Effects of Liquid Organic Fertilizers on Plant Growth and Rhizosphere Soil...

[Full title: Effects of Liquid Organic Fertilizers on Plant Growth and Rhizosphere Soil Characteristics of Chrysanthemum]

sustainability Article Effects of Liquid Organic Fertilizers on Plant Growth and Rhizosphere Soil Characteristics of Chrysanthemum Rongting Ji 1,2 , Gangqiang Dong 3 , Weiming Shi 1 and Ju Min 1, * 1 State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China; rtji@issas.ac.cn (R.J.); wmshi@issas.ac.cn (W.S.) 2 University of the Chinese Academy of Sciences, Beijing 100049, China 3 Amway (China) Botanical R&D Center, Wuxi 214115, China; tony.dong@amway.com * Correspondence: jmin@issas.ac.cn; Tel.: +86-25-8688-1577 Academic Editor: Iain Gordon Received: 31 March 2017; Accepted: 8 May 2017; Published: 18 May 2017 Abstract: Organic fertilizers are generally thought to be an effective way to sustain soil fertility and plant growth. To promote the productivity of chrysanthemum, five sources of liquid organic fertilizers (L 1–L 5), as well as a chemical fertilizer, were applied at an early stage of the growth cycle to investigate their effects on plant growth. In the short-term pot experiment, the liquid organic fertilizers significantly promoted root and aboveground growth by 10.2–77.8% and 10.7–33.3%, respectively, compared with the chemical fertilizer. The order of growth promotion was: L 1 (shrimp extracts) > L 2 (plant decomposition) > L 4 (seaweed extracts)/L 5 (fish extracts) > L 3 (vermicompost). Morphological and chemical analyses indicated that, compared with other organic fertilizers, the treatment with shrimp extract (L 1) produced the greatest increases in root dry weight, total length, surface area, volume, tips, and thick root length, respectively. Furthermore, the shrimp extract treatment significantly increased the nutrient contents and altered the soil’s functional microbial community at the rhizospheric level compared with the chemical fertilizer treatment. Thus, the shrimp extract liquid organic fertilizer could be part of an effective alternative to chemical fertilization during the early stage of chrysanthemum growth Keywords: liquid organic fertilizer; chrysanthemum; root architecture; nutrient level; microbial community 1. Introduction Chrysanthemum ( Chrysanthemum morifolium Ramat.), which originated in Asia and Northeastern Europe, has been cultivated for more than 1600 years [ 1 , 2 ]. It is widely cultivated for ornamental, culinary, and medicinal uses throughout the world. For instance, in Italy, chrysanthemum production covers 1180 ha with 437 million plants and continues to grow [ 3 ]. The current trends in the chrysanthemum industry are focused on improving flower quality and creating environmentally-friendly production systems [ 4 , 5 ]. To achieve these goals and accelerate chrysanthemum production, further innovation is needed in improving fertilization regimes and other production techniques [ 6 , 7 ]. In many areas of the world, especially in China, chrysanthemum plants are cultivated under greenhouse conditions, and the over-application of chemical fertilizers has influenced the soil quality and caused serious environmental problems [ 8 , 9 ]. In addition, chrysanthemum plants are sensitive to chemical fertilizers, and their improper use affects the plant’s reproductive growth and the secondary metabolism of chrysanthemum plants [ 10 , 11 ]. Moreover, chrysanthemum has a great demand for nutrients, especially at the early stage of the growth cycle, and the nutrient status during the first seven weeks of chrysanthemum growth has a strong effect on flower size and quality [ 4 ]. Thus, growth regulation and fertilizer optimization at the early stage play critical roles during the growth cycle of chrysanthemum Sustainability 2017 , 9 , 841; doi:10.3390/su 9050841 www.mdpi.com/journal/sustainability

Sustainability 2017 , 9 , 841 2 of 16 Organic fertilizers are effective in promoting environmental sustainability and plant growth after long-term use, but previous studies have focused primarily on the conventional solid organic fertilizer product, such as straw and manure [ 12 , 13 ]. Specialized horticultural production has fostered the emergence of new liquid organic fertilizers [ 14 ], which have usually been derived from natural products and their biological activities occur at limited doses. Compared with conventional organic fertilizer, the abundant organic matter and soluble nutrients in the liquid organic fertilizers could maintain soil sustainability and plant health [ 15 , 16 ]. In addition, the integration of watering and fertilization patterns could improve the nutrient use efficiency and decrease the risk of nutrient loss [ 17 , 18 ]. Moreover, the special compounds in liquid organic fertilizers, such as chitin, humic and fulvic acids, and other biopolymers, can be biostimulants to plants [ 19 – 22 ]. Canfora et al. reported that liquid organic fertilizers containing stillage and vermicompost promoted the root growth of tomato and improved the soil microbial communities Eubacterial and Archaeal diversity, and this was in accordance with the results of liquid residues from lipopeptide production that could promote tomato growth and increase the diversity of the soil’s microbial community, as well as the related enzyme activities and nutrient cycles [ 23 , 24 ]. Given the ecological and economic benefits of liquid organic fertilizer, evaluating plant growth under organic versus chemical fertilizer use and studying the possible mechanisms of action are promising steps in developing an effective alternative fertilizer for chrysanthemum production [ 25 , 26 ]. The root systems of terrestrial plants perform two primary functions: acquiring nutrients and water from soil-based resources and recruitment of desired microbial partners for greater mutualistic benefits [ 27 – 30 ]. In a previous study, Xu et al. found that restricted the growth of shoot-borne roots of maize decreased nutrient absorption, leaf formation, and shoot growth [ 31 ]. The rhizosphere is the thin layer of soil contacted by the root and is the habitat for soil microbial communities [ 32 ]. Ecological processes in the soil are often complex interactions between the plant’s roots, soil nutrients, and the rhizosphere’s microorganisms [ 33 , 34 ]. Soil microorganisms in the rhizosphere play critical roles in nutrient cycling and soil structure maintenance, which could further promote nutrient cycles and plant growth [ 34 , 35 ]. The Biolog microplate technique is a powerful tool for monitoring the soil bacteria’s functional diversity, although it cannot determine the total microbial community but, rather, the active microbes, which can indicate environmental changes [ 36 , 37 ]. Therefore, root growth and rhizosphere microbial community changes appear to be extremely vital to evaluating liquid organic fertilizers The present study was conducted to investigate the short-term effects [ 24 , 38 ] of five liquid organic fertilizers from different sources (shrimp extracts (L 1), plant decomposition (L 2), vermicompost (L 3), seaweed extracts (L 4), and fish extracts (L 5)), on chrysanthemum plant growth at the seedling stage using the root architecture and plant growth parameters. To understand the effects of various liquid fertilizers on the soil quality, the soil nutrient level and functional bacterial diversity, which sustains microbes at the rhizosphere level, were also studied. The chief objectives of this study were: (1) to identify the effects of liquid organic fertilizers and a chemical fertilizer on chrysanthemum growth at the seedling stage; (2) to find a suitable source of liquid organic fertilizer to be applied at the early stage of the growth cycle during chrysanthemum production; and (3) to study the effects of liquid organic fertilizers on the soil characteristics at the rhizosphere level 2. Materials and Methods 2.1. Materials, Experimental Design, and Sampling The chrysanthemum cultivar Hangju ‘No. 2 of Jinju’ was obtained from the Amway Botanical Research and Development Center, Wuxi, Jiangsu, China. The cutting seedlings of chrysanthemum were cultivated on a sterilized substrate of vermiculite and perlite (1:1, v : v ) without fertilization After rooting for 15 days, seedlings of a similar height and diameter were transplanted into plots filled with 500 g of a peat and paddy soil (2:1, v : v ) substrate and grown for 60 days, with one plant per pot.

