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To understand the effects of tree peony (Paeonia suffruticosa) on soil microbiological and biochemical properties, soil samples were collected from tree peony growing sites with 3 growth years and four tree peony cultivars as well as from an adjacent wasteland in a tree peony garden at Luoyang, Henan Province of China. With the development of the tree peony garden ecosystem, soil microbial biomass carbon (Cmic), basal respiration (Rmic), Cmic as a percent of soil organic C (Cmic/Corg), and enzyme activities first increased and then decreased. For the tree peony cultivars Yao Huang and Dou Lu, Cmic, Rmic, Cmic/Corg, catalase, invertase, cellulose, proteinase, and phosphatase decreased after 5 years of growth, whereas urease decreased after 12 years. For the cultivars Er Qiao and Shou An Hong, catalase, proteinase, and phosphatase decreased after 5 years, whereas Cmic, Rmic, Cmic/Corg, invertase, cellulose, and urease decreased after 12 years. Biolog analysis indicated that the average well color development and microbial functional diversity were significantly greater at the 5-year sites than in the wasteland but decreased significantly as growth continued. The growth duration of tree peony had a greater effect on soil microbial communities than did tree peony cultivar.
Tree peony (Paeonia suffruticosa), known as the “queen of flowers,” is one of the most famous horticultural plants and has been cultivated throughout the temperate regions of the world. China is the country of first domestication and the origin center for cultivated tree peonies, and the species continues to be planted throughout the country. To develop tourism and the commercialization of tree peony, planting areas of this species have increased year after year. However, because tree peony grows for multiple years, its growth potential becomes weak and it experiences serious diseases, which are inevitably connected with the soil environment. Plant cover type, planting years, and management are known to significantly impact soil ecological sustainability (Yao et al., 2000). Tree peony gardens have unique cultivation and management styles. To reduce diseases and insect pests and save nutrition for the growth of floral buds and flowers, weak and ill branches, deadwood, and fallen leaves are cut annually from tree peony plants and the litter layer is removed from the garden. Like with many perennial woody flowers, fertilization is a very important management measure for tree peonies. Tree peony garden soils usually receive three applications of fertilizer per year to ensure large and colorful flowers. These management measures inevitably affect soil properties and microbial ecology.
Soil biological and biochemical properties have become increasingly recognized as important for assessing the sustainability of ecosystems. In soil ecosystems, soil microorganisms play a crucial role in nutrient cycling, energy flow, and organic matter decomposition (Unger et al., 2013; Yao et al., 2000). Consequently, these microorganisms have been widely recognized as integral components of soil quality. Microbial biomass C (Cmic), basal respiration (Rmic), and enzyme activities have been widely used in soil investigations as a result of their great sensitivity to anthropogenic disturbance and environmental changes (Franco-Otero et al., 2012; Reeve and Drost, 2012; Wells, 2011). Recently, the functional diversity of soil microbial communities has frequently been used to assess soil quality and ecosystem functions (McGuire and Treseder, 2010; Sherman and Steinberger, 2012). The Biolog system, which reflects the oxidative catabolism of substrates to generate patterns of potential sole carbon source use, has been proven more advantageous for assessing the functional diversity of microbial communities in various soil ecosystems than other methods such as phospholipid fatty acid analysis and denaturing gradient gel electrophoresis (Saul-Tcherkas and Steinberger, 2009).
Most studies regarding tree peony have focused primarily on the plant itself, examining such issues as germplasm resources, cultivar classification, and tissue culture and micropropagation (Suo et al., 2005, 2008, 2012; Teixeira da Silva et al., 2012). Relatively few studies have focused on the soil environment under tree peony and the influence of tree peony growth on soil properties. Little is currently known about how soil microbiological and biochemical properties change with the growth of tree peony. The objective of our study was to evaluate the changes in soil microbiological and biochemical properties in response to growth duration and cultivars of tree peony. The results of the study will facilitate the assessment of ecological sustainability and definition of appropriate management strategies in tree peony garden ecosystems.
The study was conducted in central China at the National Peony Garden (lat. 34°42′ N, long. 112°23′ E) located in Luoyang, Henan Province, which is famous for its Luoyang peony. The area is characterized by a temperate monsoon climate with a mean annual temperature of 14.9 °C and mean annual rainfall of ≈530 to 600 mm. The soils of this area are cinnamon soils derived from carbonatite. Twelve tree peony growing sites with 3 growth years and four cultivars were selected to assess the responses of soil chemical, microbiological, and biochemical properties to growth duration and cultivars of tree peony. The four tree peony cultivars were Yao Huang, Dou Lu, Er Qiao, and Shou An Hong, and they were planted on the wasteland in 1986, 1999, and 2006. Therefore, the plants had been growing for 25, 12, and 5 years, respectively, when the soil samples were taken. Each site consisted of several plots separated by a footpath. All sites received annual applications of nitrogen–phosphorus–potassium compound fertilizer (15N–6.6P–12.4K) three times per year, averaging ≈3000 kg·ha−1 per year. A neighboring wasteland covered with sparse grasses was chosen as a control site. All of the sites had similar ecological conditions with the same soil type and topography.
