TY - JOUR
AU - Yang,, Guangyao
AB - Abstract The unbridled expansion of bamboo has imposed serious threats on ecosystem processes and functions. Considerable evidence indicates that bamboo invasions can alter plant-available soil nitrogen (N) pools and rates of N cycling, but the consequences of altered N availability for plant growth and community structure have thus far been poorly characterized. The primary soil-accessible N forms for most plants are ammonium (NH4+) and nitrate (NO3−), but plants differ in their ability to use the different N forms, and these differences can be related to their ecological characteristics and drive community structure. In this context, we evaluated the growth response, N uptake and interspecific competition in two subtropical species, Phyllostachys edulis (Carrière) J. Houzeau (Synonym Phyllostachys heterocycla Carrière) and Castanopsis fargesii Franch., dominant species of bamboo and secondary evergreen broad-leaved forests, respectively, under changing N availability in seedlings supplied with different N concentrations and NH4+/NO3− proportions, in vermiculite culture, in a controlled environment. The results show that (i) both species display an NH4+ preference at elevated N concentrations. The growth of P. edulis seedlings was strongly enhanced at increased ratios of NH4+ to NO3− especially at higher N concentrations, but to a much lesser extent in C. fargesii. (ii) NH4+ preference at the level of N uptake in both species was confirmed by the Non-invasive Micro-test Technology and by examining 15N signatures. Phyllostachys edulis had higher NH4+ net fluxes and N concentration under NH4+ treatments than C. fargesii. (iii) NH4+ at higher concentrations caused toxicity in both species as it inhibited root growth and even caused seedling death, but P. edulis had a higher NH4+-tolerance threshold (24 mM) than C. fargesii (16 mM). (iv) When mixed-species cultures were examined in an NH4+-rich medium, the growth of C. fargesii, but not P. edulis, was significantly inhibited compared with growth in monoculture. Therefore, P. edulis exhibited stronger plasticity and adaptation to changing N availability, whereas C. fargesii had low responsiveness and capacity to acclimate to soil N changes. Phyllostachys edulis displayed a significant competitive growth advantage compared with C. fargesii on NH4+-dominated substrates. Introduction Moso bamboo (Phyllostachys edulis), Phyllostachys edulis (Carrière) J. Houzeau (Synonym Phyllostachys heterocycla Carrière) a member of the Poaceae and tree-like woody bamboo, widely predominates in subtropical China, possessing ecological, economic and cultural importance (Song et al. 2016). However, P. edulis is also notorious for its invasive character in Asian forests (Okutomi et al. 1996, Kleinhenz and Midmore 2001, Lima et al. 2012, Ying et al. 2016). Secondary evergreen broad-leaved forest, the typical vegetation type in subtropical areas, is the most vulnerable to expansion and can be readily displaced by bamboo (Okutomi et al. 1996, Song et al. 2016, Ying et al. 2016). The unbridled expansion of bamboo has caused substantial impacts on colonized forests, such as altered community structure and species composition (Okutomi et al. 1996, Lima et al. 2012), reduced biodiversity (Larpkern et al. 2011), altered soil processes and microbial composition (Song et al. 2013, 2016, 2017), and broken-up and degraded forest landscapes (Okutomi et al. 1996), and these, together, have been considered among the greatest contemporary threats to woody forests in China. According to previous studies, bamboo can become a dominant species in many forest communities by virtue of shading (Suzaki and Nakatsubo 2001, Lima et al. 2012), mechanical damage (Okutomi et al. 1996), litter suppression (Larpkern et al. 2011) and allelopathy (Chou and Yang 1982). Recently, many studies have reported the influence of P. edulis expansion on soil nutrients, such as soil organic matter, available soil nitrogen (N) pools and rates of N cycling, phosphorus status and dynamics, and soil potassium (K) (Song et al. 2013, 2016, 2017, Fukushima et al. 2015, Y. Li et al. 2017, Z. Li et al. 2017, Wu et al. 2018). However, little information has been gathered on the effects of these ecological processes to community structure, and the role of N has remained surprisingly understudied. It has been proposed that soil N dynamics can be a driving force in forest succession (Kronzucker et al. 1997, 2003). Fukushima et al. (2015) assumed that P. edulis expansion into broadleaved forests changed the distribution pattern of carbon and N stored in plants and soil. It increased plant N accumulation but reduced soil N available pool, slowed the N cycling and reduced soil N mineralization rate (Song et al. 2016, 2017). Specifically, soil N mineralization changes in terms of both ammonification and nitrification occurred at similar rates, i.e., 3.82 and 3.31 mg kg−1 (30 days)−1 in secondary evergreen broad-leaved forest soil dominated by ammonification, that is 5.66 and 0.18 mg kg−1 (30 days)−1, respectively, in bamboo-dominated mixed forest after the expansion of bamboo (Song et al. 2013). Enhanced ammonification rates (soil NH4+-N) but reduced nitrification rates (soil NO3−-N) following bamboo invasion of broad-leaved forests have also been reported by other different laboratories in different sites (Y. Li et al. 2017, Z. Li et al. 2017). Changes in soil N chemistry are likely to be significant determinants of synecological changes in such forests, as they are in temperate Northern forests (Kronzucker et al. 2003). Although a considerable nnumber of reports indicate that bamboo invasions can alter N content and the chemical speciation of inorganic N in forest soils, the consequences of altered soil N availability for forest plants are poorly understood in the bamboo and tree species coexisting in the affected areas. Next to usually smaller quantities of organic N, the primary accessible forms of N for plant roots are ammonium (NH4+) and nitrate (NO3−) in most natural ecosystems (Britto and Kronzucker 2013), and species vary greatly in their ability to absorb different inorganic N forms (Britto and Kronzucker 2006, 2013). Over the course of their evolution and community construction, plants may have become adapted to the nitrogenous characteristics of their native forest soils (Kronzucker et al. 1997, 2003), that is, in environments dominated by NH4+ or NO3− in soils, many plant species develop greater biomass or accumulate greater N quantities on one form of N versus the other, showing obvious, and at times stark, NH4+ or NO3− preferences. For instance, trees occupying specific habitats of late-successional forests (mature forests) dominated by NH4+ demonstrate more efficient N capture with NH4+ relative to NO3−, in both field and laboratory studies (Kronzucker et al. 1997, Li et al. 2015). Species-specific strategies and the differential capacity of plants to respond to, take up and metabolically process different sources of N is very important for species establishment on successionally different sites (Kronzucker et al. 1997, Min et al. 1999, 2000, Kronzucker et al. 2003). For instance, in late-successional forests in the Northern temperate zone, disturbances, such as clear-cut harvesting, tend to convert soil N from NH4+ to mostly NO3− and late-successional trees, often characterized by low NO3− uptake and utilization capacities (Kronzucker et al. 1995a, 1995b), as a consequence, become poor competitors for inorganic N on such soils, whereas the sites become dominated by nitrophiles (Kronzucker et al. 1997). On the other hand, in later successional stages, differential toxicity thresholds for NH4+ can play a significant role in driving species composition in the opposite direction, away from domination by nitrophiles, as the latter succumb to NH4+ toxicity (Kronzucker et al. 2003, Britto and Kronzucker 2013, and references therein). In many of the forest soils in Asia in which bamboo incursions have been recorded, soil NH4+ tends to increase relative to NO3− during P. edulis expansion, but species-specific strategies in terms of uptake preference and growth response to different inorganic N forms have remained uncharacterized in bamboo and the evergreen broad-leaved forest species with which they compete. Table 1 Nutrient solution composition of different N concentrations [N] and NH4+/NO3− ratios. [N] NH4+/NO3− . 