Sustainability 2017 , 9 , 841 3 of 16 At transplanting and at one additional time (30 days after transplanting), the CK, NPK, and five liquid organic fertilizers (L 1: shrimp extracts; L 2: plant decomposition; L 3: vermicompost; L 4: seaweed extracts; L 5: fish extracts) were applied to the substrate of the chrysanthemum. The major characteristics of these fertilizers are presented in Table 1 . All products were applied as a diluted solution according to the instructions provided by the manufacturers. The total amount of the macro-elements applied with the mineral and organic fertilizers was as follows: NPK: 60.0 mg N pot − 1 ; 13.1 mg P pot − 1 ; 41.2 mg K pot − 1 ; L 1: 36.8 mg N pot − 1 ; 0.5 mg P pot − 1 ; 9.1 mg K pot − 1 ; L 2: 35.0 mg N pot − 1 ; 2.9 mg P pot − 1 ; 40.4 mg K pot − 1 ; L 3: 18.4 mg N pot − 1 ; 0.3 mg P pot − 1 ; 24.6 mg K pot − 1 ; L 4: 15.1 mg N pot − 1 ; 3.3 mg P pot − 1 ; 12.5 mg K pot − 1 ; and L 5: 27.4 mg N pot − 1 ; 4.1 mg P pot − 1 ; 14.6 mg K pot − 1 Table 1. Nutrient elements content of the liquid organic fertilizers applied in the experiment Product Source Biostimulants pH N 1 (g/L) P (g/L) K (g/L) Using Instructions Providers Production Organic Certification CK / / / / / / / / / NPK chemical reagent / 8.9 267.7 58.2 184.4 0.20% / / L 1 shrimp extracts chitosan 7.2 98.0 1.3 23.2 0.25% Shenbotai Biotechnology and Chemical Co., Ltd., Zhanjiang, Guangdong, China China OFDC 2 certified organic L 2 plant decomposition humic acid 10.4 140.1 11.4 169.5 0.17% Tiancibao Agrtcultural and Technology Co., Ltd., Changsha, Hunan, China / L 3 vermicompost amino acids 4.0 49.0 0.7 65.5 0.25% Wenxing Biotech Co., Ltd., Shanghai, China China OFDC certified organic L 4 seaweed extracts alginate 7.1 60.4 13.0 51.0 0. 17% Qingdao Seawin Biotech Group Co., Ltd., Qingdao, Shandong, China EU 3 certified organic L 5 fish extracts fish emulsion 3.6 91.3 13.6 48.5 0.20% Yirong Bio-engineering Co., Ltd., Ningde, Fujian, China China OFDC certified organic Notes: 1 The concentration of N, P, and K in the mineral and organic fertilizers was determined by the chemical analysis methods in Section 2.3 ; 2 OFDC, Organic Food Development Center; 3 EU, European Union To exclude the influence of chrysanthemum roots, one more treatment, Non-R (without chrysanthemum plant), was also included in our experiment to assess the non-rhizospheric effects. Each treatment consisted of three pots placed in a completely randomized design. At the end of the trial (60 days after transplanting), plant growth was monitored and the rhizospheric soil of the chrysanthemum root was gathered by removing the loose soil and collecting the remaining soil that was tightly adhered to the roots. The soil was divided into two parts: one air-dried for the soil properties analyses, and the other stored at 4 ◦ C for further microbial analysis 2.2. Root Morphology and Aboveground Growth Parameters Root morphological parameters, including total root length, root surface area, root volume, and root tip number, were analyzed using the root analysis instrument WinRhizo-LA 1600 (Regent Instruments Inc., Quebec, QC, Canada) [ 39 ]. Thick root lengths were calculated from root diameters >0.5 mm. Root weight was measured after determining of the root morphological parameters.

Sustainability 2017 , 9 , 841 4 of 16 Aboveground growth was monitored by shoot height, diameter, and dry weight, as well as leaf width, length, area, and dry weight. The SPAD values of leaves were quantified using the hand-held Minolta SPAD-502 (Minolta corporation, Ltd., Osaka, Japan) 2.3. Chemical Analyses of Fertilizer and Soil The content of N, P, and K of the mineral fertilizer and L 1–L 5 organic fertilizers was determined with an ICP (inductively-coupled plasma) spectrometer (Thermo Electron Corporation, Ltd., Waltham, MA, USA) [ 24 ]. Soil NH 4 + -N and NO 3 − -N were extracted from the fresh soil samples with 1 M KCl (1:10 soil:solution ratio) for 1 h, and their levels were determined using a continuous flow analyzer (Skalar Analytical B.V., Breda, The Netherlands). The content of soil mineral N was calculated as the sum of the soil NH 4 + -N and NO 3 − -N contents [ 40 ]. Available P was extracted using a 0.5 M NaHCO 3 solution (1:10 soil: solution ratio) and was measured using the colorimetric method with molybdenum Available K was extracted with 1 M NH 4 OAC solution (1:10 soil:solution ratio) and was determined using flame photometry [ 38 ]. The EC of air-dried soil was determined by means of an EC meter (Bante, Ltd., Shanghai, China) with a soil: water ratio of 1:5 ( w / v ). The soil pH was measured using the pH meter (Agilent technologies, Ltd., Palo Alto, CA, USA) with a soil: water ratio of 1:2.5 ( w / v ) [ 41 ]. 2.4. Community Level Physiological Profile Analyses Biolog EcoPlates (Biolog Inc., Hayward, CA, USA) and a BIOLOG Emax TM reading (Biolog Inc.) were used to determine the community level physiological profiles of the chrysanthemum rhizospheric soil. Each plate contains three sets of 31 carbon substrates and one control, and uses tetrazolium dye as the substrate utilization indicator (Janice Young, Biolog, Inc., personal communication). The substrates were classified into six substrate sources, namely, carbohydrates, carboxylic acids, phenolic compounds, amino acids, polymers, and amines. Briefly, 5 g of chrysanthemum rhizospheric soil was mixed in 45 mL of a sterile NaCl solution (0.85%, m / v ) and then oscillated at 180 rpm for 30 min. Using a sterile NaCl solution (0.85%, m / v ), a serial dilution was performed until a final 1:1000 dilution was reached. Then, 150 µ L of the supernatant was added to each well. Microplates were incubated at 25 ◦ C for 192 h, and the influence of turbidity on the OD values at 590 nm and 750 nm were recorded every 12 h and calculated by subtracting the absorbance values of the two wavelengths. The OD values at the 96 h and 192 h of incubation were used for subsequent statistical analyses. All of the treatments had three replications. The well absorbance values were adjusted by subtracting the absorbance of the control well (water only) before the data analyses. Negative readings (OD < 0) were excluded from all subsequent analyses. The microbial activity in each microplate, expressed as the average well color development (AWCD), was determined as follows: AWCD = ∑ ODi/31, where ODi is the optical density value from each well. The Shannon diversity index was calculated as follows: H = − ∑ Pi(ln Pi), where Pi is the ratio of the activity on each substrate (ODi) to the sum of activities on all substrates ( ∑ ODi). The Shannon evenness index was calculated as E = H/ln(richness), where richness refers to the number of substrates utilized [ 36 ]. 2.5. Statistical Analyses Statistical analyses of data were conducted with the SPSS software program (ver. 20.0 for Windows, Chicago, IL, USA). Variations among chrysanthemum root morphological and aboveground growth parameters, the chemical analyses of rhizospheric soil properties, carbon utilization, and diversity index were analyzed using a two-way analysis of variance. Duncan’s test was used to determine the different treatment levels. The PCA was conducted to analyze the substrate utilization pattern based on the Biolog EcoPlates data at 96-h after incubation and was performed to visualize the carbon utilization characteristics using CANOCO for Windows 4.5. All of the graphs were created with OriginPro 8.5 (OriginLab Corporation, Northampton, MA, USA).

Sustainability 2017 , 9 , 841 5 of 16 3. Results 3.1. Effects of Organic Fertilizers on the Root Architecture and Aboveground Growth of Chrysanthemum The root architecture of chrysanthemum is of great importance in nutrient uptake and transport The statistical results of the effects on the root architecture of the various fertilizers are shown in Figure 1 . Each liquid organic fertilizer had a positive effect on root growth. Compared with the CK and NPK treatments, the application of liquid organic fertilizers significantly promoted the root growth by 76.2–179.6% and 10.2–77.8%, respectively. L 1 showed the highest promotional effect on the root growth of chrysanthemum than the other liquid organic fertilizer treatments. The root dry weight, root total length, root surface area, root volume, root tips, and thick root length of chrysanthemum under the L 1 treatment was higher than the NPK treatment by 63.4%, 63.9%, 65.6%, 67.8%, 115.4%, and 90.5%, respectively. The L 2 treatment had the second highest promotional effect and the root indices were enhanced by 35.1%, 44.2%, 41.9%, 40.1%, 75.3%, and 43.6%, respectively, compared with the NPK treatment. All of the root indices under L 3, L 4, and L 5 treatments were similar to each other and slightly higher than those of the NPK treatment even at a limited mineral nutrient input rate Analyses of chrysanthemum root growth showed that the application of L 1 resulted in the greatest growth-promoting effects among the various liquid organic fertilizers Sustainability 2017 , 9 , 841 5 of 16 3. Results 3.1. Effects of Organic Fertilizers on the Root Architecture and Aboveground Growth of Chrysanthemum The root architecture of chrysanthemum is of great importance in nutrient uptake and transport. The statistical results of the effects on the root architecture of the various fertilizers are shown in Figure 1. Each liquid organic fertilizer had a positive effect on root growth. Compared with the CK and NPK treatments, the application of liquid organic fertilizers significantly promoted the root growth by 76.2–179.6% and 10.2–77.8%, respectively. L 1 showed the highest promotional effect on the root growth of chrysanthemum than the other liquid organic fertilizer treatments. The root dry weight, root total length, root surface area, root volume, root tips, and thick root length of chrysanthemum under the L 1 treatment was higher than the NPK treatment by 63.4%, 63.9%, 65.6%, 67.8%, 115.4%, and 90.5%, respectively. The L 2 treatment had the second highest promotional effect and the root indices were enhanced by 35.1%, 44.2%, 41.9%, 40.1%, 75.3%, and 43.6%, respectively, compared with the NPK treatment. All of the root indices under L 3, L 4, and L 5 treatments were similar to each other and slightly higher than those of the NPK treatment even at a limited mineral nutrient input rate. Analyses of chrysanthemum root growth showed that the application of L 1 resulted in the greatest growth-promoting effects among the various liquid organic fertilizers. Figure 1. Effects of CK, NPK, and organic liquid fertilizers (L 1: shrimp extracts; L 2: plant decomposition; L 3: vermicompost; L 4: seaweed extracts; L 5: fish extracts) treatments on the root architecture of chrysanthemum at 60 days after transplanting. ( A ) Root dry weight; ( B ) total root length; ( C ) root surface area; ( D ) root volume; ( E ) root tips; and ( F ) thick root length. Every value is expressed as the mean ± standard deviation. Different letters indicate significantly statistical differences at the p < 0.05 level as determined by Duncan’s multiple range test. The statistical results of the aboveground growth indices affected by different fertilization regimes are shown in Figure 2. The shoot and leaf growth of chrysanthemum was significantly improved by the application of liquid organic fertilizers. Each type of liquid organic fertilizer produced greater seedling growth than the NPK treatment, especially in terms of shoot height and weight, leaf length, width, area, and weight, which were enhanced by 28.9%, 30.8%, 15.9%, 18.9%, Figure 1. Effects of CK, NPK, and organic liquid fertilizers (L 1: shrimp extracts; L 2: plant decomposition; L 3: vermicompost; L 4: seaweed extracts; L 5: fish extracts) treatments on the root architecture of chrysanthemum at 60 days after transplanting. ( A ) Root dry weight; ( B ) total root length; ( C ) root surface area; ( D ) root volume; ( E ) root tips; and ( F ) thick root length. Every value is expressed as the mean ± standard deviation. Different letters indicate significantly statistical differences at the p < 0.05 level as determined by Duncan’s multiple range test The statistical results of the aboveground growth indices affected by different fertilization regimes are shown in Figure 2 . The shoot and leaf growth of chrysanthemum was significantly improved by the application of liquid organic fertilizers. Each type of liquid organic fertilizer produced greater seedling growth than the NPK treatment, especially in terms of shoot height and weight, leaf length, width, area, and weight, which were enhanced by 28.9%, 30.8%, 15.9%, 18.9%, 36.2%, and 28.2%, respectively.