Soil samples were collected using a soil sampler from three sampling plots randomly chosen within each study site. Twenty cores (5 cm diameter × 20 cm length) were taken from each sampling plot and mixed. Field-moist soils were sieved through a 2-mm mesh to remove plant debris and soil fauna. One portion of soil was stored at 4 °C for microbiological and biochemical analyses, and the other portion was air-dried for chemical analysis.
Soil pH was measured using a combination glass electrode with the ratio of soil and distilled water at 1:2.5. Total nitrogen was determined using Kjeldahl digestion (Keeney and Nelson, 1982) and quantified using a continuous flow analyzer (Skalar, Delft, The Netherlands), and total organic C was determined by dichromate oxidation (Nelson and Sommers, 1982). Total P concentration was determined colorimetrically at 440 nm using the molybdate procedure (Murphy and Riley, 1962) (Table 1).
Cmic was determined using the chloroform fumigation–extraction method (Vance et al., 1987). The K2SO4-extracted C of both fumigated and unfumigated samples was analyzed using a total organic C analyzer (Shimazu, TOC-500, Chiba, Japan), and a KEC value of 0.45 was used to convert the measured flush of C to Cmic (Yao et al., 2000). Rmic was determined by measuring CO2 evolution. A 20-g sample (oven-dry basis) of field-moist soil was incubated in an airtight 250-mL glass vessel at 25 °C for 1 d. The CO2 produced from the soil was absorbed in NaOH and determined by titration with HCl. The metabolic quotient (qCO2) was defined as the ratio of Rmic to Cmic, i.e., the amount of CO2-C produced per unit of Cmic (Anderson and Domsch, 1986).
Soil enzyme activities were determined using the methods described by Guan (1986). Catalase activity was determined by back-titrating residual H2O2 with a standard solution of 0.1 M KMnO4 in the presence of H2SO4. Invertase activity was determined with the 3,5-dinitrosalicylic acid colorimetry method using sucrose as the substrate, and the amount of glucose released was assayed colorimetrically at 508 nm. Cellulase activity was measured using carboxymethyl cellulose as the substrate, and the amount of glucose released was assayed with 3,5-dinitrosalicylic acid colorimetry at 540 nm. Urease activity was determined using urea as the substrate, and the released ammonium was assayed colorimetrically using the indophenol blue method at 578 nm. Proteinase activity was measured colorimetrically using casein as the substrate with the ninhydrin method at 500 nm. Phosphatase activity was analyzed using disodium phenyl phosphate as the substrate, and the amount of phenol released was assayed colorimetrically at 660 nm.
Biolog EcoPlates (Biolog Inc., Hayward, CA) were used to study the substrate use patterns of the soil microbial communities, as described by Girvan et al. (2003). Briefly, 10 g of fresh soil was added to 100 mL of distilled water in a 250-mL flask and shaken at 200 rpm for 10 min to achieve a 10−1 dilution. Ten-fold serial dilutions were prepared, and the 10−3 dilution was used to inoculate the Biolog EcoPlates. The plates were incubated at 25 °C for 6 d, and color development was read daily as absorbance at 590 nm with a Biolog microplate reader.
All data were expressed as means and sds and were compared statistically using Fisher's least significant difference test at the 5% level with the software package SPSS 16.0 for Windows (SPSS Inc., Chicago, IL). The average well color development (AWCD) value of the Biolog data was calculated at each time point by dividing the sum of the optical density data by 31 (number of substrates). The functional diversity of the microbial community was assessed by the Shannon index (Zak et al., 1994) calculated using the following formula:
where H′ is the value of the Shannon index and pi is the proportional color development of the ith well relative to the total color development of all wells. For the principal component analysis (PCA) of the carbon use pattern, the absorbance values at equivalent AWCD from different incubation times were compared; these values were also transformed through dividing by the AWCD to avoid bias between samples with different inoculum densities (Garland, 1997).
