Soil microbial carbon utilization, enzyme activities and nutrient availability responses to Bidens pilosa and a non-invasive congener under different irradiances

Two Bidens species (Bidens pilosa and B. bipinnata) that originate from America have been introduced widely in pan-tropics, with the former regarded as a noxious invasive weed whereas the latter naturalized as a plant resource. Whether the two species exhibit different effects on the belowground system remains rarely studied. This study was conducted to investigate soil microbial carbon (C) utilization, enzyme activities and available nitrogen, phosphorus and potassium contents under the two species in a subtropical garden soil of southern China under different levels of light intensity. Results showed that the microbial C utilization and enzyme activities were not significantly different under the two species, implying that the strong invasiveness of B. pilosa could not be due to the plant-soil microbe interactions, at least plant-induced alterations of microbial community function to utilize C substrates. Alternatively, available soil nitrogen and potassium contents were significantly higher under B. pilosa than under B. bipinnata in full sun, indicating that the strong invasiveness of B. pilosa could result from rapid nutrient mobilizations by B. pilosa. However, the differences turned non-significant as light intensity decreased, suggesting that light availability could substantially alter the plant effects on soil nutrient mobilizations.

pattern was not significantly different between plants under each of the light intensity treatments (p > 0.05 for all, Fig. 3). As light intensity decreased, however, the soil microbial community significantly or marginally significantly decreased C substrate utilization, as indicated by the lower AWCD under low light intensity (p = 0.015 for carbohydrates, p = 0.077 for carboxylic acids, p = 0.032 for amine acids, p = 0.073 for amines, p = 0.055 for polymers, and p = 0.078 for phenolic compounds; Fig. 3).
Effects on soil enzyme activities. Soil invertase activity was significantly lower under the invasive B.
pilosa than under B. bipinnata (p < 0.05 for all the three light intensity treatments). The other three soil enzyme activities, i.e., urease, catalase and cellulase activities, were not significantly different between the two species (p > 0.05 for all, Fig. 4). For the both species, soil enzyme activities were depressed when light intensity decreased (Fig. 4). However, the changes of light intensity did not significantly alter the pattern of three of the four soil enzyme activities (except catalase with p = 0.025) between species, as indicated by the non-significant interactions between plant species and light intensity treatment (p > 0.05, Table 1).
Effects on soil available nutrients. With full light (100% RI) treatment, soil available N and K contents were significantly higher under B. pilosa than under B. bipinnata (p = 0.021 and 0.035, respectively; Fig. 5a,c),  but soil available P was not significantly different between the two species (p = 0.118; Fig. 5b). Under 40% and 10% RI treatments, the differences of soil available N, P and K contents were not statistically significant between the two species (p > 0.05 for all; Fig. 5), although two-way ANOVAs showed that the interactions between plant species and light intensity were not statistically significant (p > 0.05 for all, Table 1). Regardless of plant species, a decrease in light intensity reduced soil available N content but increase soil available P and K content (Fig. 5).

Discussion
In the present study, the soil microbial C utilization pattern and three of the four extracellular enzyme activities were not significantly different by growing the two species (Figs 2-4). Although both originate from tropical   non-invasive congeners. Plant-soil interactions have been proposed as one of the potential mechanisms to explain a successful invasion of alien plant species in new habitats 4,6,23 . Although the soil microbial community has functional and structural plasticity which could make them resistant to environmental changes 9 , an increasing quantity of evidences demonstrate that plant invasions can modify soil microbial community composition 10,11,13 and consequently change ecosystem processes such as litter decomposition and N cycling 3,11,13 . From an aspect of C and N cycling, invasive and non-invasive species did not shape significantly different soil microbial communities. However, our observation did not deny the possibility that plant-soil interactions may benefit to B. pilosa invasion in other ways, such as altering soil microbial community composition, increasing soil microbial biomass (also referring to Fig. S1) and stimulating soil enzyme activities 12,39 . These may be different strategies to accelerate nutrient cycling as needed and thus to reinforce invasiveness of alien species in introduced ecosystems 8, 24 .