0.1 mM . 2 mM . 8 mM . . 0/4 . 1/3 . 2/2 . 3/1 . 4/0 . 0/4 . 1/3 . 2/2 . 3/1 . 4/0 . 0/4 . 1/3 . 2/2 . 3/1 . 4/0 . KNO3 0.1 0.075 0.05 0.025 0 2 1.5 1 0.5 0 3 1 0 0 0 Ca(NO3)2.4H2O 0 0 0 0 0 0 0 0 0 0 2.5 2.5 2 1 0 (NH4)2SO4 0 0.0125 0.025 0.0375 0.05 0 0.25 0.5 0.75 1 0 1 2 3 4 K2SO4 1.45 1.4625 1.475 1.4875 1.5 0.5 0.75 1 1.25 1.5 0 1 1.5 1.5 1.5 CaCl2.2H2O 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 0 0 0.5 1.5 2.5 MgSO4.7H2O 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 Na2HPO4.10H2O 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 Fe-EDTA 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 H3BO3 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 MnCl2.4H2O 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 ZnSO4.7H2O 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 CuSO4.5H2O 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 Na2MoO4.2H2O 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 CoCl2.6H2O 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 [N] NH4+/NO3− . 0.1 mM . 2 mM . 8 mM . . 0/4 . 1/3 . 2/2 . 3/1 . 4/0 . 0/4 . 1/3 . 2/2 . 3/1 . 4/0 . 0/4 . 1/3 . 2/2 . 3/1 . 4/0 . KNO3 0.1 0.075 0.05 0.025 0 2 1.5 1 0.5 0 3 1 0 0 0 Ca(NO3)2.4H2O 0 0 0 0 0 0 0 0 0 0 2.5 2.5 2 1 0 (NH4)2SO4 0 0.0125 0.025 0.0375 0.05 0 0.25 0.5 0.75 1 0 1 2 3 4 K2SO4 1.45 1.4625 1.475 1.4875 1.5 0.5 0.75 1 1.25 1.5 0 1 1.5 1.5 1.5 CaCl2.2H2O 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 0 0 0.5 1.5 2.5 MgSO4.7H2O 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 Na2HPO4.10H2O 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 Fe-EDTA 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 H3BO3 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 MnCl2.4H2O 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 ZnSO4.7H2O 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 CuSO4.5H2O 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 Na2MoO4.2H2O 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 CoCl2.6H2O 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 Concentrations are in mM. Open in new tab Table 1 Nutrient solution composition of different N concentrations [N] and NH4+/NO3− ratios. [N] NH4+/NO3− . 0.1 mM . 2 mM . 8 mM . . 0/4 . 1/3 . 2/2 . 3/1 . 4/0 . 0/4 . 1/3 . 2/2 . 3/1 . 4/0 . 0/4 . 1/3 . 2/2 . 3/1 . 4/0 . KNO3 0.1 0.075 0.05 0.025 0 2 1.5 1 0.5 0 3 1 0 0 0 Ca(NO3)2.4H2O 0 0 0 0 0 0 0 0 0 0 2.5 2.5 2 1 0 (NH4)2SO4 0 0.0125 0.025 0.0375 0.05 0 0.25 0.5 0.75 1 0 1 2 3 4 K2SO4 1.45 1.4625 1.475 1.4875 1.5 0.5 0.75 1 1.25 1.5 0 1 1.5 1.5 1.5 CaCl2.2H2O 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 0 0 0.5 1.5 2.5 MgSO4.7H2O 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 Na2HPO4.10H2O 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 Fe-EDTA 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 H3BO3 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 MnCl2.4H2O 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 ZnSO4.7H2O 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 CuSO4.5H2O 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 Na2MoO4.2H2O 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 CoCl2.6H2O 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 [N] NH4+/NO3− . 0.1 mM . 2 mM . 8 mM . . 0/4 . 1/3 . 2/2 . 3/1 . 4/0 . 0/4 . 1/3 . 2/2 . 3/1 . 4/0 . 0/4 . 1/3 . 2/2 . 3/1 . 4/0 . KNO3 0.1 0.075 0.05 0.025 0 2 1.5 1 0.5 0 3 1 0 0 0 Ca(NO3)2.4H2O 0 0 0 0 0 0 0 0 0 0 2.5 2.5 2 1 0 (NH4)2SO4 0 0.0125 0.025 0.0375 0.05 0 0.25 0.5 0.75 1 0 1 2 3 4 K2SO4 1.45 1.4625 1.475 1.4875 1.5 0.5 0.75 1 1.25 1.5 0 1 1.5 1.5 1.5 CaCl2.2H2O 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 0 0 0.5 1.5 2.5 MgSO4.7H2O 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 Na2HPO4.10H2O 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 Fe-EDTA 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 H3BO3 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 MnCl2.4H2O 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 ZnSO4.7H2O 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 CuSO4.5H2O 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 Na2MoO4.2H2O 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 CoCl2.6H2O 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 Concentrations are in mM. Open in new tab The objectives of this study were to investigate species-specific N-use strategies of bamboo and tree species, and to explore the influence of altered N availability on plant growth. We addressed this question by using seedlings of the P. edulis, a dominant species of bamboo-dominant forests, and Castanopsis fargesii Franch. a dominant tree species of secondary evergreen broad-leaved forests in subtropical China (Cornelissen 1993, Song et al. 2016), which has been threatened in many parts of subtropical China by the expansion of moso bamboo (Okutomi et al. 1996, Song et al. 2016, Ying et al. 2016). Phyllostachys edulis typically grows on soils where N mineralization is dominated by ammonification, and, thus, NH4+ is the even more dominant N form (Song et al. 2013, 2016, 2017, Z. Li et al. 2017). Soil ammonification and nitrification occur at similar rates in such evergreen broad-leaved forests, although soil nitrification is more pronounced during the principal growing season (from April to October), with soil NH4+ exceeding NO3− (Song et al. 2013, 2016, 2017; Z. Li et al. 2017) or soil NO3− higher than NH4+ (Y. Li et al. 2017) according to sites. Growth response and N uptake of the two species were studied by supplying seedlings with different N concentrations and NH4+/NO3− proportions in vermiculite culture, and in controlled environments. The Non-invasive Micro-test Technology (NMT) and 15N isotope tracing were employed to assess acquisition of NH4+ and NO3− in the test species. In view of soil N mineralization and the predominant N form in bamboo and evergreen broad-leaved forest soils, we hypothesized that the two species would display visible species-specific responses and competitiveness to altered N availabilities. Thus, we predicted that: (i) P. edulis will show NH4+ preference, whereas C. fargesii will have improved performance and N uptake with NO3− or mixed N diets based on co-provision of NH4+ and NO3−; (ii) in NH4+-rich conditions, P. edulis will demonstrate competitive advantages compared with C. fargesii; and (iii) at increased levels of NH4+ supply, C. fargesii will succumb to NH4+ toxicity more readily than P. edulis. Materials and methods Plants and treatments Seeds of P. edulis were collected from Guanyang, Guangxi Province (25°14′N, 110°51′E, 354 m altitude) and were stored at 4 °C. The moso bamboo seeds were soaked overnight and sterilized using 20% NaClO (V/V) for 20 min, rinsed five times in sterile water and then germinated in plastic pots filled with vermiculite and 1 month afterwards three-foliate seedlings were selected for varying N treatments. Castanopsis fargesii seeds were collected from Xiayang State Forest Farm of Nanping, Fujian Province (26°45′N, 118°02′E) and were stratified with sand in a 3:1 (V/V) in greenhouse for 3 months after collection and before sowing in sand. Seedlings were