Sustainability 2017 , 9 , 841 6 of 16 The shoot heights of all the plants under the liquid organic fertilizer treatments were higher than under the NPK treatment, and the tallest plants were exposed to the L 2 treatment, having an increase of 35.7%, but there were no significant differences among the liquid organic fertilizers treatments Treatments with L 1, L 2, and L 4 showed significant promotional effects that led to the cultivation of strong seedlings, and the shoot diameters of L 1-treated plants were 1.2 times those of the NPK-treated plants. Plants treated with L 1 achieved the greatest shoot and leaf biomasses, which were greater than the CK treatment by 70.0% and 66.7%, respectively. All of the liquid organic fertilizer treatments significantly promoted the leaf growth of chrysanthemum, and the leaf length, width, and area under the L 5 treatment were 44.7%, 29.7%, and 90.7% greater than those under the CK treatment, respectively The SPAD measurements of leaves were elevated after the application of liquid organic fertilizers and greater than under the NPK treatment (expect L 3). The SPAD value of the L 1 treatment was 29.3% greater than the CK treatment. Analyses of the aboveground indices of chrysanthemum showed that the L 1, L 2, and L 5 treatments had the greatest promotional effects on chrysanthemum shoot and leaf growth Sustainability 2017 , 9 , 841 6 of 16 36.2%, and 28.2%, respectively. The shoot heights of all the plants under the liquid organic fertilizer treatments were higher than under the NPK treatment, and the tallest plants were exposed to the L 2 treatment, having an increase of 35.7%, but there were no significant differences among the liquid organic fertilizers treatments. Treatments with L 1, L 2, and L 4 showed significant promotional effects that led to the cultivation of strong seedlings, and the shoot diameters of L 1-treated plants were 1.2 times those of the NPK-treated plants. Plants treated with L 1 achieved the greatest shoot and leaf biomasses, which were greater than the CK treatment by 70.0% and 66.7%, respectively. All of the liquid organic fertilizer treatments significantly promoted the leaf growth of chrysanthemum, and the leaf length, width, and area under the L 5 treatment were 44.7%, 29.7%, and 90.7% greater than those under the CK treatment, respectively. The SPAD measurements of leaves were elevated after the application of liquid organic fertilizers and greater than under the NPK treatment (expect L 3). The SPAD value of the L 1 treatment was 29.3% greater than the CK treatment. Analyses of the aboveground indices of chrysanthemum showed that the L 1, L 2, and L 5 treatments had the greatest promotional effects on chrysanthemum shoot and leaf growth. Figure 2. Effects of CK, NPK, and organic liquid fertilizers (L 1: shrimp extracts; L 2: plant decomposition; L 3: vermicompost; L 4: seaweed extracts; L 5: fish extracts) treatments on the aboveground growth of chrysanthemum at 60 days after transplanting. ( A ) Δ H; ( B ) shoot diameter; ( C ) shoot dry weight; ( D ) leaf length; ( E ) leaf width; ( F ) leaf area; ( G ) leaf dry weight; and ( H ) SPAD value. Δ H indicates the shoot height change during the growth stage; leaf length and width were measured using the largest leaf of the plant. Leaf area was estimated as follows: leaf length × width × Figure 2. Effects of CK, NPK, and organic liquid fertilizers (L 1: shrimp extracts; L 2: plant decomposition; L 3: vermicompost; L 4: seaweed extracts; L 5: fish extracts) treatments on the aboveground growth of chrysanthemum at 60 days after transplanting. ( A ) ∆ H; ( B ) shoot diameter; ( C ) shoot dry weight; ( D ) leaf length; ( E ) leaf width; ( F ) leaf area; ( G ) leaf dry weight; and ( H ) SPAD value ∆ H indicates the shoot height change during the growth stage; leaf length and width were measured using the largest leaf of the plant. Leaf area was estimated as follows: leaf length × width × 0.75. Different letters indicate significant differences among treatments as determined by Duncan’s multiple range test at the p < 0.05 level.

Sustainability 2017 , 9 , 841 7 of 16 3.2. Effects of Liquid Organic Fertilizers on the Nutrient Contents of Chrysanthemum Rhizospheric Soil To investigate the soil’s chemical properties influenced by the application of liquid organic fertilizers, several main nutritional parameters of rhizospheric soil were measured and the results are shown in Table 2 . The nutrient content in the non-rhizosphere (Non-R) treatment was significantly different from the rhizospheric soil. The contents of mineral nitrogen and available potassium were much higher in the Non-R treatment. In the rhizospheric soil, the mineral N content was significantly increased with the addition of L 1, whereas the content was the lowest under the NPK treatment, with a gap of 12.46 mg/kg. The available P and K contents under the L 4 treatment were greater than under other treatments, and were enhanced by 49.0% and 13.4%, respectively, compared with the CK treatment. The EC measurements of rhizospheric soil significantly increased with the application of liquid organic fertilizers, which was consistent with the change in the mineral N content. The L 1 treatment showed the highest EC value, followed by the L 3 treatment, and no significant differences were observed among the other fertilizer treatments. The pH value of rhizospheric soil changed under different fertilizer treatments, and this was partly due to the different pH values of the fertilizers themselves, and because of different influences on the root exudates of chrysanthemum. Compared with that of the NPK, the pH values of the liquid organic fertilizer treatments were closer to that of the CK Table 2. The mineral nitrogen (N), available phosphorus (Avail-P), available potassium (Avail-K), electric conductivity (EC), and pH of chrysanthemum rhizospheric soil at the end of the trial * Treatment Mineral N (mg/kg) Avail-P (mg/kg) Avail-K (mg/kg) EC (us/cm) pH Non-R 29.9 ± 2.7 a 15.6 ± 0.3 d 48.3 ± 1.9 a 228.0 ± 17.1 b 5.1 ± 0.1 e CK 11.9 ± 0.6 d 17.4 ± 0.7 cd 18.3 ± 0.3 b 139.6 ± 5.1 c 5.3 ± 0.1 b NPK 7.4 ± 0.7 e 18.7 ± 0.5 bcd 17.4 ± 1.3 b 186.8 ± 15.9 bc 5.5 ± 0.1 a L 1 19.8 ± 1.2 b 19.4 ± 1.1 bcd 17.3 ± 0.5 b 340.3 ± 39.6 a 5.2 ± 0.1 cd L 2 12.5 ± 1.1 cd 21.0 ± 0.9 bc 18.5 ± 1.1 b 194.6 ± 9.8 bc 5.4 ± 0.1 a L 3 11.4 ± 0.4 d 17.0 ± 0.7 d 18.7 ± 1.1 b 294.0 ± 10.1 bc 5.1 ± 0.1 de L 4 13.1 ± 1.3 cd 25.9 ± 2.5 a 20.8 ± 1.3 b 171.4 ± 28.9 c 5.3 ± 0.1 bc L 5 16.3 ± 1.0 bc 22.4 ± 1.3 ab 19.0 ± 0.8 b 209.3 ± 21.5 bc 5.2 ± 0.1 bcd Notes:* Treatments included: Non-R: non-rhizosphere; CK: control; NPK: chemical fertilizer; L 1: shrimp extracts; L 2: plant decomposition; L 3: vermicompost; L 4: seaweed extracts; and L 5: fish extracts. Different letters indicate significant differences among treatments as determined by Duncan’s multiple range test at the p < 0.05 level 3.3. Effects of the Organic Fertilizers on Microbial Community Functions in Chrysanthemum Rhizospheric Soil The AWCD was used as an indicator of the microbial activity in the soil. As presented in Figure 3 , the AWCD of the rhizospheric soil was almost zero over the first 50 h of incubation, and it experienced a rapidly increasing stage, subsequently. The highest AWCD values were achieved under L 1 and L 2 treatments, while the lowest AWCD values were exhibited under the Non-R and CK treatments The addition of liquid organic fertilizers significantly increased the AWCD values after incubation, and the AWCD values of rhizospheric soil treated with L 1–L 5 were 2.46, 2.43, 1.70, 1.35, and 1.83 times greater, respectively, than those of the CK soil. Thus, the addition of liquid organic fertilizers generally improved the functions of chrysanthemum rhizospheric soil’s microbial community. Additionally, treatments with L 1 and L 2 affected the increase in the AWCD. The AWCD under treatment L 5 showed no significant variation compared with the NPK treatment, whereas the AWCD values of the L 3 and L 4 treatments at the preliminary stage of incubation were lower than the NPK treatment, but the AWCD of the L 3 treatment exceeded that of the NPK treatment at the end of the incubation.

