SON between litter removal and double-litter addition. We interpret these results as strong evidence of litter manipulation influences on soil N concentrations. The amount of N in plot soils without litter gradually declines because it is taken up by plants and washed away through leaching [9]. There is a possibility that initial higher soil N availability could explain why there are no effects of litter treatments on soil inorganic N concentration over such a short time. However, previous studies found that the changes in soil inorganic N concentration after litter removal or addition can be attributed to the microbial N immobilization rate [14,17]. In our study, litter removal and addition had little impact on soil MBN and net N mineralization rates, which may explain its negligible effect on soil inorganic N concentration.

4.2. Changes in Rates of Soil Net N Mineralization and Nitrification after Litter Removal and Addition

Contrary to hypothesis 1, litter removal and addition had little impact on soil N mineralization rates (Table 2), which is consistent with other previous litter manipulation results [17,44,50]. However, other studies have demonstrated that N mineralization rates were significantly altered by litter manipulation. In broad-leaved forests, Zhang et al. [51] reported that the impact of litter removal on net N mineralization rates was significant. In Larix gmelinii forest in Northeast China, Xiao et al. [52] reported that litter addition significantly increased soil net N mineralization by 128%, while litter exclusion decreased it by 81%. Alexander and Arthur [53] confirmed red maple leaf litter addition tended to lower N mineralization rates. Our litter manipulation experiment was carried out for less than two years, which is a short time for a significant response in forests. Litter manipulation effects on N mineralization rates in mineral soil probably occur on the order of decades rather than months or years [50]. Pine litter from the P. massoniana plantation had high C:N, cellulose, and lignin, which are generally thought to be responsible for a slower litter decomposition rate [54]. It is the slow rate that causes the return of humus components to the soil although mineralization is slow [55,56]. Although new litter has been added, the old lowest litter layer has not yet decomposed; thus, N mineralization is less sensitive to the large new organic matter in the short term [50,52]. A necessary condition for N mining is that there must be an energy-rich C source available for microbial decomposers to break down SOM to release N [57]. Even though our monthly litter removal treatments succeeded in removing most of the aboveground litter inputs, three other sources still provided fresh organic matter inputs to soil microbial communities: dissolved organic matter in throughfall, roots (including root litter and exudates), and labile C released from litter between monthly raking cycles [7]. Our speculation is that after litter removal, microbial decomposers receive increased fresh organic inputs from these three sources. It is possible that microbial decomposers do not invest additional resources into producing N-acquiring enzymes to mineralize SOM and access N due to initial higher soil N availability. Our results indicate that litter input might change microbial N immobilization and thus affect N mineralization, because we did not observe differences in soil MBN under different litter treatments (Table 1).
Unexpectedly, both litter removal and addition increased nitrification rates (Table 2), which has rarely been observed in previous studies. Yan et al. [58] reported that litter addition significantly increased net nitrification rates in pure Masson pine and mixed Masson pine/Camphor tree forests, whereas litter removal significantly decreased net nitrification rates. Interestingly, both litter removal and addition decreased gross nitrification rates in a lowland tropical forest due to a lower mean relative abundance of napA, nirS, ammonia-oxidizing archaea (AOA), and ammonia-oxidizing bacteria (AOB) [5]. Matsushima and Chang (2007) [59] attributed an increased net nitrification rate after litter addition to higher soil temperature. Increasing temperature can increase microbial activity, promote nitrification, and increase net N nitrification rates. Moreover, many previous studies have confirmed litter addition increased soil fertility and supplied greater energy for soil biota, stimulating a significant increase in microbial activity, and increasing SOM decomposition [60,61,62]. Increased nitrification after litter removal may contribute to increased nitrifying bacteria activity caused by higher soil permeability and oxygen content [63]. Both litter addition and removal treatment had the same effect on nitrification in our experiment, but the potential mechanism was different. Adding litter to the soil can supply ample substrates and nutrients that support microbial growth and promote nitrification. Conversely, removing litter may trigger the N-conserving mechanism and promote nitrification as the system experiences a N shortage [52].