germinated for ~3 months before transferring the three-foliate seedlings to different N treatments. Seedlings of both species were transplanted into plastic pots (diameter 150 mm, height 130 mm) filled with vermiculite and watered three times per week with deionized water until new roots were produced. Nitrogen form preference experiment Two weeks afterwards, uniform seedlings were selected for N treatments. Fifteen N treatments consisted of three N concentrations (0.1 mM, 2 mM, 8 mM) and five ratios (NH4+/NO3− = 0/4, 1/3, 2/2, 3/1, 4/0). The nutrient solution, modified from Norisada and Kojima (2005), contained: 2.5 mM Ca2+, 3 mM K+, 0.25 mM MgSO4·7H2O, 0.6 mM Na2HPO4·10H2O, 0.01 mM Fe-EDTA, 0.02 mM H3BO3, 2 μM MnCl2·4H2O, 2 μM ZnSO4·7H2O, 2 μM CuSO4·5H2O, 0.5 μM Na2MoO4·2H2O, 0.5 μM CoCl2·6H2O (Table 1). Different N forms and concentrations were supplied by KNO3, Ca(NO3)2·4H2O, (NH4)2SO4 and K2SO4. The pH of each treatment nutrient solution was adjusted to pH 4.0 with 0.1 M NaOH or 0.1 M HCl. In total, 1 ml of 7 mM C2H4N4 (294 mg C2H4N4 was dissolved in 500 ml deionized water) was added to 1000 ml of N-treatment nutrient solutions to prevent nitrification. Two seedlings per pot, and four pots placed inside another bigger pot as saucer, formed one replicate, and there were three replicates of 24 seedlings for each treatment. Seedlings were watered with nutrient solution twice a week from August to October in 2014. Plants were grown with 25/15 °C ± 3 °C day/night temperature, 65–70% relative humidity and 14/10 h day/night photoperiod in a greenhouse at Jiangxi Agricultural University (28°41′N, 115°52′E). Pots were rotated every week to eliminate location effects. After 2 months, growth characteristics and root morphology were analyzed. Growth analysis and root morphology Height was measured with a ruler. Leaf number was determined at the end of the treatments. The chlorophyll content of the leaves was determined with a chlorophyll meter (SPAD-502, Minolta, Osaka, Japan). Root morphology characteristic indices, including total root length (RL), average diameter (AD), root surface (RS) area, root volume (RV) and leaf surface were determined by a root automatic scanning apparatus (EPSON color image scanner LA1600+, Toronto, Canada), equipped with WinRHIZO 2012 software (Regent Instruments, Quebec, Canada) after 2 months of treatments. When treatments were completed, plants were harvested and separated into foliage, stems and roots, dried at 105 °C for 30 min, and then to a constant weight at 60 °C for biomass determination. Measurement of net NH4+ and NO3− fluxes with the NMT system Net NH4+ and NO3− fluxes were measured using the NMT (NMT100 Series, Younger USA LLC, Amherst, MA, Xuyue (Beijing) Science and Technology Co., Ltd, Beijing, China) and imFluxes V2.0 (Younger USA, LLC, Amherst, MA, USA) software. The NMT system and its application in ion flux detection are described in detail elsewhere (Li et al. 2010, Luo et al. 2013). Uniform three-foliate seedlings of P. edulis and C. fargesii were selected and transplanted to plastic pots (diameter 150 mm, height 130 mm) with vermiculite. Two weeks after, seedlings were supplied with 8 mM of NH4+ or NO3−. After treatment for 2 weeks from June to July in 2016, the white fine roots were selected and cut from the root system of P. edulis and C. fargesii. The excised roots were immediately equilibrated in measuring solution for 20–30 min. Eight plants for NH4+ and another eight plants for NO3− were used for ion flux analyses. Each plant was measured once. The equilibrated roots were then transferred to the measuring chamber containing 2–3 ml of fresh measuring solution. The measuring solution was composed of 2.5 mM CaCl2·2H2O, 1.5 mM K2SO4, 0.6 mM Na2HPO4·10H2O, 0.25 mM MgSO4·7H2O, 4 mM (NH4)2SO4 for NH4+ and 2.5 mM CaCl2·2H2O, 1.5 mM K2SO4, 0.6 mM Na2HPO4·10H2O, 0.25 mM MgSO4·7H2O, 8 mM NaNO3 for NO3− and pH 4.0 was adjusted by HCl-NaOH. The final flux values at each point were the means of eight individual plants from a given treatment. Pre-pulled and silanized microsensor (Φ9 ± 1 μm, XY-CGQ-01, Younger USA) were first filled with a backfilling solution (NH4+,100 mM NH4Cl, NO3−, 10 mM KNO3) to a length of ~1.0 cm from the tip. Then, the micropipettes were front-filled with 40–50 μm or 80 μm columns of selective liquid ion-exchange cocktails (NH4+ LIX, XY-SJ-NH4, NO3− LIX, XY-SJ-NO3, Younger USA) for NH4+ and NO3−, respectively. YG003-Y11 (Younger USA) was used as the reference microsensor. Before each flux measurement, the microelectrodes were calibrated using NH4+ (NO3−) at concentrations of 0.5, 1.0, 5.0 and 10 mM. Only microsensors with a Nernstian slope >50 mV/decade for NH4+ and <−50 mV/decade for NO3− were used in the study. 15N-tracer experiments and determination of total N and δ15N Uniform three-foliate seedlings of P. edulis and C. fargesii were selected and transplanted to plastic pots (diameter 150 mm, height 130 mm) with vermiculite. Two weeks after, seedlings were supplied with one of six 15N isotope-labeled solutions two times per week from September to November, in 2016. The six N solutions all contained 8 mM of N, with the three N sources provided in equimolar amounts (8 mM NaNO3, 4 mM (NH4)2SO4 or 4 mM NH4NO3), but differing in terms of which N source was labeled: (15NH4)2SO4, Na15NO3, 15NH4 NO3, NH415NO3, or unlabeled N, provided as (NH4)2SO4 and NaNO3. There were three replicates of 36 seedlings per treatment. After 2 months of N treatment, seedlings were harvested. The roots were washed to eliminate vermiculite and traces of 15N, twice with tap water and once with distilled water. Then, all fractions including the foliage, stems and roots were oven-dried at 60 °C for 48 h, weighed and finely ground with a mortar and pestle to pass through 0.5 mm sieve before measurement. The concentration of N and 15N abundance was measured using a Flash 2000 HT elemental analyzer interfaced to an Isotope Ratio Mass Spectrometer (DELTA V Advantage, Thermo Fisher Scientific, Waltham, MA, USA), as described by Piao et al. (2017). NH4+-tolerance experiments Uniform three-foliate seedlings of P. edulis and C. fargesii were selected and transplanted to plastic pots (diameter 150 mm, height 130 mm) containing vermiculite. After 15 days of growth, seedlings were treated with normal nutrient solution supplemented with varying concentrations of NH4+ (2, 8, 16, 24, 32, 40 mM) supplied as (NH4)2SO4 two times per week from July to September, in 2015. There were three replicates of 24 seedlings per treatment. The height of seedlings was monitored every 2 weeks. After 2 months, growth and root morphology were analyzed. Monoculture and mixed culture experiments Uniform three-foliate seedlings of P. edulis and C. fargesii were selected and transplanted to plastic pots (length 435 mm, width 200 mm, height 140 mm) with vermiculite. There were eight seedlings per pot to form the following three planting systems: P. edulis monoculture (seedlings of the same species grown separately in containers), C. fargesii monoculture, and P. edulis and C. fargesii mixed culture (seedlings of each species mixed in containers) to simulate the growth of two species in natural conditions and evaluate interspecific competitive responses in an NH4+-rich environment. Two weeks after production of new roots, seedlings were treated with normal nutrient solution supplemented 4 mM (NH4)2SO4 once a week from September 2017 to August 2018. The growth of seedlings was monitored by recording height monthly. Data analysis Data were statistically analyzed with SPSS version 13.0 (SPSS, Chicago, IL, USA). One-way analysis of variance (ANOVA) along with a Fisher’s least significance (LSD) test was applied to identify differences. Significant differences (P < 5%) between