Although most of microbial community functions were comparable, available soil N and K contents were significantly higher under B. pilosa than under B. bipinnata in the 100% RI control (Fig. 5). This is likely associated with changes in multiple aspects of change in soil and plant characteristics, such as soil microbial biomass (Fig. S1) and nutrient contents in litter and fine root 40,41 . Growing B. pilosa could substantially increase soil enzyme activities 39 and similar positive effects were also reported for other invasive species 42 . Soil microbial biomass and enzyme activities may contribute to higher soil nutrient supplies under B. pilosa. More specifically, the alkali-hydrolyzable N content was significantly higher under the invasive B. pilosa than under the non-invasive B. bipinnata in the 100% RI control, which is accompanied with relatively higher soil microbial biomass and urea activity (Figs S1 and 4c).
Invasive species often have greater productivity than those non-invasive counterparts with higher capacity to tolerate environmental stresses 25 and consequently could produce higher nutrient inputs into the soil. These processes could result in increases in soil nutrient contents such as soil N and P 3, 40, 41 , which is supported by our observations. When living in a resource-limited habitat with native and non-invasive species, invasive species may outcompete their competitors through rapid nutrient absorptions due to their fast-growth property 41 . Therefore, limited resource availability stresses and excludes those non-invasive species that have relatively lower reproduction and growth rate. This is a potential explanation for the invasion success of B. pilosa in southern China. At the preliminary stage, establishment of B. pilosa could change soil conditions (e.g., to activate more soil nutrients for its growth) to facilitate its invasion 8 . Invasive species could outcompete their competitors and invade successfully because they often have higher phenotypic plasticity and growth potential 28 . Nutrients fixed in plant tissues are returned to the soil through litterfall and root exudates, thus leading to increased contents of soil nutrients as observed in previous 22,41 and the present studies. This highlights the importance to consider potentially different effects of plant invasions with various severity on ecosystem processes and functions 16,18 .
We expected that differences in those investigated variables would be smaller between the two species, because both species could prefer to invest more resources for light competition 29 and therefore exhibit less effects on the belowground part. In the present study, the total and specific soil microbial activity and three of the four soil enzyme activities obviously decreased as light intensity declined (Figs 1, 3 and 4), partially supporting our expectation. This is likely attributable to the decreased soil microbial biomass under low light condition (Fig. S1). Soil under low light intensity could receive lower substrate inputs due to declined plant growth 31 and therefore cannot maintain a comparable soil microbial community with that in full sun, or at least the active soil microbial community 26 . Previous literature reported that size and functions of the soil microbial community were greatly affected by substrate supply 43 . However, our observation that light intensity exhibits little interactions with species (Table 1) suggests that low light intensity did not alter species-induced differences in the soil microbial community functions.
Soil had significantly higher available soil N content but lower available soil K content under high RI condition, regardless of the species (Fig. 5). The soil N pattern is consistent with that of soil urease activity (Fig. 4c), implying light-induced changes in soil enzyme activities could alter soil nutrient condition as needed. Nitrogen is a critical to plant growth, because it can be utilized to produce chlorophyll 44 that fixes atmospheric C via photosynthesis. Therefore, plants could up-regulate N need to generate more chlorophyll for C assimilation as light intensity increases 45 . Greater N need stimulates plant to affect the associated soil system to produce more soil urease that converts organic N as inorganic N 46 . Unlike N that can be fixed from the atmosphere and then returned into the soil to increase soil N content, P and K cannot be easily produced and activated by plants and soil microorganisms 47,48 . High light intensity stimulate plant growth 31 and thus increase the P and K needs from soils. As a result, available soil P and K contents will decline under high light intensity when the P and K elements cannot be activated as rapid as they are absorbed. Moreover, the soil beneath the invasive B. pilosa had significantly higher available N and K contents than that beneath its congener in full sun (Fig. 5a,c). This pattern turned non-significant when light intensity decreased, suggesting that light availability substantially impacts the species effects on available soil nutrient condition.