4.3. Changes in Root C Exudation Rates after Litter Removal and Addition

We hypothesized that litter removal reduced N availability to plants, which would promote microbial N mining and increase root C exudation rates. There is now sufficient evidence that litter removal reduces soil total and available N concentrations [3,7]. Numerous studies have documented that root C exudation rates under low soil N availability would increase to acquire N [19,64,65]. Trees under lower N availability may increase C exudation rates to stimulate microbial production of more exoenzymes, thus accelerating the mineralization of both organic C and N [66]. Our results do not conform to our initial hypothesis and showed that experimental litter removal decreased root C exudation rates in April 2021 and annual root C exudation rates (Figure 3). This may be related to decreased plant growth after litter removal treatment, limiting the distribution of C photosynthetic products to root exudates [67]. It has been reported that removing all forest residues decreased wood productivity by approximately 40% [68]. Reducing the input of root exudate C by removing litter is a plant strategy aimed at maximizing short-term forest growth, because more photosynthetic C can be retained for plant growth [36]. It is possible that lower exudation rates under litter removal resulted from changes in root morphological characteristics. Several studies have found that root length is positively correlated with exudation rates [69]. Root mass and length were reduced with decreased litter input [70,71], which may have contributed to the exudation rate decrease. Contrary to our initial hypothesis, litter addition had little effect on root C exudation rates. In Pinus armandii plantations, N addition, to some extent, mitigated N limitation by reducing root exudation rate per root biomass by 36.43% [72]. In an alpine shrub-dominated ecosystem, three consecutive years of N addition at two levels reduced annual root exudate C inputs by 44% and 66%, respectively [64]. We initially hypothesized that increased soil N availability after litter addition will cause a decrease in root C exudation rates and encourage plants to adopt a “low C cost–N benefit” nutrient acquisition strategy rather than a “high C cost–N benefit” strategy [72]. Our results indicated that litter addition had little effect on soil N availability, which explained the root C exudation rates after litter addition. However, the long-term effects of litter addition need to be continuously observed.

4.4. Soil N Transformations and Root Exudates

Comprehending the mechanisms by which potential modifications in root-derived C impact the microbial regulation of soil N cycling under litter manipulation is crucial in predicting biotic feedbacks to litter input. Our results indicated that root C exudation rates were positively correlated with urease, which is involved in N depolymerization from SOM (Figure 3), and this has been confirmed in previous studies in which urease activity was strongly associated with the root exudation rate [20]. C-containing compounds exudated can be C and energy sources for target soil microbes to synthesize extracellular enzymes, and stimulate plant-N acquisition via organic matter decomposition [73]. Previous studies have also reported microbes that are heterotrophic and live in soil, such as actinomycetes, which utilize energy obtained from exudates to produce extracellular enzymes that break down SOM and release N [74]. Importantly, root C exudation rates were negatively correlated with hydroxylamine reductase and nitrite reductase activities (Figure 3), indicating that root C exudation affects soil nitrification and denitrification. We interpret these results as strong evidence of root C exudation influencing the microbial regulation of soil N transformations in forest plantations. Root exudation from trees under experimental litter manipulation is an important physiological adjustment that stimulates N transformations in forest plantations.
Root C exudation can significantly affect soil denitrification and microbial activity via the rhizosphere priming effect [75]. However, denitrification spatiotemporal variability and many factors affecting plant–microbe interactions in rhizosphere soils limit the understanding of denitrification [76]. We found that root C exudation rates had direct positive correlations with nitrate reductase activity (Figure 3). This suggests that labile C efflux from exudates promotes extracellular enzyme activity and denitrification rates. Previous studies demonstrate that increasing root exudate release promotes denitrification, and increases N2O emission [77,78]. Langarica-Fuentes et al. [79] confirmed that root exudates can change the size and structure of denitrifying bacterial communities and increase the denitrification rate. Root exudates can regulate the expression of N transformation-related genes and affect denitrification (e.g., narG/napA, nirK/nirS, and norB/nosZ [80]). Root exudates and root respiration could also promote denitrification through reducing oxygen availability [81,82].
We also demonstrated that increases in labile C flux from the roots to the soil under litter experimental manipulation conditions inhibit the soil nitrification rate (Figure 4). Root exudates have been identified as nitrification inhibitors and as signaling compounds facilitating N-acquisition symbioses [83]. Root exudate inhibition of soil nitrification has been definitively demonstrated in sorghum [31], Brachiaria humidicola [84], rice [32,85], and wheat [34]. Nitrification inhibitors in root exudates affect soil nitrification by impeding ammonia monooxygenase, hydroxylamine oxidoreductase, and ammonia monooxygenase [83]. Linoleic acid and alpha-linolenic acid in biological nitrification inhibitors likely have the structure and chain length required to inhibit nitrification, especially ammonia monooxygenase and hydroxylamine reductase [86]. Additionally, biological nitrification inhibitors affect the metabolic function of the ammonia oxidation process by disrupting the electron transport pathway from hydroxylamine reductase to ubiquinone and cytochrome [87]. Allelochemicals in root exudates also inhibit soil nitrification by affecting microorganisms [88]. Our results suggest that litter manipulation accelerates the N nitrification rate. A nitrification reduction via root exudation is expected to improve N-use efficiency by reducing N losses via leaching, runoff, and denitrification [83]. Although the inhibition of soil nitrification by root exudates has been demonstrated in agroecosystems, there are few reports in forest ecosystems. The degree to which root exudates mediate forest nitrification through litter manipulation warrants further study.