treatments are indicated by different letters. Sigma Plot 13.0 was used for the generation of graphs and Photoshop for photocomposition. Results Growth analysis For seedlings of both species, negligible growth differences were found between N forms (ratios of NH4+ to NO3−) at 0.1 mM N, whereas, at greater N concentrations, seedlings displayed superior growth with NH4+ or mixed N compared with that of NO3−. For P. edulis, an increment in NH4+ levels and the proportion of NH4+ to NO3− at 8 mM N in the treatments significantly increased growth, root morphology and chlorophyll content (SPAD value) (Figures 1–3). The biomass of P. edulis seedlings treated with 8 mM NH4+ was significantly larger (3.03- and 8.80-fold) than in seedlings grown with the same N form at 2 and 0.1 mM, respectively, as opposed to seedlings treated with 8 mM NO3−, which were similar (1.06- and 1.61-fold) to the seedlings with NO3− at 2 and 0.1 mM, respectively (Figure 1G). The growth of C. fargesii seedlings was significantly less affected by N form at identical N concentrations in terms of most morphological parameters, except for biomass and chlorophyll content, which were higher with 8 mM NH4+ (Figures 1H and 3B). The biomass of C. fargesii seedlings treated with 8 mM NH4+ were 1.78- and 1.66-fold, respectively, of seedlings grown on the same N form of 2 and 0.1 mM, as compared with the biomass of seedlings treated with 8 mM NO3−, which was 1.29- and 1.24-fold, respectively, of that of seedlings treated with 2 mM and 0.1 mM NO3− (Figure 1H). Two-way ANOVA indicated that the effects of N concentration, N form and their interaction were significant for all morphological parameters of P. edulis (Table 2). By contrast, the effect of N concentration was significant for most morphological parameters of C. fargesii except for total RL, whereas the effects of N form and the interaction were significant only for biomass, RV and chlorophyll content (Table 2). Figure 1. Open in new tabDownload slide Nitrogen concentrations and NH4+/NO3− ratios on the growth of P. edulis and C. fargesii. Height (A and B), leaf number (C and D), leaf surface area (E and F) and biomass (G and H) of P. edulis (left) and C. fargesii (right) as affected by different N treatments. Data are means from three replicates of 24 seedlings, and bars represent the standard error (SE). Different letters indicate statistical differences among treatments for each species (Fisher’s LSD, P < 0.05). To facilitate treatment comparison, mass scale is different for each species. This applies to following figures. Figure 1. Open in new tabDownload slide Nitrogen concentrations and NH4+/NO3− ratios on the growth of P. edulis and C. fargesii. Height (A and B), leaf number (C and D), leaf surface area (E and F) and biomass (G and H) of P. edulis (left) and C. fargesii (right) as affected by different N treatments. Data are means from three replicates of 24 seedlings, and bars represent the standard error (SE). Different letters indicate statistical differences among treatments for each species (Fisher’s LSD, P < 0.05). To facilitate treatment comparison, mass scale is different for each species. This applies to following figures. Figure 2. Open in new tabDownload slide Nitrogen concentrations and NH4+/NO3− ratios on root morphology of P. edulis and C. fargesii. Total RL (A and B), AD (C and D), RS area (E and F) and RV (G and H) of P. edulis (left) and C. fargesii (right) as affected by different N treatments. Data are means from three replicates of 24 seedlings, and bars represent the SE. Different letters indicate statistical differences among treatments for each species (Fisher’s LSD, P < 0.05). Figure 2. Open in new tabDownload slide Nitrogen concentrations and NH4+/NO3− ratios on root morphology of P. edulis and C. fargesii. Total RL (A and B), AD (C and D), RS area (E and F) and RV (G and H) of P. edulis (left) and C. fargesii (right) as affected by different N treatments. Data are means from three replicates of 24 seedlings, and bars represent the SE. Different letters indicate statistical differences among treatments for each species (Fisher’s LSD, P < 0.05). Figure 3. Open in new tabDownload slide Nitrogen concentrations and NH4+/NO3− ratios on chlorophyll content (SPAD value) of fully developed leaves in P. edulis and C. fargesii. Chlorophyll content is shown as value from SPAD meter. Data are means from three replicates of 24 seedlings, and bars represent the SE. Different letters indicate statistical differences among treatments for each species (Fisher’s LSD, P < 0.05). Figure 3. Open in new tabDownload slide Nitrogen concentrations and NH4+/NO3− ratios on chlorophyll content (SPAD value) of fully developed leaves in P. edulis and C. fargesii. Chlorophyll content is shown as value from SPAD meter. Data are means from three replicates of 24 seedlings, and bars represent the SE. Different letters indicate statistical differences among treatments for each species (Fisher’s LSD, P < 0.05). Table 2 Summary of two-way ANOVA (P-value) for N concentrations and NH4+/NO3− ratios on growth indices of P. edulis and C. fargesii seedlings. Growth index . P-value (P. edulis) . P-value (C. fargesii) . . [N] . NH4+/NO3− . [N] × NH4+/NO3− . [N] . NH4+/NO3− . [N] × NH4+/NO3− . Seedling height <0.001 <0.001 <0.001 <0.001 0.148 0.101 Leaf number <0.001 <0.001 <0.001 <0.001 0.619 0.285 Leaf surface area <0.001 <0.001 <0.001 <0.001 0.671 0.21 Biomass <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Total RL <0.001 <0.001 <0.001 0.524 0.077 0.034 RS area <0.001 <0.001 <0.001 0.011 0.095 0.176 AD <0.001 <0.001 <0.001 <0.001 0.052 0.026 RV <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 SPAD value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Growth index . P-value (P. edulis) . P-value (C. fargesii) . . [N] . NH4+/NO3− . [N] × NH4+/NO3− . [N] . NH4+/NO3− . [N] × NH4+/NO3− . Seedling height <0.001 <0.001 <0.001 <0.001 0.148 0.101 Leaf number <0.001 <0.001 <0.001 <0.001 0.619 0.285 Leaf surface area <0.001 <0.001 <0.001 <0.001 0.671 0.21 Biomass <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Total RL <0.001 <0.001 <0.001 0.524 0.077 0.034 RS area <0.001 <0.001 <0.001 0.011 0.095 0.176 AD <0.001 <0.001 <0.001 <0.001 0.052 0.026 RV <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 SPAD value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Nitrogen concentrations (0.1, 2 and 8 mM) and ratios of NH4+/NO3− (0/4, 1/3, 2/2, 3/1, 4/0) are the same as shown in Table 1. [N] × NH4+/NO3− indicates the interaction effect of N concentrations and NH4+/NO3− ratios. Open in new tab Table 2 Summary of two-way ANOVA (P-value) for N concentrations and NH4+/NO3− ratios on growth indices of P. edulis and C. fargesii seedlings. Growth index . P-value (P. edulis) . P-value (C. fargesii) . . [N] . NH4+/NO3− . [N] × NH4+/NO3− . [N] . NH4+/NO3− . [N] × NH4+/NO3− . Seedling height <0.001 <0.001 <0.001 <0.001 0.148 0.101 Leaf number <0.001 <0.001 <0.001 <0.001 0.619 0.285 Leaf surface area <0.001 <0.001 <0.001 <0.001 0.671 0.21 Biomass <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Total RL <0.001 <0.001 <0.001 0.524 0.077 0.034 RS area <0.001 <0.001 <0.001 0.011 0.095 0.176 AD <0.001 <0.001 <0.001 <0.001 0.052 0.026 RV <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 SPAD value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Growth index . P-value (P. edulis) . P-value (C. fargesii) . . [N] . NH4+/NO3− . [N] × NH4+/NO3− . [N] . NH4+/NO3− . [N] × NH4+/NO3− . Seedling height <0.001 <0.001 <0.001 <0.001 0.148 0.101 Leaf number <0.001 <0.001 <0.001 <0.001 0.619 0.285 Leaf surface area <0.001 <0.001 <0.001 <0.001 0.671 0.21 Biomass <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Total RL <0.001 <0.001 <0.001 0.524 0.077 0.034 RS area <0.001 <0.001 <0.001 0.011 0.095 0.176 AD <0.001 <0.001 <0.001 <0.001 0.052 0.026 RV <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 SPAD value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Nitrogen concentrations (0.1, 2 and 8 mM) and ratios of NH4+/NO3− (0/4, 1/3, 2/2, 3/1, 4/0) are the same as shown in Table 1. [N] × NH4+/NO3− indicates the interaction effect of N concentrations and NH4+/NO3− ratios. Open in new tab NH4+ and NO3− net fluxes The results show different net fluxes for NH4+ and NO3− at the RS along the root tip in both species when exposed to different N forms (Figure 4). When treated with 8 mM NH4+, net NH4+ flux varied from −2839.11 (influx) to 992.22 (efflux) pmol cm−2 s−1 for P. edulis and −609.20 to 601.46 pmol cm−2 s−1 for C. fargesii, and the maximal net NH4+ uptake was detected at 0.3 mm from the root tip for both species (Figure 4A and B). When treated with 8 mM NO3−, net NO3− flux ranged from −153.81 to 697.78 pmol cm−2 s−1 for P. edulis, −72.23 to 119.32 pmol cm−2 s−1 for C. fargesii and, at 1 mm from the root tip, net NO3− displayed maximum influx in both species (Figure 4C and D). The maximal net fluxes of NH4+ were 18.46- and 8.43-fold of those of NO3−, respectively, in P. edulis and C. fargesii. The maximal net NH4+ and NO3− fluxes of P. edulis were 4.66- and 2.13-fold, respectively, of those C. fargesii. Figure 4. Open in new tabDownload slide Effects of 8 mM different N treatment on NH4+ and NO3− flux along the root tip of P. edulis and C. fargesii. Three-foliate seedlings of P. edulis and C. fargesii were treated with 8 mM NH4+ (A and B) or NO3− (C and D) for 2 weeks and then subjected to NH4+ or NO3− flux measurements at 0–40 mm from the root cap junction. Data are means from two replicates of eight seedlings, and bars represent the SE. Different letters on the error bars in each panel indicate significant difference among the measured positions for each species. The measuring solution contained 2.5 mM CaCl2·2H2O, 1.5 mM K2SO4, 0.6 mM Na2HPO4·10H2O, 0.25 mM MgSO4·7H2O, pH 4.0, to which either 4 mM (NH4)2SO4 for NH4+ or 8 mM NaNO3 for NO3− flux measurements were added. Figure 4. Open in new tabDownload slide Effects of 8 mM different N treatment on NH4+ and NO3− flux along the root tip of P. edulis and C. fargesii. Three-foliate seedlings of P. edulis and C. fargesii were treated with 8 mM NH4+ (A and B) or NO3− (C and D) for 2 weeks and then subjected to NH4+ or NO3− flux measurements at 0–40 mm from the root cap junction. Data are means from two replicates of eight seedlings, and bars represent the SE. Different letters on the error bars in each panel indicate significant difference among the measured positions for each species. The measuring solution contained 2.5 mM CaCl2·2H2O, 1.5 mM K2SO4, 0.6 mM Na2HPO4·10H2O, 0.25 mM MgSO4·7H2O, pH 4.0, to which either 4 mM (NH4)2SO4 for NH4+ or 8 mM NaNO3 for NO3− flux measurements were added. Nitrogen concentration and δ15N in seedlings The N concentration of plant fractions and total seedlings varied with species and the ratio of NH4+ to NO3− but did not differ between treatments with 15N-labeled solutions and unlabeled controls with the same N form for both species (Figure 5A and B). In P. edulis seedlings, N concentration of plant fractions and total seedlings markedly increased with an increased proportion of NH4+ to NO3−, and N concentration in leaves was higher than in stem and root under the same treatment especially with NH4+ or mixed N (NH4NO3) (Figure 5A). Moreover, variation in whole-plant N concentration was attributed to changes in N concentration in aboveground (stem and foliage), which was different among N forms, rather than root N concentration. For C. fargesii, seedlings grown with mixed N or NH4+ showed higher N concentration than those NO3− treated seedlings (Figure 5B). In addition, N concentration in leaves was similar to that of root but larger than stem under the same treatment. Moreover, variation in plant fractions N concentration followed the same trend as that described at the plant level. Figure 5. Open in new tabDownload slide Nitrogen content and δ15N in root, stem, and leaf of P. edulis and C. fargesii under different NH4+/NO3− ratios of 8 mM N. (A and B) Nitrogen content in whole plant and root, stem and leaf of P. edulis and C. fargesii, whole plant N concentration was calculated as the weighted average of N concentration in leaves, stems and roots taking into account the mass of the three compartments. (C and D) The δ15N signatures in plant roots, stem and leaves for species from P. edulis and C. fargesii. Data are means from three replicates of 36 seedlings, and bars represent SE. The different lowercase letters indicate significant differences in the means among treatments for either root, stem or foliar samples for each species using one-way ANOVA and Fisher’s LSD (P < 0.05). Figure 5. Open in new tabDownload slide Nitrogen content and δ15N in root, stem, and leaf of P. edulis and C. fargesii under different NH4+/NO3− ratios of 8 mM N. (A and B) Nitrogen content in whole plant and root, stem and leaf of P. edulis and C. fargesii, whole plant N concentration was calculated as the weighted average of N concentration in leaves, stems and roots taking into account the mass of the three compartments. (C and D) The δ15N signatures in plant roots, stem and leaves for species from P. edulis and C. fargesii. Data are means from three replicates of 36 seedlings, and bars represent SE. The different lowercase letters indicate significant differences in the means among treatments for either root, stem or foliar samples for each species using one-way ANOVA and Fisher’s LSD (P < 0.05). For both species, δ15N was higher in 15NH4+-treated seedlings than in 15NO3−-treated seedlings when supplied as either sole N-sources or in co-provision with 15NH4NO3 and NH415NO3 (Figure 5C and D). δ15N of 15NH4+-treated P. edulis seedlings was 1.31-, 1.37- and 1.91-fold of 15NO3−-treated seedlings in, respectively, root, stem and leaf tissue when different N-sources were provided with (15NH4)2SO4 and Na15NO3, compared with a 2.18-, 1.65- and 2.22-fold in root, stem and leaf tissue when N co-provision as 15NH4NO3 and NH415NO3 was imposed (Figure 5C). For C. fargesii, δ15N of 15NH4+-treated seedlings was 1.19-, 1.50- and 1.58-fold in roots, stem and leaf tissue of that of 15NO3−-treated seedlings with singular N sources, whereas δ15N in roots, stem, and leaf tissue was 5.22-, 3.45- and 5.35-fold of that with 15NO3− when NH4+ and NO3− were provided together (Figure 5D). By analyzing δ15N between (15NH4)2SO4 and 15NH4NO3, NH415NO3 and Na15NO3, it emerged that the increased differences in δ15N were brought about not only by increased 15NH4+ uptake in the presence of NO3− but also by decreased uptake of 15NO3−, and 15NO3− uptake in the presence of NH4+ was reduced when compared with the single-N nutrient solutions in both species (Figure 5C and D). NH4+ tolerance Seedling growth differences began being evident after the 28th day of treatment (Figure 6). For P. edulis, differences in height growth among NH4+ treatments were visible from the 42nd day and accentuated with time, and, on the 56th day, the seedlings treated with 16–40 mM NH4+ were significantly larger than the seedlings treated with 2 mM NH4+ (Figure 6A). By contrast, differences in height growth of C. fargesii seedlings among NH4+ treatments were also accentuated with time, but there were no significant differences during the whole experiments (Figure 6B). For both species, the growth of aboveground parts such as leaf number, biomass, and SPAD value were increased with rising NH4+ concentrations (Figure 7). Figure 6. Open in new tabDownload slide Effects of NH4+ concentrations on height increment of P. edulis and C. fargesii along