Sustainability 2017 , 9 , 841 8 of 16 Sustainability 2017 , 9 , 841 8 of 16 Figure 3. Changes in the AWCD of 31 carbon sources utilized in chrysanthemum rhizospheric soil treated with the following: Non-R: non-rhizosphere soil; CK: control; NPK: chemical fertilizer treatment; L 1: shrimp extracts; L 2: plant decomposition; L 3: vermicompost; L 4: seaweed extracts; and L 5: fish extracts As demonstrated by the PCA (Figure 4), 59.1% of the total variance was explained, with the first principal component explaining 43.3% of the variance. The microbial community primarily clustered into six distinct groups: the CK, L 1, L 3/L 4, L 2/L 5, NPK, and Non-R, and, as revealed by the PCA analysis, the liquid organic fertilizer treatments, could significantly influence the bacteria’s carbon source utilization in the rhizospheric soil. The NPK treatment was separated from the liquid organic fertilizer treatments on the PC 1 axis, and the CK treatment could be separated from the liquid organic fertilizer treatments on the PC 2 axis. The metabolic activities in the rhizospheric soil under different fertilizers indicated that the microbial community was capable of growing by ingesting diverse types of carbon sources under different conditions. For example, the main carbon substrates utilized under the CK treatment was 2-hydroxybenzoic acid, whereas under the NPK treatment it was 4-hydroxybenzoic acid. Under the L 5 treatment D -mannitol and N -acetyl- D -glucosamine were the main carbon sources. Moreover, significant differences were detected in the utilization of the six main carbon sources (carbohydrates, carboxylic acid, phenolic compounds, amino acid, polymer, and amines) (Figure 5). Carbohydrates and carboxylic acid were the major carbon sources for different treatments. The highest utilization of carbohydrates was achieved under the L 1 treatment, and the optical density (OD) value was 2.29 times that of the CK treatment. This was followed by L 2, and the lowest utilization values occurred under the CK and Non-R treatments. The highest utilization of carboxylic acid was achieved under the L 2 treatment, and the OD value was 3.89 times that of the CK treatment. There were no significant differences in the utilization of the other fertilizer treatments. The utilization of phenolic compounds was highest under the Non-R treatment and lowest under the CK treatment, while the utilization of amines from the rhizospheric soil under fertilizer treatments (except L 3 and L 4) was higher than that observed in other unfertilized treatments. There was a similar pattern for the utilization of amino acids and polymers, where the highest utilization occurred under the L 1 and L 2 treatments and there were no significant differences among the other fertilizer treatments. Figure 3. Changes in the AWCD of 31 carbon sources utilized in chrysanthemum rhizospheric soil treated with the following: Non-R: non-rhizosphere soil; CK: control; NPK: chemical fertilizer treatment; L 1: shrimp extracts; L 2: plant decomposition; L 3: vermicompost; L 4: seaweed extracts; and L 5: fish extracts As demonstrated by the PCA (Figure 4 ), 59.1% of the total variance was explained, with the first principal component explaining 43.3% of the variance. The microbial community primarily clustered into six distinct groups: the CK, L 1, L 3/L 4, L 2/L 5, NPK, and Non-R, and, as revealed by the PCA analysis, the liquid organic fertilizer treatments, could significantly influence the bacteria’s carbon source utilization in the rhizospheric soil. The NPK treatment was separated from the liquid organic fertilizer treatments on the PC 1 axis, and the CK treatment could be separated from the liquid organic fertilizer treatments on the PC 2 axis. The metabolic activities in the rhizospheric soil under different fertilizers indicated that the microbial community was capable of growing by ingesting diverse types of carbon sources under different conditions. For example, the main carbon substrates utilized under the CK treatment was 2-hydroxybenzoic acid, whereas under the NPK treatment it was 4-hydroxybenzoic acid. Under the L 5 treatment D -mannitol and N -acetyl- D -glucosamine were the main carbon sources. Moreover, significant differences were detected in the utilization of the six main carbon sources (carbohydrates, carboxylic acid, phenolic compounds, amino acid, polymer, and amines) (Figure 5 ). Carbohydrates and carboxylic acid were the major carbon sources for different treatments. The highest utilization of carbohydrates was achieved under the L 1 treatment, and the optical density (OD) value was 2.29 times that of the CK treatment. This was followed by L 2, and the lowest utilization values occurred under the CK and Non-R treatments. The highest utilization of carboxylic acid was achieved under the L 2 treatment, and the OD value was 3.89 times that of the CK treatment. There were no significant differences in the utilization of the other fertilizer treatments The utilization of phenolic compounds was highest under the Non-R treatment and lowest under the CK treatment, while the utilization of amines from the rhizospheric soil under fertilizer treatments (except L 3 and L 4) was higher than that observed in other unfertilized treatments. There was a similar pattern for the utilization of amino acids and polymers, where the highest utilization occurred under the L 1 and L 2 treatments and there were no significant differences among the other fertilizer treatments.

Sustainability 2017 , 9 , 841 9 of 16 Sustainability 2017 , 9 , 841 9 of 16 Figure 4. PCA of diverse uses of carbon sources after the 96-h incubation of the chrysanthemum non-rhizospheric (Non-R) soil and rhizospheric soil treated with CK: control; NPK: chemical fertilizer treatment; L 1: shrimp extracts; L 2: plant decomposition; L 3: vermicompost; L 4: seaweed extracts; and L 5: fish extracts. Carbon substrates of the Biolog TM Plate include: A 2: β -methyl- D -glucoside; A 3: D -galactonic acid g-lactone; A 4: L -arginine; B 1: pyruvic acid methylester; B 2: D -xylose; B 3: galacturonic acid; B 4: L -asparagine; C 1: Tween 40; C 2: i-erytritol; C 3: 2-hydroxybenzoic acid; C 4: L -serine; D 1: Tween 80; D 2: D -mannitol; D 3: 4-hydroxybenzoic acid; D 4: L -phenylalanine; E 1: α -cyclodextrine; E 2: N -acetyl- D -glucosamine; E 3: γ -hydroxybutiric acid; E 4: L -threonine; F 1: glycogen; F 2: D -glucosaminic acid; F 3: itaconic acid; F 4: glycyl- L -glutamic acid; G 1: D -cellobiose; G 2: glucose-1-phosphate; G 3: α -ketobutiric acid; G 4: phenylethylamine ; H 1: α -lactose; H 2: D , L - α -glycerol phosphate; H 3: D -malic acid; and H 4: putrescine . Figure 4. PCA of diverse uses of carbon sources after the 96-h incubation of the chrysanthemum non-rhizospheric (Non-R) soil and rhizospheric soil treated with CK: control; NPK: chemical fertilizer treatment; L 1: shrimp extracts; L 2: plant decomposition; L 3: vermicompost; L 4: seaweed extracts; and L 5: fish extracts. Carbon substrates of the Biolog TM Plate include: A 2: β -methyl- D -glucoside; A 3: D -galactonic acid g-lactone; A 4: L -arginine; B 1: pyruvic acid methylester; B 2: D -xylose; B 3: galacturonic acid; B 4: L -asparagine; C 1: Tween 40; C 2: i-erytritol; C 3: 2-hydroxybenzoic acid; C 4: L -serine; D 1: Tween 80; D 2: D -mannitol; D 3: 4-hydroxybenzoic acid; D 4: L -phenylalanine; E 1: α -cyclodextrine; E 2: N -acetyl- D -glucosamine; E 3: γ -hydroxybutiric acid; E 4: L -threonine; F 1: glycogen; F 2: D -glucosaminic acid; F 3: itaconic acid; F 4: glycyl- L -glutamic acid; G 1: D -cellobiose; G 2: glucose-1-phosphate; G 3: α -ketobutiric acid; G 4: phenylethylamine;H 1: α -lactose; H 2: D , L - α -glycerol phosphate; H 3: D -malic acid; and H 4: putrescine Sustainability 2017 , 9 , 841 9 of 16 Figure 4. PCA of diverse uses of carbon sources after the 96-h incubation of the chrysanthemum non-rhizospheric (Non-R) soil and rhizospheric soil treated with CK: control; NPK: chemical fertilizer treatment; L 1: shrimp extracts; L 2: plant decomposition; L 3: vermicompost; L 4: seaweed extracts; and L 5: fish extracts. Carbon substrates of the Biolog TM Plate include: A 2: β -methyl- D -glucoside; A 3: D -galactonic acid g-lactone; A 4: L -arginine; B 1: pyruvic acid methylester; B 2: D -xylose; B 3: galacturonic acid; B 4: L -asparagine; C 1: Tween 40; C 2: i-erytritol; C 3: 2-hydroxybenzoic acid; C 4: L -serine; D 1: Tween 80; D 2: D -mannitol; D 3: 4-hydroxybenzoic acid; D 4: L -phenylalanine; E 1: α -cyclodextrine; E 2: N -acetyl- D -glucosamine; E 3: γ -hydroxybutiric acid; E 4: L -threonine; F 1: glycogen; F 2: D -glucosaminic acid; F 3: itaconic acid; F 4: glycyl- L -glutamic acid; G 1: D -cellobiose; G 2: glucose-1-phosphate; G 3: α -ketobutiric acid; G 4: phenylethylamine ; H 1: α -lactose; H 2: D , L - α -glycerol phosphate; H 3: D -malic acid; and H 4: putrescine . Figure 5. Cont .

