N deposition affects allelopathic potential of Amaranthus retroflexus with different distribution regions

This study aims to determine the allelopathic potential of Amaranthus retroflexus (Ar) with different climatic zones on seed germination and growth of A. tricolor (At) treated with a gradient N addition. Ar leaf extracts only displayed significantly allelopathic potential on the underground growth of Ar but not the aboveground growth of At. The allelopathic potential of Ar leaf extracts on root length of At were enhanced under N addition and there may be a N-concentration-dependent relationship. The effects of the extracts of Ar leaves that collected from Zhenjiang on seed germination and growth of At may be higher than that collected from Jinan especially on root length of At under medium N addition. This reason may be the contained higher concentration of secondary metabolites for the leaves of plants that growths in high latitudes compare with that growth in low latitudes. This phenomenon may also partly be attributed to the fact that Ar originated in America and/or south-eastern Asia which have higher similarity climate conditions as Zhenjiang rather than Jinan. The allelopathic potential of Ar on seed germination and growth of acceptor species may play an important role in its successful invasion especially in the distribution region with low latitudes.


INTRODUCTION
At present, biological invasion has triggered notable changes on the structure and functioning of native ecosystems (Gurevitch et al. 2011, Powell et al. 2013).Numerous studies have suggested that some plants can invade certain environments successfully via the released toxic compounds, a mechanism referred to as allelopathy (Callaway andRidenour 2004, Yuan et al. 2013).Allelopathics compounds can pose a remarkably allelopathic effect on seed germination and growth of native species (Mlakar et al. 2012, Yuan et al. 2013, Wang et al. 2016a).Meanwhile, seed germination and growth are highly important for plant recruitment and resource competition (Turk and Tawaha 2003).Thus, the inhibition of seed germination and growth of native species mediated by the allelopathic effects of invasive species may obviously decrease the competitiveness of native species.
Currently, one of the most significant global environmental problems has been an increase in atmospheric nitrogen (N) deposition (Galloway et al. 2004(Galloway et al. , 2008)).Meanwhile, the rapidly increased deposition of atmospheric N is expected to continue in the coming decades (Galloway et al. 2004, 2008, Liu et al. 2013).East Asia (mainly China) is currently one of the world's three largest areas of N deposition (Galloway et al. 2004, Wang et al. 2007, Liu et al. 2013).However, different regions in China may suffer different levels of atmospheric N deposition (Wang et al. 2007, Zhan et al. 2014).As the world's third largest country, meanwhile, China covers five climatic zones: cold-temperate, temperate, warm-temperate, subtropical, and tropical (Weber et al. 2008, Li et al. 2009).A wide range of habitats and environmental conditions allow China obviously vulnerable to the successful plantation of the invasive species that originating from diverse regions due to the potential invasive species from most areas of the world might searched out the suitable habitat somewhere in China eventually (Weber et al. 2008, Yan et al. 2014).Thus, the invasion of many invasive plants may cross multiple distribution regions (Yan et al. 2014).To illustrate, the notorious invasive plant Amaranthus retroflexus L. has been distributed in many regions of China (Weber et al. 2008, Yan et al. 2014).Thus, understanding the allelopathic potential of A. retroflexus L. with different distribution regions on seed germination and growth of native species is important to elucidate the mechanism underlying its successful invasion especially under the condition with increasing amounts of anthropogenic N being deposited into ecosystems gradually.
The present study aims to determine the allelopathic potential of the invasive plant A. retroflexus with different distribution regions that belong to different climatic zones on seed germination and growth of acceptor species A. tricolor in the presence of a gradient of simulated N deposition (control, 0 g m -2 yr -1 ; low N, 2.5 g m -2 yr -1 ; medium N, 5 g m -2 yr -1 ; and high N, 10 g m -2 yr -1 ).Both are members of the Amaranthus genus.A. retroflexus is a summer annual weed (Horak and Loughlin 2000) native to America (Mandák et al. 2011, Yang et al. 2011) and/or south-eastern Asia (Yang et al. 2011).At present, A. retroflexus is a common weed that grows vigorously in orchards, roadside verges, fields, woods, and scrubland in China and has been listed as a destructive and widespread invasive species in China (Yang et al. 2011).Allelopathy is invoked to explain the successful invasion of A. retroflexus (Mlakar et al. 2012, Konstantinović et al. 2014).
This study addressed the following hypotheses: (1) A. retroflexus exhibits allelopathic potential on seed germination and growth of A. tricolor; (2) the allelopathic potential of A. retroflexus on seed germination and growth of A. tricolor will be changed under exogenous N addition; and (3) there may be significant difference in the allelopathic potential of A. retroflexus between different distribution regions that belong to different climatic zones on seed germination and growth of A. tricolor.Approximately 10 g of air-dried leaves of A. retroflexus were placed in flasks containing 500 mL of distilled water and soaked for 48 h at room temperature.A. retroflexus leaf extracts were strained using a cheese cloth and then through two layers of filter paper to remove solid particles.The stock solution was stored at 4°C for further study.The concentrations of A. retroflexus leaf extracts from Jinan and Zhenjiang (abbreviated as JN and ZJ hereafter, respectively) were both set at 20 g L -1 with distilled water as control (CK, 0 g L -1 ).