with time course. Seedlings were grown with supply of different concentrations of NH4+. Heights are expressed relative to initial size. Data are means from three replicates of 24 seedlings, and bars represent the SE. Asterisk indicates statistical differences compared with the 0 day, different letters indicate statistical differences among treatments at the same time, and NS indicates no significant differences (Fisher’s LSD, P < 0.05). Figure 6. Open in new tabDownload slide Effects of NH4+ concentrations on height increment of P. edulis and C. fargesii along with time course. Seedlings were grown with supply of different concentrations of NH4+. Heights are expressed relative to initial size. Data are means from three replicates of 24 seedlings, and bars represent the SE. Asterisk indicates statistical differences compared with the 0 day, different letters indicate statistical differences among treatments at the same time, and NS indicates no significant differences (Fisher’s LSD, P < 0.05). Figure 7. Open in new tabDownload slide Growth, biomass and chlorophyll content of aboveground parts in P. edulis and C. fargesii seedlings supplied with different concentrations of NH4+ (mM). Data are means from three replicates of 24 seedlings at the end of experiment, and bars represent SE. Different letters indicate statistical differences among treatments for each species (Fisher’s LSD, P < 0.05). Figure 7. Open in new tabDownload slide Growth, biomass and chlorophyll content of aboveground parts in P. edulis and C. fargesii seedlings supplied with different concentrations of NH4+ (mM). Data are means from three replicates of 24 seedlings at the end of experiment, and bars represent SE. Different letters indicate statistical differences among treatments for each species (Fisher’s LSD, P < 0.05). Although aboveground growth in both species proceeded well at soil NH4+ concentrations from 2 to 40 mM, root growth suffered NH4+ toxicity symptoms in both P. edulis and C. fargesii with increasing NH4+ levels. Total RL, RS area and RV were significantly inhibited in P. edulis at NH4+ concentrations >24 mM compared with seedlings treated with 2–16 mM NH4+. By contrast, in C. fargesii, these root morphology indices were significantly inhibited at NH4+ concentrations >16 mM compared with seedlings treated at 2 mM NH4+ (Figure 8). Figure 8. Open in new tabDownload slide Root biomass and morphology of P. edulis and C. fargesii seedlings supplied with different concentrations of NH4+ (mM). Data are means from three replicates of 24 seedlings at the end of the experiment, and bars represent the SE. Different letters indicate statistical differences among treatments for each species (Fisher’s LSD, P < 0.05). Figure 8. Open in new tabDownload slide Root biomass and morphology of P. edulis and C. fargesii seedlings supplied with different concentrations of NH4+ (mM). Data are means from three replicates of 24 seedlings at the end of the experiment, and bars represent the SE. Different letters indicate statistical differences among treatments for each species (Fisher’s LSD, P < 0.05). The results show different survival rates in the two species at increased NH4+ concentrations (Figure 9). In P. edulis, the survival rate of all NH4+-treated seedlings was 100%, whereas, in C. fargesii, the survival rate sharply decreased with increasing NH4+, and the survival rate of 16–40 mM NH4+ treated seedlings was significantly lower than that treated with 2 mM NH4+. Figure 9. Open in new tabDownload slide Effects of NH4+ concentrations on survival rate of P. edulis and C. fargesii. Seedlings were grown with different concentrations of NH4+, survival rates were surveyed at the end of experiments. Data are means from three replicates of 24 seedlings, and bars represent the SE. Different letters indicate statistical differences among treatments (Fisher’s LSD, P < 0.05). Figure 9. Open in new tabDownload slide Effects of NH4+ concentrations on survival rate of P. edulis and C. fargesii. Seedlings were grown with different concentrations of NH4+, survival rates were surveyed at the end of experiments. Data are means from three replicates of 24 seedlings, and bars represent the SE. Different letters indicate statistical differences among treatments (Fisher’s LSD, P < 0.05). Interspecific competition As both species demonstrated a similar preference for NH4+, it was interesting to examine the niche complementarity of these species. Interspecific competition between P. edulis and C. fargesii under NH4+-rich conditions was detected by comparing results from monoculture and mixed culture experiments, as shown in Figure 10A and B. In P. edulis, the seedlings showed very similar height growth between the monoculture and mixed culture experiments (Figure 10C): during September 2017–February 2018, the growth rate was relatively flat, then growth suddenly accelerated, in March 2018; after that, the growth rate decreased, a suppression that lasted for the rest of experiment. In C. fargesii, height growth was recorded from April 2018, accelerated, and lasted from May 2018 to August 2018 in the monoculture experiment, but growth was clearly inhibited in the mixed culture for the whole experiment (Figure 10C). Moreover, P. edulis displayed similar tillers, biomass and ratio of root to shoot between the monoculture and mixed culture experiments; however, biomass and branches of C. fargesii were significantly inhibited, whereas the ratio of root/shoot was higher in the mixed culture trial compared with monoculture (Figure 10D–F). Figure 10. Open in new tabDownload slide Growth and response variables for seedlings of P. edulis and C. fargesii grown in monoculture and mixed culture experiments supplied with 8 mM NH4+. (A) Layout of the monoculture and mixed culture experiment with spatial distribution of seedlings, (B) seedlings of C. fargesii and P. edulis in monoculture and mixed culture, (C) height increment of P. edulis and C. fargesii seedlings in response to different plantation systems, (D) tiller and branches, (E) biomass and (F) ratios of root/shoot in P. edulis and C. fargesii seedlings at the end of the experiment. Data are means from three replicates of 12 to 36 seedlings, and bars represent the SE. Different letters indicate statistical differences among treatments and species (Fisher’s LSD, P < 0.05). Figure 10. Open in new tabDownload slide Growth and response variables for seedlings of P. edulis and C. fargesii grown in monoculture and mixed culture experiments supplied with 8 mM NH4+. (A) Layout of the monoculture and mixed culture experiment with spatial distribution of seedlings, (B) seedlings of C. fargesii and P. edulis in monoculture and mixed culture, (C) height increment of P. edulis and C. fargesii seedlings in response to different plantation systems, (D) tiller and branches, (E) biomass and (F) ratios of root/shoot in P. edulis and C. fargesii seedlings at the end of the experiment. Data are means from three replicates of 12 to 36 seedlings, and bars represent the SE. Different letters indicate statistical differences among treatments and species (Fisher’s LSD, P < 0.05). Discussion Both P. edulis and C. fargesii display NH4+ preference Our data support the view that substrate N concentration affects plant preference for N form (Britto and Kronzucker 2006), whereas the effects of N form were negligible at low concentrations for both species (Figures 1–3). Similar results were reported by Uscola et al. (2014) in two ecologically distinct Mediterranean forest trees that were fertilized with 1 mM N, and by Warren and Adams (2002), who found negligible growth differences between N forms in seedlings of Pinus pinaster, fertilized with 0.125 and 0.5 mM N. The insignificant effects of N form at low concentrations