Sustainability 2017 , 9 , 841 10 of 16 Sustainability 2017 , 9 , 841 10 of 16 Figure 5. The OD values of diverse carbon substrates after the incubation of chrysanthemum rhizospheric soil treated with CK: control; NPK: chemical fertilizer treatment; L 1: shrimp extracts; L 2: plant decomposition; L 3: vermicompost; L 4: seaweed extracts; and L 5: fish extracts. Non-R: non-rhizosphere soil. The main carbon sources were as follows: ( A ) carbohydrate; ( B ) carboxylic acid; ( C ) phenolic compounds; ( D ) amino acid; ( E ) polymer; and ( F ) amine. Different letters indicate significant differences among treatments as determined by Duncan’s multiple range test at the p < 0.05 level The Shannon diversity and evenness indices of the soil microbial community after different fertilization treatments were calculated from the 96-h Biolog data and are shown in Figure 6. The Shannon diversity index for the liquid organic fertilizer treatments (except L 3 and L 4) was significantly higher than for the Non-R, CK, and NPK treatments. The Shannon diversity index ranged from 2.58 to 3.04, and the averaged index value of L 1, L 2, and L 5 was 11.36% and 7.7% higher than those of the CK and NPK treatments, respectively. There was no significant difference between the Shannon diversity index under the L 1, L 2, and L 5 treatments. The Shannon evenness index under L 1, L 2, and L 5 was significantly higher than that found under the other fertilized and non-fertilized treatments. The averaged Shannon evenness index of L 1, L 2, and L 5 was 10.9% and 6.5% higher than those of the CK and NPK treatments, respectively. Thus, the application of liquid organic fertilizers (especially L 1, L 2, and L 5) had a significantly positive effect on the diversity and evenness of the soil microbial community. Figure 6. Effects of the various fertilization treatments on the Shannon diversity index ( A ); and Shannon evenness index ( B ) of the rhizospheric soil of chrysanthemum. Treatments were as follows: CK: control; NPK: chemical fertilizer treatment; L 1: shrimp extracts; L 2: plant decomposition; L 3: vermicompost; L 4: seaweed extracts; and L 5: fish extracts. Non-R: non-rhizospheric soil. Every value shown in the bar is expressed as the mean ± standard deviation. Different letters indicate significantly statistical differences at the p < 0.05 level as determined by Duncan’s multiple range test. Figure 5. The OD values of diverse carbon substrates after the incubation of chrysanthemum rhizospheric soil treated with CK: control; NPK: chemical fertilizer treatment; L 1: shrimp extracts; L 2: plant decomposition; L 3: vermicompost; L 4: seaweed extracts; and L 5: fish extracts. Non-R: non-rhizosphere soil. The main carbon sources were as follows: ( A ) carbohydrate; ( B ) carboxylic acid; ( C ) phenolic compounds; ( D ) amino acid; ( E ) polymer; and ( F ) amine. Different letters indicate significant differences among treatments as determined by Duncan’s multiple range test at the p < 0.05 level The Shannon diversity and evenness indices of the soil microbial community after different fertilization treatments were calculated from the 96-h Biolog data and are shown in Figure 6 . The Shannon diversity index for the liquid organic fertilizer treatments (except L 3 and L 4) was significantly higher than for the Non-R, CK, and NPK treatments. The Shannon diversity index ranged from 2.58 to 3.04, and the averaged index value of L 1, L 2, and L 5 was 11.36% and 7.7% higher than those of the CK and NPK treatments, respectively. There was no significant difference between the Shannon diversity index under the L 1, L 2, and L 5 treatments. The Shannon evenness index under L 1, L 2, and L 5 was significantly higher than that found under the other fertilized and non-fertilized treatments. The averaged Shannon evenness index of L 1, L 2, and L 5 was 10.9% and 6.5% higher than those of the CK and NPK treatments, respectively. Thus, the application of liquid organic fertilizers (especially L 1, L 2, and L 5) had a significantly positive effect on the diversity and evenness of the soil microbial community Sustainability 2017 , 9 , 841 10 of 16 Figure 5. The OD values of diverse carbon substrates after the incubation of chrysanthemum rhizospheric soil treated with CK: control; NPK: chemical fertilizer treatment; L 1: shrimp extracts; L 2: plant decomposition; L 3: vermicompost; L 4: seaweed extracts; and L 5: fish extracts. Non-R: non-rhizosphere soil. The main carbon sources were as follows: ( A ) carbohydrate; ( B ) carboxylic acid; ( C ) phenolic compounds; ( D ) amino acid; ( E ) polymer; and ( F ) amine. Different letters indicate significant differences among treatments as determined by Duncan’s multiple range test at the p < 0.05 level The Shannon diversity and evenness indices of the soil microbial community after different fertilization treatments were calculated from the 96-h Biolog data and are shown in Figure 6. The Shannon diversity index for the liquid organic fertilizer treatments (except L 3 and L 4) was significantly higher than for the Non-R, CK, and NPK treatments. The Shannon diversity index ranged from 2.58 to 3.04, and the averaged index value of L 1, L 2, and L 5 was 11.36% and 7.7% higher than those of the CK and NPK treatments, respectively. There was no significant difference between the Shannon diversity index under the L 1, L 2, and L 5 treatments. The Shannon evenness index under L 1, L 2, and L 5 was significantly higher than that found under the other fertilized and non-fertilized treatments. The averaged Shannon evenness index of L 1, L 2, and L 5 was 10.9% and 6.5% higher than those of the CK and NPK treatments, respectively. Thus, the application of liquid organic fertilizers (especially L 1, L 2, and L 5) had a significantly positive effect on the diversity and evenness of the soil microbial community. Figure 6. Effects of the various fertilization treatments on the Shannon diversity index ( A ); and Shannon evenness index ( B ) of the rhizospheric soil of chrysanthemum. Treatments were as follows: CK: control; NPK: chemical fertilizer treatment; L 1: shrimp extracts; L 2: plant decomposition; L 3: vermicompost; L 4: seaweed extracts; and L 5: fish extracts. Non-R: non-rhizospheric soil. Every value shown in the bar is expressed as the mean ± standard deviation. Different letters indicate significantly statistical differences at the p < 0.05 level as determined by Duncan’s multiple range test. Figure 6. Effects of the various fertilization treatments on the Shannon diversity index ( A ); and Shannon evenness index ( B ) of the rhizospheric soil of chrysanthemum. Treatments were as follows: CK: control; NPK: chemical fertilizer treatment; L 1: shrimp extracts; L 2: plant decomposition; L 3: vermicompost; L 4: seaweed extracts; and L 5: fish extracts. Non-R: non-rhizospheric soil. Every value shown in the bar is expressed as the mean ± standard deviation. Different letters indicate significantly statistical differences at the p < 0.05 level as determined by Duncan’s multiple range test.