5. Conclusions

In our manipulative experiment in a P. massoniana plantation, we found that litter removal and addition had little impact on C and N concentrations, microbial biomass, soil enzyme activity, and the net N mineralization rate. However, net N nitrification rates were strongly increased by litter removal and addition. Furthermore, litter removal slightly decreased root C exudation rates and was closely associated with soil enzymes involved in soil N transformations and the net N nitrification rate. Overall, root exudates played a critical role in regulating underground N biochemical processes in response to changes in litter input caused by global environmental changes. We provide new evidence from root exudates for understanding the potential influence of litter inputs on soil N cycling. Root exudates and N transformation are closely linked, offering new insights into the role of rhizosphere nutrient cycling in maintaining the stability and productivity of forest ecosystems amid changing environments.

Author Contributions

Q.Z. and T.H. contributed to the ideas and designed methodology. Laboratory analyses and data collection were performed by Y.C. and T.Z. Data analysis was performed and the main manuscript text was written by C.Z. and L.Z. helped in formal analysis and visualization. All authors have read and agreed to the published version of the manuscript.

Funding

This study received financial assistance from the National Natural Science Foundation of China (No. 32060339), Guizhou Provincial Basic Research Program (No. QKHJC[2020]1Y177), Science and Technology Planning Project of Guizhou Province (No. QKHZC[2022]YB 200), Starting Foundation for the Innovation Sector Research of Guizhou Academy of Sciences (No. QKYCZ[2021]), and Provincial Special Foundation for Scientific Research of Guizhou Academy of Sciences (No. QKYKZHZ[2022]03).

Data Availability Statement

The information exhibited in this study can be obtained upon request from the corresponding author.

Conflicts of Interest

The authors attest that they do not possess any recognized competitive financial interest or personal relationships that may have seemed to bias the findings presented in this article.