is probably the consequence of plant depletion of supplied N (Uscola et al. 2014) and the fact that low N availability produces similar deficiency in seedlings at 0.1 mM regardless of N form, limiting growth in a similar fashion (Nacry et al. 2013). This is supported at least in part by the observation that increasing N concentrations in the nutrient solutions, especially in the case of NH4+, significantly accelerated growth in both species (Figures 1–3, Table 2). At increased N concentrations such as 8 mM, the growth of both species displayed an NH4+ preference, but species had distinct performance to altered N availability: P. edulis responded very strongly to different ratios of NH4+/NO3−, and an increase in the proportion of NH4+, even when NH4+ was the only N source, significantly promoted growth, biomass, leaf chlorophyll and N concentration compared with seedlings on NO3− (Figures 1–3 and 5). For example, seedlings treated with NH4+ displayed an increase of 61.47 and 449%, respectively, in biomass compared with seedlings with NH4+/NO3−(2/2) and NO3− at 8 mM N (Figure 1G), similar to the species classified as ‘ammonium specialists’, such as late-successional Northern conifers (Kronzucker et al. 1997, Britto and Kronzucker 2006). By contrast, different ratios of NH4+/NO3− had minor effects in C. fargesii, although biomass production (Figure 1H), leaf chlorophyll content (Figure 3B) and N concentration (Figure 5B) were higher with NH4+ when supplied at 8 mM. For example, the biomass of 8 mM NH4+ treated seedlings was increased only 14.73 and 23.33%, respectively, compared with NH4+/NO3−(2/2) and NO3− treated seedlings at the same N concentration (Figure 1H). Although it was significantly larger than at 2 mM N (0/4, NO3− alone), the growth of P. edulis at 2 mM N (4/0, NH4+ alone) was inhibited compared with growth at 2 mM N (3/1, NH4+/NO3−) (Figures 1 and 2). We attributed the enhanced response to mixed N at 1.5 mM NH4+ + 0.5 mM NO3− to a stimulatory effect of NO3−, for which there is much literature precedent (Kronzucker et al. 1999b, and references therein), whereas, at 2 mM of sole NH4+ supply, growth appears to still be suboptimal in P. edulis (Figures 1–3 and 6–8). Although there appear to not be any stimulatory effects by NO3− at concentrations of NO3− <0.4 mM (Gu et al. 2016; Figures 1–3), or >2 mM, as seen when analyzing the treatments ranging from 2 mM N (0/4) and 8 mM N (0/4), growth was promoted with increased NH4+ concentrations. It is not unusual for NH4+/NO3− ratios to have optimal response points that differ with overall N concentration (Kronzucker et al. 1999b, Zhang et al. 2019). Ueda (1960) has reported that the application of ammonium sulfate strongly increased the production of new culms in the first year after fertilization in the groves of P. edulis and P. reticulata. Therefore, the data support a more generic NH4+ preference in bamboo. A higher growth response to NH4+ than NO3− both in P. edulis and C. fargesii might be associated with higher nutrient activation and increased absorption associated with NH4+ uptake than NO3− because of increased H+ extrusion and altered ion balance (van Beusichem et al. 1988, Britto and Kronzucker 2002); this could include phosphate, which is limiting for the growth of forest plants and net primary productivity both in bamboo and broad-leaved forest ecosystems (Yan et al. 2008, Du et al. 2016). In addition, similar to the report by Norisada and Kojima (2005), superior performance with NH4+ treatment in both species might also be associated with the higher photosynthetic capacity of a larger overall photosynthetic apparatus (leaf number, leaf area), higher leaf chlorophyll and higher leaf N concentration of NH4+-treated seedlings (Figures 1, 3 and 5). Furthermore, the size and architecture of the root system are significant factors in determining the ability of a plant to interception and acquisition of N (Nacry et al. 2013). In our experiments, increased NH4+/NO3− at higher N concentrations significantly promoted root growth in P. edulis seedlings as seen in root biomass and root system architecture (Figures 1G and 2A, C, E and G, Table 2), and seedlings achieved higher aboveground growth and biomass accumulation under such treatments (Figure 1A, C, E and G). However, the architecture of the straight root of C. fargesii was less affected by N forms (Figure 2B, D, F and H, Table 2), compared with the fibrous adventitious root of P. edulis. Nitrogen deposition in China has been dramatically enhanced by anthropogenic emissions. In Jiangxi red soil forestland, one typical site of moso bamboo and broad-leaved forest, the total inorganic N deposition was 83.7 kg ha−1 year−1 in 2004 and 81.3 kg ha−1 year−1 in 2005, with reduced N compounds accounting for 78.3% of total N deposition (Fan et al. 2009). The increased deposition of atmospheric NH3/NH4+ would lead to the accumulation of excess NH4+ in forest soil. Nitrogen concentrations of NH4+ and NO3− in soil solutions >2.0 mM have been reported, [NH4+] in forest-floor soil solutions can range from 0.4 to 4 mM (Britto and Kronzucker 2006), and, in some soils, NH4+ concentrations of ~40 mM are achieved (Britto and Kronzucker 2002), whereas [NO3−] may range 1000-fold over a distance of centimeters and over the course of hours (Bloom 2015). Furthermore, N availability is highly heterogeneous both spatially and temporally, and varies greatly among sites and with the seasons (Britto and Kronzucker 2006, Wang et al. 2007, Song et al. 2016, Y. Li et al. 2017, Z. Li et al. 2017). Therefore, such N concentrations are likely on some soils where P. edulis and C. fargesii are found, in subtropic forest ecosystems. Increased NH4+ would benefit both C. fargesii and P. edulis given their N-source preference. Due to the advantages of P. edulis in interspecific competition and the higher NH4+-tolerance threshold compared with C. fargesii, increased soil ammonification accompanying bamboo invasion coupled with NH4+-dominated N deposition can be inferred as an important driving force for the bamboo expansion success in subtropical China. Due to NH4+ preference, NH4+-N should also be the main form of fertilizer in the seedling cultivation of the two species. Both species display NH4+ preference but P. edulis has higher NH4+ net fluxes and N concentration than C. fargesii By using NMT, we observed that the spatial variability and net influxes of NH4+ and NO3− were largest at 0.3 and 1 mm, respectively, from the root tips of P. edulis and C. fargesii (Figure 4), and distinct net fluxes of NH4+ and NO3− in different zones have also been seen in other woody plants (Luo et al. 2013). The apical region of the root is characterized by the root cap, the meristematic region, the cell elongation zone and the maturation zone (Li et al. 2010), and the net fluxes of NH4+ and NO3− can reflect results of N assimilation and uptake kinetics of these ions into root cells (Britto and Kronzucker 2006, Luo et al. 2013). So, our results suggest that the root tip and the meristematic and cell elongation zones (0.3 ~ 1 mm) are more important than the maturation zone for uptake of NH4+ and NO3−, possibly because of cytosolic concentrations of NH4+ and NO3− in those zones falling below the thresholds needed for N assimilation to support the fast-growing in the species. Although effects by root-cutting on ions fluxes cannot be discounted, the more efficient N capture with NH4+ relative to NO3− in both species and higher maximal net NH4+ fluxes in P. edulis than C. fargesii displayed by NMT were also consistent with and supported by δ15N experiments and N concentration in both plants (Figure 4). Both bamboo-dominated forests and C. fargesi-dominated secondary evergreen broad-leaved forests contained more NH4+ than