Sustainability 2017 , 9 , 841 11 of 16 4. Discussion 4.1. Effects of Liquid Organic Fertilizers on the Growth of Chrysanthemum The root systems of plants perform important roles in plant growth [ 42 ]. Here, the various liquid organic fertilizer treatments significantly promoted root growth by 76.2–179.6% and 10.2–77.8% compared with the CK and NPK treatments, respectively (Figure 1 ). The improved root growth of chrysanthemum could be due to the liquid organic fertilizers’ abilities to supply soluble organic nutrients and biostimulants more quickly to the plant, which supported its growth [ 43 ]. Among the five liquid organic fertilizers, plants treated with L 1 achieved greater root dry weights, root total lengths, root surface areas, root volumes, root tips, and thicker root lengths than with the other organic fertilizer treatments by 35.4%, 31.6%, 36.3%, 39.4%, 57.5%, and 34.5%, respectively (Figure 1 ), followed by the L 2 and then the L 4 treatments. The L 3 and L 5 treatments only slightly increased the root growth of chrysanthemum. The shoot and leaf growth of chrysanthemum showed the same trend, and the liquid organic fertilizer treatments significantly promoted the aboveground growth by 30.2–56.5% and 10.7–33.3% compared with the CK and NPK treatments, respectively (Figure 2 ). These results are consistent with those of Mart í nez-Alc á ntara et al., in which either an animal or plant-based liquid organic fertilizer produced a higher total biomass of citrus trees than mineral fertilizers because of the more profuse development of new organs under the organic treatments [ 26 ]. Additionally, the shoot diameter, SPAD value and aboveground biomass of chrysanthemum significantly increased with the application of L 1 (Figure 2 ), suggesting that the L 1 had a positive effect on seedling growth, leaf chlorophyll concentrations, photosynthetic activities, and nutrient uptake efficiency [ 44 ]. In addition, the L 2 treatment had a positive effect on shoot height, which may be due to the modified availability of resources [ 45 ], whereas the application of L 5 significantly enhanced the leaf growth of chrysanthemum, which contributed toward an advanced photosynthetic efficiency [ 46 ]. Thus, compared with NPK, the addition of L 1 significantly improved the root and plant growth of chrysanthemum under the experimental conditions. The L 2 treatment had the next greatest effect, followed by the L 4 and L 5 treatments. The addition of L 3 resulted in only a slight increase in seedling growth L 1 was derived from shrimp extract, which is the most abundant natural resource on earth, with an estimated chitin yield of 10 10 –10 11 tons per year [ 22 , 47 ]. Additionally, seafood processing wastes do not contain known toxic or carcinogenic materials like liquid wastes from other industries [ 48 ]. Therefore, the shrimp extract is a low-cost and environmentally-friendly resource. The chitosan compounds deacetylated from chitin that are extracted from shrimp are effective in promoting seed germination, and root and shoot growth, and can induce resistance to abiotic stresses, as well as acting as biopesticides [ 14 , 22 , 49 – 51 ]. Therefore, the positive effect of L 1 may be partly due to the presence of chitosan. In the conventional monoculture production system, chrysanthemum is generally affected by Fusarium wilt and other diseases [ 6 ]. The addition of L 1 from this experiment may promote root growth and ecological adaptability and reduce the occurrence of plant diseases and insect pests, thereby improving chrysanthemum growth, and additional experiments are required to verify the hypothesis [ 52 , 53 ]. 4.2. Effects of Liquid Organic Fertilizers on Chrysanthemum Rhizospheric Soil’s Characteristics Organic amendments are frequently used to improve the soil structure, microbial diversity, and plant nutrient status [ 12 , 54 ]. In our study, the applications of various liquid organic fertilizers significantly improved the nutrient level (mineral N, available P, and available K contents) by 2.9–28.3% and 8.1–134.2% compared with the CK and NPK treatments, respectively. Increases in the available nutrient content of rhizospheric soil after the application of liquid organic fertilizers have been attributed to the improved diversity of the microbial community, which enhances the nutrient cycle in the soil, increasing the soil nutrients available for plant growth [ 23 , 55 ]. The addition of L 1 significantly stimulated the N mineralization process (Table 2 ), which was different from the findings of Gutser et al. in which the input of organic fertilizers on arable land significantly reduced the mineral-N

Sustainability 2017 , 9 , 841 12 of 16 content and increased the stability of the organic matter [ 56 ]. This was mainly because the organic fertilizer form used by Gutser et al. was compost, whereas the liquid organic fertilizer used in our experiment contained a large amount of soluble nutrients and tended to be more quickly available to plants compared with the traditional substrate-incorporated fertilizers [ 57 ]. In our experiment, the L 1 treatment produced the highest EC values, which were 15.8–143.8% higher than those of the other treatments (Table 2 ). The increase in the soil EC value could be correlated with the mineralization of organic matter after the application of liquid organic fertilizers [ 58 ], which was in accordance with the change in the mineral N content (Table 2 ). The carbon substrate utilization presented by the Biolog-Eco plates was sensitive enough to detect short-term changes in the soil environment, and the appropriate fertilizer regimes could increase the soil’s functional microbial diversity [ 38 , 59 ]. In our study, the application of liquid organic fertilizers improved the AWCD of rhizospheric soil by 35.4–146.2% and − 13.5–57.3% compared with the CK and NPK treatments, respectively (Figure 3 ). The addition of L 1 exhibited the highest carbon substrate utilization, and the AWCD of L 1 at the end of the incubation was 1.4–81.9% higher than those of the other liquid organic fertilizer treatments. In agreement with our results, Gomez et al. found that organic soil amendments significantly stimulated the carbon substrate utilization [ 60 ], and this mainly depended on the availability of soil carbon. The PCA showed that the fertilizer regimes significantly influenced the rhizospheric soil’s functional diversity (Figure 4 ). Liquid organic fertilizers may induce changes in soil properties which, in turn, influence the soil’s microbial functional diversity, resulting in differences in the soil carbon substrate utilization and plant growth [ 38 , 61 ]. Under the liquid organic fertilizer treatments, the utilization of carbohydrate, carboxylic acid, phenolic compounds, amino acid, and polymer was much greater than in the CK and NPK treatments, and an enhancement of these carbon sources was observed by 59.4–191.7% and 0.2–56.0% compared with the CK and NPK treatments, respectively (Figure 5 ). This was in accordance with earlier work of Romaniuk et al. in which microbial functional diversity could indicate the influence of fertilizer and management practices in organic and conventional horticulture systems [ 62 ]. The utilization of phenolic compounds showed a different tendency in our experiment and it was highest under the Non-R treatment and lowest for CK treatment. This phenomenon was possible partly for the different pH values among treatments and the pH value of the Non-R treatment was the lowest (Table 2 ). Additionally, phenolic compounds were the main autotoxicity substance in rhizosphere soil of many plants and it would influence the growth of plants; thus, the utilization of phenolic compounds was higher in Non-R treatment than the rhizosphere soil [ 63 , 64 ]. The application of L 1 significantly improved the utilization of the six main carbon sources (carbohydrate, carboxylic acid, phenolic compounds, amino acid, polymer, and amine) by 76.6, 40.1, 22.4, 81.6, 29.7, and 2.9% than the NPK treatment, respectively. The Shannon index analysis also showed that the application of liquid organic fertilizer treatments (especially L 1, L 2, and L 5) significantly improved the diversity and evenness of the soil bacterial community (Figure 6 ), which would be beneficial in resisting stress [ 61 , 65 ]. Thus, the application of liquid organic fertilizers, especially L 1, significantly increased the microbial population’s diversity in the rhizosphere 5. Conclusions Fertilizer management is of great importance in chrysanthemum production. In this study, five sources of liquid organic fertilizer were applied to promote chrysanthemum growth at the early stage. The application of liquid organic fertilizers significantly promoted the root architecture and plant growth of chrysanthemum compared with the CK and inorganic fertilizer treatment over the short term, even with a limited amount of mineral nutrient input. Among the five liquid organic fertilizers treatments applied in our experiment, the L 1 liquid fertilizer proved to be effective in both root development and aboveground growth promotion, especially the root tips, SPAD value of leaves, and the aboveground biomass. The addition of L 1 liquid organic fertilizer significantly stimulated the soil’s microbial activity and functional diversity through the enhancement the N mineralization process at the rhizospheric level. Treating with L 1 liquid organic fertilizer indicated its potential as

Sustainability 2017 , 9 , 841 13 of 16 an effective fertilizer regime in the chrysanthemum production system. Moreover, the successful application of liquid organic fertilizers in our study suggested a rational way to reuse agricultural wastes and was effective in sustaining plant growth and the health of the soil system. In the future, more attention should be paid to quantifying the optimal fertilizer rate of the shrimp extract liquid organic fertilizer in diverse growth periods of chrysanthemum production, and further analysis is also required to clarify the key microbiologic population in the process Acknowledgments: This work was funding from the National Natural Science Foundation of China (31672236) and enterprise academician workstation in Amway (China) Botanical Research and Development Center of the herbal chrysanthemum planting project (BC 20160005 Z) Author Contributions: Ju Min, Weiming Shi, and Gangqiang Dong conceived and designed the experiments; Rongting Ji performed the experiments; Rongting Ji analyzed the data; Gangqiang Dong contributed reagents/materials/analysis tools; and Rongting Ji and Ju Min wrote the paper Conflicts of Interest: The authors declare no conflict of interest Abbreviations NPK chemical fertilizer CK no fertilizer control EC electric conductivity SPAD Soil and Plant Analyzer Development AWCD Average Well Color Development References 1 Tsukiboshi, T.; Chikuo, Y.; Ito, Y.; Matsushita, Y.; Kageyama, K. Root and stem rot of chrysanthemum caused by five pythium species in Japan J. Gen. Plant Pathol 2007 , 73 , 293–296. [ CrossRef ] 2 Zhao, H.E.; Liu, Z.H.; Hu, X.; Yin, J.L.; Li, W.; Rao, G.Y.; Zhang, X.H.; Huang, C.L.; Anderson, N.; Zhang, Q.X.; et al. Chrysanthemum genetic resources and related genera of chrysanthemum collected in China Genet. Resour. Crop Evol 2009 , 56 , 937–946. [ CrossRef ] 3 Pecchia, S.; Franceschini, A.; Santori, A.; Vannacci, G.; Myrta, A. Efficacy of dimethyl disulfide (dmds) for the control of chrysanthemum verticillium wilt in Italy Crop Prot 2017 , 93 , 28–32. [ CrossRef ] 4 MacDonald, W.N.; Blom, T.J.; Tsujita, M.J.; Shelp, B.J. Review: Improving nitrogen use efficiency of potted chrysanthemum: Strategies and benefits Can. J. Plant Sci 2013 , 93 , 1009–1016. [ CrossRef ] 5 Wandl, M.T.; Haberl, H. Greenhouse gas emissions of small scale ornamental plant production in Austria—A case study J. Clean. Prod 2017 , 141 , 1123–1133. [ CrossRef ] 6 Zhao, S.; Chen, X.; Deng, S.P.; Dong, X.N.; Song, A.P.; Yao, J.J.; Fang, W.M.; Chen, F.D. The effects of fungicide, soil fumigant, bio-organic fertilizer and their combined application on chrysanthemum fusarium wilt controlling, soil enzyme activities and microbial properties Molecules 2016 , 21 , 526. [ CrossRef ] [ PubMed ] 7 Rostami, M.; Zamani, A.; Goldasteh, S.; Shoushtari, R.; Kheradmand, K. Influence of nitrogen fertilization on biology of Aphis gossypii (hemiptera: Aphididae) reared on Chrysanthemum lindicum (asteraceae) J. Plant Prot. Res 2012 , 52 , 118–121. [ CrossRef ] 8 Han, J.P.; Luo, Y.H.; Yang, L.P.; Liu, X.M.; Wu, L.S.; Xu, J.M. Acidification and salinization of soils with different initial pH under greenhouse vegetable cultivation J. Soils Sediments 2014 , 14 , 1683–1692. [ CrossRef ] 9 Min, J.; Lu, K.P.; Sun, H.J.; Xia, L.L.; Zhang, H.L.; Shi, W.M. Global warming potential in an intensive vegetable cropping system as affected by crop rotation and nitrogen rate Clean Soil Air Water 2016 , 44 , 766–774. [ CrossRef ] 10 Macz, O.; Paparozzi, E.T.; Stroup, W.W.; Leonard, R.; Nell, T.A. Effect of nitrogen and sulfur applications on pot chrysanthemum production and postharvest performance. II. Plant growth responses J. Plant Nutr 2001 , 24 , 131–146. [ CrossRef ] 11 Liu, D.H.; Zhu, D.W.; Guo, L.P.; Liu, W.; Zuo, Z.T.; Jin, H.; Yang, Y. Effects of nitrogen fertilization on growth,yield and quality of Chrysanthemum morifolium Plant Nutr. Fertil. Sci 2012 , 18 , 188–195 12 Sun, J.; Zhang, Q.; Zhou, J.; Wei, Q.P. Pyrosequencing technology reveals the impact of different manure doses on the bacterial community in apple rhizosphere soil Appl. Soil Ecol 2014 , 78 , 28–36. [ CrossRef ]