In summary, the soil microbial community beneath the invasive B. pilosa was not significantly different from that beneath the non-invasive congener B. bipinnata in this study, as indicated by the non-significant soil microbial C utilization and enzyme activities. However, soil contained higher available N and K contents under B. pilosa than under B. bipinnata under full sunlight but the differences turned non-significant when light intensity decreased. This result indicates that nutrient mobilizations could have contributed to the strong invasiveness of B. pilosa, which depends greatly on light availability in invaded ecosystems. As an important resource for plant growth, light availability substantially also changed the soil microbial community functions and available soil N and K contents. Our results suggest that nutrient mobilizations could contribute to the strong invasiveness of B. pilosa relative to its non-invasive congeners. Nevertheless, plant-induced alterations of microbial C utilization pattern may not be the reasons for B. pilosa invasion.

Methods
Site description and experiment preparations. This study was conducted at the experimental and teaching farm of South China Agricultural University in Guangzhou. This region has the typical subtropical monsoon climate, with annual air temperature being 21.8 °C and annual precipitation being 1694 mm 49 . Most of the precipitation occurs from April to September (the wet season). This results in an obvious dry-wet season cycling each year in the study site.
On December 2011, seeds of B. pilosa and B. bipinnata were collected from wild populations at South China Agricultural University (N 23°16′, E 113°37′) and South China Botanical Garden (N 23°18′, E 113°36′) in Guangzhou, respectively. They were dried in the sun and then stored in the sealed plastic bags at 4 °C until used for incubation (around 4 months). On March 31 2012, seeds were sowed to raise seedlings using breeding beds in a greenhouse which located at College of Agriculture, South China Agricultural University. During the period, seedlings were thinned to leave enough space for the growth of each seedling after 20 days of culturing seedlings. Ten days after thinning, uniform individuals of B. pilosa and B. bipinnata which were approximately 10 cm high were transplanted in pots for the following study.
Experimental design. Sun-shelters with a size of 4 × 4 m 2 were established for light intensity treatments, by means of covering black shading net with different light transmissions in the experimental and teaching farm of South China Agricultural University. Finally, three levels of relative light intensity, i.e., two sheltering treatments with 40% and 10% relative intensity (RIs) of full sunlight and the full sunlight control (100% RI), were established to explore potential roles of light availability on plant-induced effects on the soil microbial community functions and nutrient contents.
Under each light intensity treatment, three pots (40 cm diameter × 33 cm height) were used to grow seedlings of the invasive B. pilosa and another three used to grow seedlings of its congener B. bipinnata. Each pot contained 7.5 kg soil, with two individuals of each species grown. The used soil was collected from the experimental and teaching farm of South China Agricultural University in which both of the two species grew and then composited completely for the following plant cultivation. The soil organic matter was 2.1% and soil available nitrogen (N), phosphorus (P) and potassium (K) were 120.8, 96.0 and 99.7 mg kg −1 , respectively. Plantation duration was 64 days and through the period, all the pots were watered per day to maintain soil water content. At the end of conditioning phase, soil samples were collected to analyze for soil microbial properties and available N, P and K contents.