EXPERIMENTAL
A mixed N solution was prepared with a 1:1:1 ratio of KNO 3 -N : NH 4 Cl-N : urea-N.The ratio of different N forms in the mixed N was similar to the global average ratio of natural atmospheric N deposition (Neff et al. 2002, Cornell et al. 2003).The concentrations were made with distilled water prior to use to create a series with gradient contents, namely, control 0 g m -2 yr -1 (CK, fertilized with sterile distilled water only), low N, 2.5 g m -2 yr -1 (N-2.5);medium N, 5 g m -2 yr -1 (N-5); and high N, 10 g m -2 yr -1 (N-10).N-5 corresponded to levels of N deposition already recorded in the Yangtze River Delta region of China (Wang et al. 2007).N-10 represented a high deposition level that may be reached in the future.
The experiment included twelve treatments, each consisting of a combination of N form and aqueous extract regime.five replicates were performed per treatment.

SEED GERMINATION EXPERIMENT
Experiments were performed through incubation in Petri dishes (Wang et al. 2016a).The A. tricolor seeds were surface sterilized (1% NaClO for approximately 15 min) and then thoroughly washed thrice with deionized water.Thirty healthy seeds of A. tricolor were placed in a 9 cm Petri dish and covered with two layers of filter paper.The seeds were treated with deionized water, N solutions, and/or A.retroflexus leaf extracts.The Petri dishes were placed in a climate-controlled incubator at 25 °C for 7 d with 12 h light per day (light intensity was set at 27.5 µmol • m -2 • s -1 ).The number of germinated seeds was counted every day at incubation time, and each seed was considered to have germinated when its radicle protruded (Wang et al. 2016a).

DATA MEASUREMENTS
Ten seedlings per Petri dish were randomly selected for growth measurement on the same day.The plant height (PH), root length (RL), seedling biomass (BM, fresh weight), germination rate (GR), germination potential (GP), germination index (GI), germination rate index (GRI) and vigor index (VI) of A. tricolor were determined.PH and RL were measured using a ruler (Wang et al. 2016a).BM was determined using an electronic balance with an accuracy of 0.001 g (Wang et al. 2016a).GR was calculated using the ratio of the final number of germinated seeds to the total number of seeds when no new germination occurred after 7 d of incubation (Wang et al. 2016a).GP was determined by dividing the number of germinated seeds on the third day by the total number of seeds (Wang et al. 2016a).GI was calculated using the following GRI was calculated using the following equation: GRI = GR × GI (Steinmaus et al. 2000).VI was determined using the following equation: VI = BM × GI (Lin et al. 2000).

STATISTICAL ANALYSES
Differences among various dependent variables were assessed using analysis of variance between groups followed by a multiple comparisons correction using the Student-Newman-Keuls test.
All statistical analyses were performed using SPSS Statistics (version 22.0; IBM, Armonk, NY, USA).
Statistical significance was set at P values equal to or lower than 0.05.

ALLELOPATHIC POTENTIAL Of A. RETROFLEXUS LEAf EXTRACTS ON SEED GERMINATION AND GROWTH Of A. TRIcOLOR
A. retroflexus leaf extracts exerted significant effects on seed germination and growth of A. tricolor (Table I).In particular, the PH, BM, and VI of A. tricolor under JN and ZJ were significantly higher than those of A. tricolor under CK (Table I, P < 0.05).While, the RL of A. tricolor under JN and ZJ were significantly lower than those of A. tricolor under CK (Table I, P < 0.05).Both JN and ZJ did not significantly affect the GR, GP, GI, and GRI of A. tricolor (Table I, P > 0.05).There was no significant difference in seed germination and growth of A. tricolor between JN and ZJ (Table I, P > 0.05).