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Forests 14 01305 g001 550
Figure 1. Seasonal variation in the (a) urease, (b) hydroxylamine reductase, (c) nitrate reductase, and (d) nitrite reductase under different litter treatments. Values are mean ± standard error (n = 3). LR, litter removal; CT, control; LA, litter addition.
Figure 1. Seasonal variation in the (a) urease, (b) hydroxylamine reductase, (c) nitrate reductase, and (d) nitrite reductase under different litter treatments. Values are mean ± standard error (n = 3). LR, litter removal; CT, control; LA, litter addition.
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Figure 2. Seasonal variation in the mass-specific root C exudation rate (μg C g−1 root biomass h−1) for the P. massoniana under different litter treatments. Values are mean ± standard error (n = 3). LR, litter removal; CT, control; LA, litter addition. Different lowercase letters indicate significant differences among the litter treatments (p < 0.05).
Figure 2. Seasonal variation in the mass-specific root C exudation rate (μg C g−1 root biomass h−1) for the P. massoniana under different litter treatments. Values are mean ± standard error (n = 3). LR, litter removal; CT, control; LA, litter addition. Different lowercase letters indicate significant differences among the litter treatments (p < 0.05).
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Figure 3. Correlations of mass-specific root C exudation rate with (a) urease, (b) hydroxylamine reductase, (c) nitrate reductase, and (d) nitrite reductase (n = 36).
Figure 3. Correlations of mass-specific root C exudation rate with (a) urease, (b) hydroxylamine reductase, (c) nitrate reductase, and (d) nitrite reductase (n = 36).
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Figure 4. Correlations of mass-specific root C exudation rate with (a) Net mineralization rate and (b) Net nitrification rate (n = 9).
Figure 4. Correlations of mass-specific root C exudation rate with (a) Net mineralization rate and (b) Net nitrification rate (n = 9).
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Table
Table 1. Soil properties in response to litter addition and removal in a P. massoniana plantation. Values are mean ± standard error (n = 3). SOC, soil organic C; TN, total N; SON, soil organic N; MN, mineral N; MBC, microbial biomass C; MBN, microbial biomass N; LR, litter removal; CT, control; LA, litter addition. Different lowercase letters indicate significant differences among the litter treatments. * p < 0.05, ** p < 0.01.
Table 1. Soil properties in response to litter addition and removal in a P. massoniana plantation. Values are mean ± standard error (n = 3). SOC, soil organic C; TN, total N; SON, soil organic N; MN, mineral N; MBC, microbial biomass C; MBN, microbial biomass N; LR, litter removal; CT, control; LA, litter addition. Different lowercase letters indicate significant differences among the litter treatments. * p < 0.05, ** p < 0.01.
SeasonLitter TreamentpHSOC (g·kg−1)TN (g·kg−1)C:NSON (g·kg−1)NH4+-N (mg·kg−1)NO3−-N (mg·kg−1)NO3−-N (mg·kg−1)MN (mg·kg−1)MBC (mg·kg−1)MBN (mg·kg−1)
July 2020LR4.76 ± 0.13 a22.96 ± 1.78 a2.48 ± 0.54 a10.10 ± 1.96 a2.63 ± 0.46 a8.29 ± 1.51 a12.62 ± 1.36 a20.91 ± 1.55 a353.39 ± 101.75 a78.37 ± 23.70 a
CT4.75 ± 0.04 a21.13 ± 0.61 a2.65 ± 0.468.44 ± 1.36 a2.46 ± 0.54 a9.87 ± 2.40 a12.66 ± 3.66 a22.53 ± 5.08 a264.95 ± 43.55 a72.81 ± 11.48 a