Soil analyses. The assayed soil microbial community properties include microbial carbon (C) utilization pattern and soil enzyme activities. Soil microbial C utilization pattern was determined using BIOLOG EcoPlate TM SCIENTIfIC REPORTS | 7: 11309 | DOI:10.1038/s41598-017-11707-x (Biolog Inc., CA, USA) which contains 31 types of C substrate commonly used by soil microorganisms and one substrate-free control 50 . For each sample, 10 g fresh soil was placed into a sterilized glass flask to mix with 100 ml of 0.85% sterilized NaCl solution on a reciprocal shaker for 0.5 h and then let stand for 1 h. The supernatant was diluted 1000 times and then 150 μl of the diluted soil suspension were added into each well of the Biolog Eco micro-plate. The micro-plates were incubated for 7 days at 25 o C and optical density was read at 590 nm using a Biolog Gen III Microstation (Biolog Inc., CA, USA) per day to record the color development of soil samples 51 . Finally, average well color development (AWCD) was calculated to indicate microbial C utilization for each sample. This method can clearly exhibit changes in soil microbial community function to utilize C substrates 50 and the results to some extent indicate the similar changes of soil microbial community, e.g, profiled by phospholipid fatty acid analysis 52 .
The activities of four soil enzymes including cellulase, invertase, urease and catalase were also analyzed to indicate soil microbial community functions 10, 53 , following the methods proposed by Guan 53 and Yao and Huang 54 . Briefly, soil cellulase and invertase activities were determined by the dinitrosalicylic acid (DNS) reduction and colorimetric method, with 1% carboxymethylcellulose and 8% sucrose solutions as substrates, respectively 53,54 . Soils mixed with the according substrate and phosphate buffer solution were incubated at 37 o C for 72 h to analyze soil cellulose activity and for 24 h to analyze soil invertase activity. The DNS solution was then added to develop color for 15 min. Finally, color density was read at 540 and 508 nm to calculate soil cellulase and invertase activity, respectively. Urease activity was analyzed using the phenol-hypochlorite reaction and colorimetric method, with 10% urea solution as a substrate 54 . After incubated at 37 o C for 24 h, phenol and hypochlorite solutions were added to develop color and then color density was read at 578 nm. Catalase activity was tested by the KMnO 4 oxidation and titration method, with 0.3% H 2 O 2 solution as a substrate and 0.02 mol L −1 KMnO 4 as an oxidizing reagent 53 .
Soil available N, P and K contents were determined as described by Bao 55 . Soil available N was assayed using the alkaline hydrolysis-diffusion method, i.e., the available N was reduced to NH 3 at 40 o C for 24 h after adding FeSO 4 powder and a NaOH solution and then the NH 3 was absorbed using H 3 BO 3 and titrated using H 2 SO 4 to determine soil available N content. Available P content was determined using a spectrophotometer at the wavelength of 700 nm, with 0.05 mol L −1 HCl-0.025 mol L −1 (1/2 H 2 SO 4 ) as the extractant, ascorbic acid as the reduction agent and a H 2 SO 4 -(NH 4 ) 6 Mo 7 O 24 solution as the color development agent. Soil available K was extracted by 1 mol L −1 NH 4 OAc solution and K concentration in the extracts was determined by the flame spectrometry method. Soil available N, P and K contents were presented as mg kg −1 soil in this study.
Statistical analyses. Principal component analysis (PCA) was conducted to reveal overall treatment effects on the microbial C utilization pattern originated from BIOLOG analysis. The PCA results could visually present the treatment effects. Moreover, the 31 types of C substrate in BIOLOG EcoPlate were pooled into six groups according to their properties 50 . For each group of the C substrates, AWCDs were compared between species and among light intensity treatments using independent-samples t test and one-way analysis of variance (ANOVA), respectively. The independent-samples t test and one-way ANOVA were also used to detect significant differences in soil enzyme activities and available N, P and K contents among treatments. Two-way ANOVA was employed to test the main and interactive effects of plant species and light intensity on microbial C utilization pattern, soil enzyme activities or available nutrient contents. For all the statistical analyses, significance level was set at p < 0.05. All these analyses were conducted in IBM SPSS Statistics 22 (IBM Corp., NY, USA) and graphs were made in SigmaPlot 10.0 (Systat Software Inc., CA, USA).
Data availability statement. The datasets generated during the current study are available from the corresponding author on reasonable request.