Soil Cmic, Rmic, and the ratio of microbial biomass C to soil organic C (Cmic/Corg) showed similar trends as growth duration increased (Fig. 1). For the tree peony cultivars Yao Huang and Dou Lu, soil Cmic, Rmic, and Cmic/Corg were higher at the 5-year sites than in the wasteland; these values then decreased through the 12- and 25-year sites. For ‘Er Qiao’ and ‘Shou An Hong’, soil Cmic, Rmic, and Cmic/Corg first increased and then decreased with the maximum values found at the 12-year sites. In addition, the soil under the cultivar Dou Lu had comparatively lower Cmic, Rmic, and Cmic/Corg than those under the other cultivars. Moreover, the soil under all four cultivars at the 25-year sites had significantly lower Cmic/Corg than that of the wasteland.
Citation: HortScience horts 49, 11; 10.21273/HORTSCI.49.11.1408
The conversion from wasteland to growing sites of tree peony resulted in a significant decrease in qCO2. However, as the tree peony garden ecosystem developed, qCO2 increased from the 5- to the 25-year sites for ‘Yao Huang’ and ‘Dou Lu’ and from the 12- to the 25-year sites for ‘Er Qiao’ and ‘Shou An Hong’.
Catalase, proteinase, and phosphatase activities increased significantly after the wasteland was reclaimed as growing sites of tree peony and decreased from the 5- to the 25-year sites for all four peony cultivars (Fig. 2). Both invertase and cellulase were significantly higher for the four cultivars at the 5-year sites than in the wasteland, then decreased from the 5- to the 25-year sites for ‘Yao Huang’ and ‘Dou Lu’ and from the 12- to the 25-year sites for ‘Er Qiao’ and ‘Shou An Hong’. Urease activity followed the same pattern for the four tree peony cultivars, increasing from the wasteland through 12 years of growth and then decreasing.
Citation: HortScience horts 49, 11; 10.21273/HORTSCI.49.11.1408
After 48 h of incubation, the AWCD of the C sources for the four tree peony cultivars followed the same pattern with increasing growth duration, i.e., 5-year sites > 12-year sites > wasteland > 25-year sites (Fig. 3).
Citation: HortScience horts 49, 11; 10.21273/HORTSCI.49.11.1408
The functional diversity of the microbial communities measured by the Shannon index is shown in Figure 4. The growth duration of tree peony had significant effects on the Shannon index of soil microbial communities. For all four peony cultivars, the Shannon index increased significantly from the wasteland to the 5-year sites and decreased significantly from the 5- to the 25-year sites. Significant differences were also observed between the Shannon index of the different tree peony cultivars: the index for ‘Dou Lu’ was significantly lower than those of the other cultivars.
Citation: HortScience horts 49, 11; 10.21273/HORTSCI.49.11.1408
The PCA based on carbon use pattern revealed a separation of soil microbial communities in response to growth duration and cultivar (Fig. 5). Moreover, the PCA clearly separated the soil microbial communities into four groups based on years of growth and irrespective of cultivar, indicating that growth duration had a greater effect on these soil microbial communities than cultivars. The first principal component (PC1) accounted for 67.3% of the variance, and the second principal component (PC2) accounted for 13.2% of the variation. The factor loadings of PCA indicated that the microbial communities at the 5-year sites had higher uses of L-threonine, glucose-1-phosphate, L-asparagine, α-cyclodextrin, L-phenylalanine, glycyl-L-glutamic acid, L-arginine, and N-acetyl-D-glucosamine compared with those at the wasteland. After 5 years of tree peony growth, the use of these C sources decreased as growth continued.
Citation: HortScience horts 49, 11; 10.21273/HORTSCI.49.11.1408
All of the studied soils were originally wastelands covered with sparse grasses before the planting of tree peony. Consequently, the wasteland is typical of unmanaged soils and can be considered as a control for the study sites. We found that the planting of tree peony on wasteland and its subsequent growth markedly affected soil chemical, microbiological, and biochemical properties, which suggested that the soil processes at the tree peony sites were profoundly different from those occurring in the wasteland and varied as tree peony growth progressed. Our results also indicated that vegetative cover had fundamental effects on soil properties (Yao et al., 2000).