NO3− (Song et al. 2016); therefore, NH4+-uptake preference appears to reflect adaptation to the most abundant N form in the soil habitat. The present study shows an inhibition of NO3− uptake by NH4+, as is classically observed (Kronzucker et al. 1999a, Luo et al. 2013), and a promotion of NH4+ uptake by NO3− (Figure 5C and D), similar to what has been reported for the NH4+ specialist rice (Kronzucker et al. 1999b) and poplar trees (Luo et al. 2013). Possibly, the enhancement of net NH4+ uptake by the co-presence of NO3− overcomes the suppressive effects of NH4+ on NO3− uptake in C. fargesii but not in P. edulis, resulting in improved N acquisition and accumulation in C. fargesii under mixed N, as compared with provision with NO3− or NH4+ alone (Figure 5A and B). Therefore, the balance in soil N mineralization between ammonification and nitrification is shifted toward ammonification in bamboo-dominated forests after the expansion of bamboo (Song et al. 2013, 2016), which would be a disadvantage for N capture by C. fargesii rather than P. edulis, taking into consideration the interactions between the inorganic N ions. NH4+ toxicity differs between the species, and P. edulis has superior NH4+ tolerance than C. fargesii at elevated soil NH4+ Despite the fact that NH4+ is the preferred N source for many plants, toxicity symptoms, such as a stunted root system and leaf chlorosis, emerge readily in most species, even those frequently labeled as ‘NH4+ specialists’ at increased levels of NH4+ supply (Britto and Kronzucker 2006, Li et al. 2014). In our study, NH4+ at higher concentrations also caused toxicity in both species, but P. edulis was distinctly more NH4+-tolerant than C. fargesii: NH4+ toxicity thresholds on belowground were higher in P. edulis (≧24 mM) than in C. fargesii (≧16 mM) (Figure 8); in addition, C. fargesii was more susceptible, and survival rate significantly decreased with increased NH4+ concentrations compared with that of P. edulis (Figure 9). Therefore, results support our second hypothesis that P. edulis had stronger NH4+ tolerance than C. fargesii. Recent studies have demonstrated that shoot-supplied NH4+ and root-supplied NH4+ sources suppress plant growth differentially by targeting different tissues (Li et al. 2014). Specifically, root-supplied NH4+ principally targets root system development, and such responses seemed to be controlled by independent genes and pathways that are enriched in root tips (Li et al. 2010, 2011, 2013). In agreement with this view, in our root-supplied NH4+ experimental system, although the root system was suppressed under increased NH4+ concentrations (Figure 8), aboveground variables such as leaf number and leaf chlorophyll content were not inhibited but even increased, in both species (Figure 7). Phyllostachys edulis has a significant competitive growth advantage compared with C. fargesii under co-existence conditions in an NH4+-rich environment The similarity in NH4+ preference implies the absence of fundamental niche complementarity for N uptake, whereas a consistency in the growth of P. edulis between monoculture and mixed culture systems suggests negligible effects of interspecific competition on P. edulis’ overall performance (Figure 10B–F). On the other hand, C. fargesii exhibited inhibited height growth, branching number and biomass in mixed culture systems compared with monoculture (Figure 10B–F). Therefore, P. edulis had higher competitiveness compared with C. fargesii in the NH4+-rich environment, which supported our third hypothesis. Fast-growth properties of P. edulis compared with C. fargesii are a general characteristic of the species under suitable growth conditions. In P. edulis, strong height growth occurred in February and March (Figure 10C), similar to the observed annual growth dynamic of aboveground culms (Kleinhenz and Midmore 2001). However, in C. fargesii, maximum growth occurred from May to July (Figure 10C), largely consistent with the reports of Hou (2015), who showed that C. fargesii seedlings grew rapidly from April to September during the growth season (Hou 2015, Song et al. 2016). It has been reported that changes in plant height have important consequences for capturing light resources on undergrowth plants (Stevens and Carson 1999). In addition, when light intensity changes, soil humidity also changes. Therefore, in addition to soil nutrient competition, accompanying environmental factors, such as low light intensity and high soil moisture, in the presence of competing and shading plant canopies may also contribute to the low competitiveness of C. fargesii in mixed culture with P. edulis. Particularly, we have to mention that the seedlings we used to test the influences of N availability on plant growth are not entirely representative of that from adult bamboo and trees in natural conditions. In natural settings, P. edulis’ active rhizomes, and their vigorous lateral expansion, further contribute to rapid invasion into neighboring forests. In addition, young shoots developed from buds on rhizomes quickly reach full heights of 10–20 m, matching those of well-developed canopy trees, are connected with mother plant (Fukushima et al. 2015), competing with trees both aboveground and belowground. Furthermore, N, phosphorus and carbohydrates can readily translocate within clonal fragments inform mother bamboo (Li et al. 2000, Saitoh et al. 2006, Wang et al. 2016, Sun et al. 2019), not only improving the adaptability of bamboo to adverse environments, but also facilitating group competitiveness compared with trees. In conclusion, this study demonstrates species-specific strategies and the differential capacity of P. edulis and C. fargesii to altered soil N availabilities. The growth of both species shows a clear NH4+ preference at higher N concentrations, as verified both by the NMT approach and by examining 15N signatures. However, the responses of growth and root system architecture were much stronger in P. edulis than those of C. fargesii when confronted with increased proportions of NH4+/NO3− and with increased NH4+ levels. An increase in the ratio of NH4+/NO3− significantly increased N concentration in P. edulis but not in C. fargesii, as compared with the NH4NO3 treated seedlings, at 8 mM N. Phyllostachys edulis demonstrated a significant competitive growth advantage compared with C. fargesii on NH4+-rich substrates. In addition, P. edulis was more NH4+-tolerant than C. fargesii. Therefore, it can be inferred that, in broad-leaved forests, soil N is converted from a mixture of NH4+ and NO3− to mostly NH4+ in the process of bamboo invasion, and the previously dominant tree species are unable to compete with the ‘NH4+ specialist’ bamboo, which, intern becomes dominant. The differential toxicity thresholds for NH4+ in P. edulis (24 mM) and C. fargesii (16 mM) may play a role in community structure succession, as the latter species is more susceptible to NH4+ toxicity and its survival rate sharply decreases with increasing NH4+. Our experiment also provides important insights for the nursery cultivation of these species. Acknowledgments We are grateful to Professor Xianquan Weng (Xiayang State Forest Farm of Nanping of Fujian Province) for the kind provision of C. fargesii seeds. 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TI - Superior growth, N uptake and NH4+ tolerance in the giant bamboo Phyllostachys edulis over the broad-leaved tree Castanopsis fargesii at elevated NH4+ may underlie community succession and favor the expansion of bamboo
JO - Tree Physiology
DO - 10.1093/treephys/tpaa086
DA - 2020-10-29
UR - https://www.deepdyve.com/lp/oxford-university-press/superior-growth-n-uptake-and-nh4-tolerance-in-the-giant-bamboo-y2ghPYrHuM
SP - 1606
EP - 1622
VL - 40
IS - 11
DP - DeepDyve
ER -