Sustainability 2017 , 9 , 841 14 of 16 13 Atiyeh, R.M.; Edwards, C.A.; Subler, S.; Metzger, J.D. Pig manure vermicompost as a component of a horticultural bedding plant medium: Effects on physicochemical properties and plant growth Bioresour. Technol 2001 , 78 , 11–20. [ CrossRef ] 14 Pichyangkura, R.; Chadchawan, S. Biostimulant activity of chitosan in horticulture Sci. Hortic 2015 , 196 , 49–65. [ CrossRef ] 15 Hou, J.Q.; Li, M.X.; Mao, X.H.; Hao, Y.; Ding, J.; Liu, D.M.; Xi, B.D.; Liu, H.L. Response of microbial community of organic-matter-impoverished arable soil to long-term application of soil conditioner derived from dynamic rapid fermentation of food waste PLoS ONE 2017 , 12 , e 0175715. [ CrossRef ] [ PubMed ] 16 Dordas, C.A.; Lithourgidis, A.S.; Matsi, T.; Barbayiannis, N. Application of liquid cattle manure and inorganic fertilizers affect dry matter, nitrogen accumulation, and partitioning in maize Nutr. Cycl. Agroecosyst 2007 , 80 , 283–296. [ CrossRef ] 17 Toonsiri, P.; Del Grosso, S.J.; Sukor, A.; Davis, J.G. Greenhouse gas emissions from solid and liquid organic fertilizers applied to lettuce J. Environ. Qual 2016 , 45 , 1812–1821. [ CrossRef ] [ PubMed ] 18 Ceretta, C.A.; Girotto, E.; Lourenzi, C.R.; Trentin, G.; Vieira, R.C.B.; Brunetto, G. Nutrient transfer by runoff under no tillage in a soil treated with successive applications of pig slurry Agric. Ecosyst. Environ 2010 , 139 , 689–699. [ CrossRef ] 19 Canellas, L.P.; Olivares, F.L.; Aguiar, N.O.; Jones, D.L.; Nebbioso, A.; Mazzei, P.; Piccolo, A. Humic and fulvic acids as biostimulants in horticulture Sci. Hortic 2015 , 196 , 15–27. [ CrossRef ] 20 Du Jardin, P. Plant biostimulants: Definition, concept, main categories and regulation Sci. Hortic 2015 , 196 , 3–14. [ CrossRef ] 21 Sharp, R.G. A review of the applications of chitin and its derivatives in agriculture to modify plant-microbial interactions and improve crop yields Agronomy 2013 , 3 , 757–793. [ CrossRef ] 22 Tang, H.; Zhang, L.Y.; Hu, L.Y.; Zhang, L.N. Application of chitin hydrogels for seed germination, seedling growth of rapeseed J. Plant Growth Regul 2013 , 33 , 195–201. [ CrossRef ] 23 Zhu, Z.; Zhang, F.G.; Wang, C.; Ran, W.; Shen, Q.R. Treating fermentative residues as liquid fertilizer and its efficacy on the tomato growth Sci. Hortic 2013 , 164 , 492–498. [ CrossRef ] 24 Canfora, L.; Malusa, E.; Salvati, L.; Renzi, G.; Petrarulo, M.; Benedetti, A. Short-term impact of two liquid organic fertilizers on solanum lycopersicum l. Rhizosphere eubacteria and archaea diversity Appl. Soil Ecol 2015 , 88 , 50–59. [ CrossRef ] 25 Chaudhry, V.; Rehman, A.; Mishra, A.; Chauhan, P.S.; Nautiyal, C.S. Changes in bacterial community structure of agricultural land due to long-term organic and chemical amendments Microb. Ecol 2012 , 64 , 450–460. [ CrossRef ] [ PubMed ] 26 Martinez-Alcantara, B.; Martinez-Cuenca, M.R.; Bermejo, A.; Legaz, F.; Quinones, A. Liquid organic fertilizers for sustainable agriculture: Nutrient uptake of organic versus mineral fertilizers in citrus trees PLoS ONE 2016 , 11 , e 0161619. [ CrossRef ] [ PubMed ] 27 Zobel, R.W. Fine roots—Functional definition expanded to crop species? New Phytol 2016 , 212 , 310–312 [ CrossRef ] [ PubMed ] 28 Pregitzer, K.S. Fine roots of trees—A new perspective New Phytol 2002 , 154 , 267–270. [ CrossRef ] 29 Hodge, A.; Berta, G.; Doussan, C.; Merchan, F.; Crespi, M. Plant root growth, architecture and function Plant Soil 2009 , 321 , 153–187. [ CrossRef ] 30 Saleem, M.; Law, A.D.; Moe, L.A. Nicotiana roots recruit rare rhizosphere taxa as major root-inhabiting microbes Microb. Ecol 2015 , 71 , 469–472. [ CrossRef ] [ PubMed ] 31 Xu, L.Z.; Niu, J.F.; Li, C.J.; Zhang, F.S. Growth, nitrogen uptake and flow in maize plants affected by root growth restriction J. Integr. Plant Biol 2009 , 51 , 689–697. [ CrossRef ] [ PubMed ] 32 Ridl, J.; Kolar, M.; Strejcek, M.; Strnad, H.; Stursa, P.; Paces, J.; Macek, T.; Uhlik, O. Plants rather than mineral fertilization shape microbial community structure and functional potential in legacy contaminated soil Front. Microbiol 2016 , 7 . [ CrossRef ] [ PubMed ] 33 Loranger-Merciris, G.; Barthes, L.; Gastine, A.; Leadley, P. Rapid effects of plant species diversity and identity on soil microbial communities in experimental grassland ecosystems Soil Biol. Biochem 2006 , 38 , 2336–2343 [ CrossRef ] 34 Saleem, M.; Moe, L.A. Multitrophic microbial interactions for eco and agro-biotechnological processes: Theory and practice Trends Biotechnol 2014 , 32 , 529–537. [ CrossRef ] [ PubMed ]