Soil Cmic represents the living portion of soil organic C and is among the most sensitive soil organic C fraction. In general, the accumulation of soil organic C enhances both Cmic and Cmic/Corg (Liang et al., 2012; Yao et al., 2000). However, in this study, we found that both Cmic and Cmic/Corg were lower at the 25-year sites than at the 12-year sites for all tree peony cultivars, although the 25-year sites had higher organic C. Together with the similar results obtained for the Rmic and AWCD of the C sources, which indicate the whole activity of the microbial community, this result convincingly demonstrated that the soil organic C available to microbes did not increase proportionately with the accumulation of soil organic C over the development of the tree peony garden system (Anderson and Domsch, 2010). In addition, soil Cmic generally accounts for 1% to 4% of total soil organic C (Sparling, 1992). Most of the tested soils in present study exhibited Cmic/Corg contents approximately within this range. However, as a result of the development of the tree peony garden system, soil Cmic composed much less than 1% of the soil organic C at the 25-year sites for all four tree peony cultivars. The continued growth of tree peony increased pH and decreased soil total N, which adversely impacted microbial activity and resulted in a low capacity of organic C mineralization (Cho et al., 2008). Moreover, the sustained rhizodeposition of tree peony caused the accumulation of soil organic C, consequently leading to lower Cmic/Corg at the 25-year sites. These results also suggested that soil Cmic and Cmic/Corg responded more rapidly to changes resulting from tree peony growth than did soil organic C; these factors may therefore be used as rapid markers for detecting changes in soil quality and provide early indications of soil ecosystem degradation resulting from the continued growth of tree peony.
Measurements of qCO2 provide further information on the ecophysiological state of microbes and have been used in several investigations to assess microbial stress (Anderson and Domsch, 2010); qCO2 is generally considered to be higher under unfavorable conditions than under favorable conditions (Anderson and Domsch, 2010). We observed that qCO2 first decreased and then increased after the wasteland was reclaimed by all four tree peony cultivars. This result indicated that soil ecosystems may be made unsuitable for microbial growth by the continued growth of tree peony. As a result, microbes divert more C to maintain respiration rather than to synthesize new microbial biomass during the mineralization of organic matter (Franco-Otero et al., 2012).
Soil enzyme activities in natural soils are positively related to organic matter content (Guan, 1986; Fu et al., 2012). Moreover, considerable evidence also suggests that soil enzyme activities are sensitive to environmental conditions, including soil chemistry, microbial populations, the presence or absence of toxic compounds, and the quantity and quality of organic matter (Dai et al., 2013; Paudel et al., 2012). In the present study, significant differences in variation were observed for all six examined enzyme activities (catalase, invertase, cellulose, urease, proteinase, and phosphatase) and organic matter. These results suggested that both positive and negative factors influenced enzyme activities in tree peony garden ecosystems, causing soil enzyme activities to first increase and then decrease with continued tree peony growth.
According to McGuire and Treseder (2010), the functional diversity of the microbial community is useful in assessing ecosystem functions. Before the present study, little information was available regarding the influence of tree peony growth on microbial functional diversity. The Shannon index based on community-level substrate use in the Biolog plates indicated the functional diversity of the soil microbial community. In the present study, the functional diversity of the soil microbial community increased significantly with the conversion from wasteland to growing sites of tree peony; however, as tree peony growth continued, the functional diversity of microbial community decreased significantly from the 5- to the 25-year sites. This trend may be explained by the following reasons. Tree peony can continually provide soil microbes with nutritive materials by releasing root exudates and rhizodeposits. In addition, the changes in soil chemical properties resulting from continued tree peony growth have lasting impacts on soil microbial communities and functional diversity (Chaerun et al., 2011). Moreover, the reduction in the functional diversity of the microbial community resulting from continued growth may also be ascribed to differences in root exudation, because the quantity and chemical composition of root exudates change over the course of tree peony growth (Marschner et al., 2002). The higher functional diversity of the soil microbial community at the 5-year sites reflects the combination of many factors. The PCA of the C use pattern for all four tree peony cultivars showed decreasing PC1 scores as growth continued after 5 years. This result may be associated with shifts in microbial community structure and decreases in the use of specific C or other compounds caused by the development of tree peony garden ecosystems.
The present study provided evidence that the sustained growth of tree peonies significantly affected soil chemical, microbiological, and biochemical properties. Tree peony growth resulted in the increase of organic matter content and the improvement of soil nutrients; therefore, the conversion from wasteland to growing sites of tree peony may be conducive to soil microorganisms. However, tree peony growth also increased soil pH and decreased N-supplying capacity, which adversely impacted soil microorganisms. The combined effects of tree peony growth ultimately led to the degradation of soil quality after 5 years. The growth duration of tree peony should thus be considered in managing the ecological stability of tree peony garden systems.
Contributor Notes
This work was supported by the National Natural Science Foundation of China (No. 41101222) and the Project of Science and Technology Department (No. 112300410139, 132102210225) and Education Department (No. 2011A210024, 13A610788) of Henan Province.
The authors greatly appreciate the support provided by National Peony Garden of Luoyang city in China.
To whom reprint requests should be addressed; e-mail xuedong78@163.com.