LA4.70 ± 0.10 a23.36 ± 3.00 a2.96 ± 0.62 a8.22 ± 1.01 a2.93 ± 0.63 a9.68 ± 2.11 a11.16 ± 4.14 a20.84 ± 2.05 a230.37 ± 74.60 a41.59 ± 8.85 a
October 2020LR5.36 ± 0.56 a21.90 ± 0.74 a1.83 ± 0.13 a12.12 ± 1.17 a1.75 ± 0.09 b7.43 ± 1.41 a6.74 ± 0.97 a14.16 ± 1.38 a312.64 ± 93.10 a39.05 ± 11.37 a
CT4.97 ± 0.03 a24.69 ± 0.92 a1.77 ± 0.09 a14.09 ± 1.17 a1.82 ± 0.13 ab9.46 ± 0.88 a4.23 ± 0.22 a13.69 ± 0.69 a347.60 ± 23.08 a53.08 ± 6.12 a
LA4.90 ± 0.04 a24.28 ± 1.75 a2.29 ± 0.17 a10.64 ± 0.50 a2.27 ± 0.18 a8.89 ± 1.08 a6.44 ± 0.88 a15.34 ± 0.88 a373.68 ± 76.71 a55.62 ± 10.81 a
December 2020LR4.77 ± 0.08 a24.94 ± 3.50 a2.34 ± 0.30 a10.66 ± 0.79 a2.18 ± 0.02 a5.45 ± 0.47 a3.34 ± 0.73 a8.79 ± 0.53 b376.09 ± 48.64 a57.25 ± 10.99 a
CT4.91 ± 0.02 a25.07 ± 0.65 a2.19 ± 0.03 a11.46 ± 0.24 a2.33 ± 0.30 a5.63 ± 1.33 a3.36 ± 0.30 a8.99 ± 1.42 b303.62 ± 19.82 a44.73 ± 4.49 a
LA4.85 ± 0.07 a23.44 ± 3.60 a2.36 ± 0.28 a9.99 ± 1.03 a2.35 ± 0.28 a7.67 ± 0.92 a6.13 ± 1.32 a13.80 ± 1.63 a303.38 ± 33.71 a53.29 ± 6.11 a
April 2021LR4.86 ± 0.05 a21.03 ± 2.77 a2.32 ± 0.10 a9.00 ± 0.80 a2.15 ± 0.09 a9.40 ± 0.70 a6.49 ± 1.23 a15.89 ± 0.75 ab370.25 ± 50.10 a52.00 ± 11.27 a
CT4.81 ± 0.04 a18.27 ± 2.15 a2.16 ± 0.08 a8.40 ± 0.69 a2.30 ± 0.10 a8.74 ± 0.67 a5.06 ± 0.22 a13.79 ± 0.85 b357.82 ± 25.50 a45.18 ± 5.67 a
LA5.02 ± 0.16 a20.56 ± 0.74 a2.12 ± 0.07 a9.73 ± 0.46 a2.10 ± 0.07 a11.91 ± 1.80 a7.02 ± 0.97 a18.93 ± 1.66 a368.75 ± 55.30 a50.05 ± 6.33 a
Variance analysis of F-statistics
Litter0.2250.0850.6431.0060.6421.8620.6062.0490.5500.809
Season1.9092.5072.9527.079 **2.8884.085 *11.075 **16.581 **2.2396.314
Litter×season0.730.4530.3371.2390.3400.4330.4360.7881.3460.592
Table
Table 2. Net N mineralization and nitrification rates under different litter treatments in the P. massoniana plantation 0–10 cm soil layers. Values are mean ± standard error (n = 3). LR, litter removal; CT, control; LA, litter addition. Different lowercase letters indicate significant differences among the litter treatments (p < 0.05).
Table 2. Net N mineralization and nitrification rates under different litter treatments in the P. massoniana plantation 0–10 cm soil layers. Values are mean ± standard error (n = 3). LR, litter removal; CT, control; LA, litter addition. Different lowercase letters indicate significant differences among the litter treatments (p < 0.05).
TreatmentNet Mineralization Rate
(mg N kg−1 d−1)		Net Nitrification Rate
(mg N kg−1 d−1)
LR0.15 ± 0.03 a0.18 ± 0.01 a
CT0.09 ± 0.05 a0.03 ± 0.01 b
LA0.16 ± 0.02 a0.14 ± 0.03 a
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MDPI and ACS Style

Zhang, C.; Zhao, Q.; Cai, Y.; Zhang, T.; Zhang, L.; He, T. Effect of Litter Removal and Addition on Root Exudation and Associated Microbial N Transformation in a Pinus massoniana Plantation. Forests 2023, 14, 1305. https://doi.org/10.3390/f14071305

AMA Style

Zhang C, Zhao Q, Cai Y, Zhang T, Zhang L, He T. Effect of Litter Removal and Addition on Root Exudation and Associated Microbial N Transformation in a Pinus massoniana Plantation. Forests. 2023; 14(7):1305. https://doi.org/10.3390/f14071305

Chicago/Turabian Style

Zhang, Chengfu, Qingxia Zhao, Yinmei Cai, Tao Zhang, Limin Zhang, and Tengbing He. 2023. "Effect of Litter Removal and Addition on Root Exudation and Associated Microbial N Transformation in a Pinus massoniana Plantation" Forests 14, no. 7: 1305. https://doi.org/10.3390/f14071305

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