EffECTS Of N ADDITION ON SEED GERMINATION AND GROWTH Of A. TRIcOLOR
N addition remarkably affected seed germination and growth of A. tricolor (Table I).In particular, the PH, BM, and VI of A. tricolor under N addition were significantly higher than those of A. tricolor under CK (Table I, P < 0.05).By contrast, the RL of A.tricolor decreased markedly with increasing concentrations of N addition (Table I, P < 0.05).
There was no significant difference in the GR, GP, GI, and GRI of A. tricolor between N addition and CK (Table I The allelopathic potential of A. retroflexus leaf extracts on A. tricolor could be changed in the presence of different levels of N addition (Table I).In particular, RL of A. tricolor exposed to both of JN and ZJ with N addition, regardless of concentration, were significantly lower than those of A. tricolor exposed only to A. retroflexus leaf extracts (Table I, P < 0.05).Meanwhile, the RL of A. tricolor under both JN and ZJ with N addition decreased significantly with increasing concentrations of N fertilizers (Table I, P < 0.05).Moreover, the PH of A. tricolor under N-5*ZJ and N-10*ZJ was significantly higher than that of A. tricolor exposed only to the extracts of A. retroflexus leaves that collected from Zhenjiang (Table I, P < 0.05).In addition, under the addition of equal N concentration, the PH of A. tricolor under ZJ with was significantly higher than that under JN (Table I, P < 0.05).furthermore, under the addition of medium N, the RL of A. tricolor under ZJ with was also significantly higher than that under JN (Table I, P < 0.05).
N plays an important role in the growth of plants, and enhanced nutrient levels in plant communities lead to increased plant productivity (Jin et al. 2016, Wang et al. 2016b).Meanwhile, exogenous N addition could stimulate seed germination and growth of plant species (Qiu and Zhang 2008).The results of this study showed that the increased levels of exogenous N addition obviously stimulated seed germination and growth of A. tricolor, especially the plant height, seedling biomass, and vigor index of A. tricolor significantly increased under N addition.The result was consistent with previous studies (Qiu and Zhang 2008).The enhanced seedling growth under exogenous N addition may be due to the elevated levels of soil available N (Bai et al. 2010, Zhang et al. 2013).Meanwhile, the root length of A. tricolor decreased significantly with increasing concentrations of exogenous N addition.This implied that exogenous N addition could inhibit the underground growth of A. tricolor.Thus, exogenous N addition promoted aboveground growth of A. tricolor but decreased its underground growth and thereby changed its biomass allocation pattern.The result was consistent with previous studies (Janssens et al. 2010, Wang et al. 2015).
The allelopathic potential of invasive species on seed germination and growth of native species may shift and change under the conditions with increased levels of N deposition.The results of this study showed that there may be a synergistic effect between exogenous N addition and A. retroflexus leaf extracts on the root length of A.tricolor, especially the root length of A. tricolor.Thus, the allelopathic potential of A. retroflexus leaf extracts on seed germination and growth of A. tricolor were enhanced under exogenous N addition and there may be a N-concentration-dependent relationship.However, the combined treatment of the extracts of A. retroflexus leaves that collected from Zhenjiang and low as well as medium N treatment showed significant positive effects on the plant height of A. tricolor.The reason may be due the fact that exogenous N addition resulted in a shift in biomass allocation from underground to aboveground (Janssens et al. 2010, Wang et al. 2015).Consequently, the result was consistent with the study's second hypothesis.Meanwhile, the effects of the extracts of A. retroflexus leaves that collected from Zhenjiang on seed germination and growth of A. tricolor may be higher than that collected from Jinan especially on root length of A. tricolor under medium N addition.The reason could be a result of the contained higher concentration of secondary metabolites for the leaves of plants that growths in high latitudes compare with that growth in low latitudes (Wright et al. 2004). Gatti et al. (2014) also found that climate conditions can affect plant secondary metabolism (such as precipitation and temperature) and then alter the allelopathic activity of plant species via alter the production of allelochemicals.The result may also be attributed in part to the fact that A. retroflexus originated in America (Mandák et al. 2011, Yang et al. 2011) and/ or south-eastern Asia (Yang et al. 2011) which have higher similarity climate conditions as Zhenjiang rather than Jinan.The result was consistent with the study's third hypothesis.Thus, the allelopathic potential of A. retroflexus on seed germination and growth of native species may play an important role in its successful invasion especially in the distribution region with low latitudes.
922CONGYAN WANG et al. equation: GI = ∑G i / I, where G i represents the number of germinated seeds, and I represents the time after cultivation (day) (Schmer et al. 2012).
The collected leaves of A. retroflexus were washed, air dried at room temperature for 48 h, and then stored at 4°C for further study.A. tricolor seeds were chosen as the acceptor seeds and bought from a local vegetable market.
of A. tricolor under CK (TableI, P < 0.05).Whereas, the RL of A. tricolor under A. retroflexus leaf extracts and N addition, regardless of concentration, was significantly lower than that of A. tricolor under CK (TableI, P < 0.05).The GI of A. tricolor under N-10*JN was also significantly lower than that of A. tricolor under CK (TableI, P < 0.05) but not the rest of the combined treatment of A. retroflexus leaf extracts and N addition (TableI, P > 0.05).The combined treatment of A. retroflexus leaf extracts and N addition, regardless of concentration, did not pose significant effects on the GR, GP, and GRI of A. tricolor compared with CK (TableI, P > 0.05).

TABLE I Differences in the indices of A. tricolor with different treatments.
CONGYAN WANG et al.