Sustainability 2017 , 9 , 841 15 of 16 35 Suzuki, C.; Kunito, T.; Aono, T.; Liu, C.T.; Oyaizu, H. Microbial indices of soil fertility J. Appl. Microbiol 2005 , 98 , 1062–1074. [ CrossRef ] [ PubMed ] 36 Guo, P.P.; Zhu, L.S.; Wang, J.H.; Wang, J.; Liu, T. Effects of alkyl-imidazolium ionic liquid [omim]cl on the functional diversity of soil microbial communities Environ. Sci. Pollut. Res 2015 , 22 , 9059–9066. [ CrossRef ] [ PubMed ] 37 Chen, L.J.; Feng, Q.; Wei, Y.P.; Li, C.S.; Zhao, Y.; Li, H.Y.; Zhang, B.G. Effects of saline water irrigation and fertilization regimes on soil microbial metabolic activity J. Soils Sediments 2016 , 17 , 376–383. [ CrossRef ] 38 Yu, C.; Hu, X.M.; Deng, W.; Li, Y.; Xiong, C.; Ye, C.H.; Han, G.M.; Li, X. Changes in soil microbial community structure and functional diversity in the rhizosphere surrounding mulberry subjected to long-term fertilization Appl. Soil Ecol 2015 , 86 , 30–40. [ CrossRef ] 39 Arsenault, J.L.; Pouleur, S.; Messier, C.; Guay, R. Winrhizo TM , a root measuring system with a unique overlap correction method HortScience 1995 , 30 , 906 40 Min, J.; Zhao, X.; Shi, W.M.; Xing, G.X.; Zhu, Z.L. Nitrogen balance and loss in a greenhouse vegetable system in southeastern China Pedosphere 2011 , 21 , 464–472. [ CrossRef ] 41 Shi, W.M.; Yao, J.; Yan, F. Vegetable cultivation under greenhouse conditions leads to rapid accumulation of nutrients, acidification and salinity of soils and groundwater contamination in south-eastern China Nutr. Cycl. Agroecosyst 2008 , 83 , 73–84. [ CrossRef ] 42 Li, X.X.; Zeng, R.S.; Liao, H. Improving crop nutrient efficiency through root architecture modifications J. Integr. Plant Biol 2016 , 58 , 193–202. [ CrossRef ] [ PubMed ] 43 Nelson, P.V.; Pitchay, D.S.; Niedziela, C.E.; Mingis, N.C. Efficacy of soybean-base liquid fertilizer for greenhouse crops J. Plant Nutr 2010 , 33 , 351–361. [ CrossRef ] 44 Uddling, J.; Gelang-Alfredsson, J.; Piikki, K.; Pleijel, H. Evaluating the relationship between leaf chlorophyll concentration and spad-502 chlorophyll meter readings Photosynth. Res 2007 , 91 , 37–46. [ CrossRef ] [ PubMed ] 45 Collet, C.; Colin, F.; Bernier, F. Height growth, shoot elongation and branch development of young quercus petraea grown under different levels of resource availability Annal. Sci. For 1997 , 54 , 65–81. [ CrossRef ] 46 Lee, J.H.; Heuvelink, E. Simulation of leaf area development based on dry matter partitioning and specific leaf area for cut chrysanthemum Ann. Bot 2003 , 91 , 319–327. [ CrossRef ] [ PubMed ] 47 Gallert, C.; Winter, J. Solid and liquid residues as raw materials for biotechnology Naturwissenschaften 2002 , 89 , 483–496. [ CrossRef ] [ PubMed ] 48 Pierantozzi, P.; Zampini, C.; Torres, M.; Isla, M.I.; Verdenelli, R.A.; Meriles, J.M.; Maestri, D. Physico-chemical and toxicological assessment of liquid wastes from olive processing-related industries J. Sci. Food Agric 2012 , 92 , 216–223. [ CrossRef ] [ PubMed ] 49 Fitza, K.N.E.; Payn, K.G.; Steenkamp, E.T.; Myburg, A.A.; Naidoo, S. Chitosan application improves resistance to fusarium circinatum in pinus patula S. Afr. J. Bot 2013 , 85 , 70–78. [ CrossRef ] 50 Katiyar, D.; Hemantaranjan, A.; Singh, B. Chitosan as a promising natural compound to enhance potential physiological responses in plant: A review Indian J. Plant Physiol 2015 , 20 , 1–9. [ CrossRef ] 51 Winkler, A.J.; Dominguez-Nuñez, J.A.; Aranaz, I.; Poza-Carri ó n, C.; Ramonell, K.; Somerville, S.; Berrocal-Lobo, M. Short-chain chitin oligomers: Promoters of plant growth Mar. Drugs 2017 , 15 , 40 [ CrossRef ] [ PubMed ] 52 Hallmann, J.; Rodriguez-Kabana, R.; Kloepper, J.W. Chitin-mediated changes in bacterial communities of the soil, rhizosphere and within roots of cotton in relation to nematode control Soil Biol. Biochem 1999 , 31 , 551–560. [ CrossRef ] 53 Debode, J.; De Tender, C.; Soltaninejad, S.; Van Malderghem, C.; Haegeman, A.; Van der Linden, I.; Cottyn, B.; Heyndrickx, M.; Maes, M. Chitin mixed in potting soil alters lettuce growth, the survival of zoonotic bacteria on the leaves and associated rhizosphere microbiology Front. Microbiol 2016 , 7 . [ CrossRef ] [ PubMed ] 54 Ling, N.; Zhu, C.; Xue, C.; Chen, H.; Duan, Y.H.; Peng, C.; Guo, S.W.; Shen, Q.R. Insight into how organic amendments can shape the soil microbiome in long-term field experiments as revealed by network analysis Soil Biol. Biochem 2016 , 99 , 137–149. [ CrossRef ] 55 Lee, J.J.; Park, R.D.; Kim, Y.W.; Shim, J.H.; Chae, D.H.; Rim, Y.S.; Sohn, B.K.; Kim, T.H.; Kim, K.Y. Effect of food waste compost on microbial population, soil enzyme activity and lettuce growth Bioresour. Technol 2004 , 93 , 21–28. [ CrossRef ] [ PubMed ]

Sustainability 2017 , 9 , 841 16 of 16 56 Gutser, R.; Ebertseder, T.; Weber, A.; Schraml, M.; Schmidhalter, U. Short-term and residual availability of nitrogen after long-term application of organic fertilizers on arable land J. Plant Nutr. Soil Sci 2005 , 168 , 439–446. [ CrossRef ] 57 Burnett, S.E.; Mattson, N.S.; Williams, K.A. Substrates and fertilizers for organic container production of herbs, vegetables, and herbaceous ornamental plants grown in greenhouses in the united states Sci. Hortic 2016 , 208 , 111–119. [ CrossRef ] 58 Gulser, C.; Demir, Z.; Ic, S. Changes in some soil properties at different incubation periods after tobacco waste application J. Environ. Biol 2010 , 31 , 671–674. [ PubMed ] 59 Fliessbach, A.; Mader, P. Carbon source utilization by microbial communities in soils under organic and conventional farming practice. In Microbial Communities—Functional versus Structural Approaches ; Insam, H., Rangger, A., Eds.; Springer: Berlin/Heidelberg, Germany, 1997; pp. 109–120 60 Gomez, E.; Ferreras, L.; Toresani, S. Soil bacterial functional diversity as influenced by organic amendment application Bioresour. Technol 2006 , 97 , 1484–1489. [ CrossRef ] [ PubMed ] 61 Saleem, M.; Pervaiz, Z.H.; Traw, M.B. Theories, mechanisms and patterns of microbiome species coexistence in an era of climate change. In Microbiome Community Ecology ; Saleem, M., Ed.; Springer: New York, NY, USA, 2015; pp. 125–152 62 Romaniuk, R.; Giuffre, L.; Costantini, A.; Nannipieri, P. Assessment of soil microbial diversity measurements as indicators of soil functioning in organic and conventional horticulture systems Ecologic. Indic 2011 , 11 , 1345–1353. [ CrossRef ] 63 Dong, Y.; Dong, K.; Tang, L.; Zheng, Y.; Yang, Z.X.; Xiao, J.X.; Zhao, P.; Hu, G.B. Relationship between rhizosphere microbial community functional diversity and faba bean fusarium wilt occurrence in wheat and faba bean intercropping system Acta Ecol. Sin 2013 , 33 , 7445–7454. [ CrossRef ] 64 Asaduzzaman, M.; Asao, T. Autotoxicity in beans and their allelochemicals Sci. Hortic 2012 , 134 , 26–31 [ CrossRef ] 65 Degens, B.P.; Schipper, L.A.; Sparling, G.P.; Duncan, L.C. Is the microbial community in a soil with reduced catabolic diversity less resistant to stress or disturbance? Soil Biol. Biochem 2001 , 33 , 1143–1153. [ CrossRef ] © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Other Environmental Sciences Concepts:

[back to top]

Discover the significance of concepts within the article: ‘Effects of Liquid Organic Fertilizers on Plant Growth and Rhizosphere Soil...’. Further sources in the context of Environmental Sciences might help you critically compare this page with similair documents:

Faba bean, Maize, Mulberry, Chrysanthemum, Bean, Organic fertilizer, Early stage, Soil quality, Medicinal use, Microbial activity, Fulvic acid, Amino acid, Inorganic fertilizer, Humic acid, Organic matter, Metabolic activities, Plant growth, Sustainable agriculture, Vermicompost, Soil fertility, Nutrient content, Environmental sustainability, Rhizosphere soil, Rhizospheric soil, Nutrient uptake, Carbon source, Dry matter, Seed germination, Carboxylic acid, Chitosan application, Soil conditioner, Microbial Communities, Phenolic compound, Chemical fertilizer, Natural compound, Carbon source utilization, Environmental problem, Shoot growth, Leaf growth, Bacterial communities, Soil properties, Soil bacteria, Secondary metabolism, Greenhouse gas emission, Liquid waste, Plant biostimulant, Seedling growth, Electric Conductivity, Microbial interactions, Chitin, Plant species diversity, Food waste, Microbial community, Nutrient level, Vegetable cultivation, Culinary use, Shannon diversity index, Microbial community structure, Available phosphorus, Available potassium, Growth Cycle, Nitrogen balance, Carbon utilization, EC values, Shoot elongation, Seaweed extract, Total variance, CK, Height growth, L 3, Principal component, Soil microbial diversity, Branch development, PCA Analysis, Nitrogen Uptake, Ornamental use, Bacterial community structure, Soil microbial community, Soil microbial communities, Soil sustainability, Solid residue, Organic Amendment, Arable soil, Root growth, Mineral nitrogen, Environmentally-friendly production, Root dry weight, Root surface area, CK treatment, L1, Microbial functional diversity, Nitrogen accumulation, Liquid fertilizer, Soil microbiome, Soil microbial community structure, Food waste compost, Bacterial functional diversity.