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Hydrolysis and nitrification are the key steps of biological transformation of urea in soils, which control the loss of nitrogen (N) in terrestrial ecosystem. The inhibitory effects of plant materials on N transformation provide potential benefits for agricultural production and environmental protection. In this study, water-soluble and ethanol-soluble compounds were extracted from 24 tropical medicinal plant species in Hainan Island, southern China, with the aim of measuring their effects on urease activity (UA), nitrification potential (NP), and ammonium oxidizer number in a paddy soil, by a series of short (5 hours) and long-term (14 days) incubation experiments. Total solid yields and phenols contents of 48 medicinal plant extracts were also analyzed in this study. There were big differences in total solid yield and phenols content among the extracts of the selected tropical medicinal plant extracts. The phenols content of the plant extracts varied from 15.1 to 273.4 mg GAE/g extract (milligrams of gallic acid equivalents per gram of extract). A significant relationship between UA and NP of the soil treated with plant extracts and their phenols content was found, indicating that phenolic compounds are the major contributors to the inhibitory properties of these plants. Further studies indicated that the water extracts of Epimeredi indica aerial and Melia azedarach leaf, containing a high content of phenols, exhibited notable urease and nitrification inhibitory effects and reduced the number of soil ammonia–oxidizing bacteria, being promising sources of natural inhibitors for soil N transformation. Urea has become one of the most commonly used N fertilizers in the world due to its high N content, stable nature, easy application, and other characteristics (Subbarao et al., 2012). However, urea applied to the soil surface can be rapidly hydrolyzed into ammonia (NH3) or ammonium (NH4 ) by soil urease, which easily causes NH3 volatilization and leads to high concentration of NH3 in soil microzone, thus harming germinating seedlings and young plants (Subbarao et al., 2013). In addition, NH4 + can be easily transformed into nitrate (NO3 ) via nitrification and NO3 – may be lost in harmful forms of nitrous oxide (N2O) and nitric oxide (NO) by denitrifying bacteria or leaching through the soil to contaminate groundwater (Arango et al., 2014). Overall, the losses of N fertilizers may exceed 70% of the total application amount and cause serious environmental issues (Upadhyay et al., 2011). Therefore, to ensure continuous and optimal N supply, improve use efficiency of N fertilizers, and protect the environment, regulation strategies should be adopted based on reducing urea hydrolysis and nitrification. Adding urease inhibitors into urea or nitrification inhibitors into NH4 form and amide N-form fertilizers is an appropriate choice (Zaman et al., 2009). A large number of studies have been conducted on urease inhibitors such as N-(n-butyl)thiophosphoric triamide (NBPT), phenyl phosphorodiamidate (PPD), and hydroquinone (HQ), as well as nitrification inhibitors such as dicyandiamide (DCD), nitrapyrin (N-serve), and 3,4-dimethylpyrazole phosphate (DMPP), some of which have been confirmed to have excellent potential to improve use efficiency of N and reduce environmental risks of applying N fertilizers (Arora and Srivastava, 2013). However, due to their high prices, poor availability, and adverse influences on beneficial microorganisms in the soil, most of synthesized urease and nitrification inhibitors are limited to the experimental stage and are not suitable to be popularized in agricultural production practice, especially in developing countries. Phenolic compounds isolated from certain plants influence N cycling, which results in a decrease of N losses and represents a key issue for environmental protection worldwide. Plant metabolites and plant-based biochemical metabolic inhibitors have been considered as the best alternatives to synthetic urease and nitrification inhibitors due to their minimal environmental impact and biological damage (Patra et al., 2006). Therefore, the search for inhibitors from natural sources has received much attention, and efforts have been made to identify new natural resources for N efficiency improving compounds. Previous studies have shown that a number of extracts derived from higher plants can effectively inhibit UA and NP. Many empirical studies have pointed out that some plants and their by-products can inhibit urea hydrolysis or nitrification or both. To be specific, karanja (Pongamia glabra), neem (Azadirachta indica), and mint (Mentha spicata) are important sources of natural inhibitors (Abbasi and Manzoor, 2013; Kiran and Patra, 2003; Opoku et al., 2014). A wide variety of tropical medicinal plants that are traditionally used to cure diseases, or to maintain and improve health, have been developed and applied to disinfection, sterilization, virus inactivation, antioxidation, and cosmetics (Schmidt, 2012). The aim of this study was to evaluate the inhibitory effects of extracts obtained from a small part of medicinal plant species that grow in Hainan Island, southern China, on urea hydrolysis and nitrification in soil, to obtain some biodegradable, eco-friendly, and inexpensive natural compounds capable of increasing N-use efficiency in agricultural soils. Moreover, this study is focused on the relationship between phenols in the extracts of selected medicinal plants and their urease/nitrification inhibitory effects. The phenolic compounds constitute a large group of secondary plant metabolites that are ubiquitous among higher plants. They are natural compounds, which generally help plants for defense, and widely known antibacterial agents that inhibit the activity of bacteria and fungi (Rauha et al., 2000). Materials and Methods Soil. In the present study, a paddy soil originated in granite latosol was used. The soil was collected from Miaolin Farm at Fenghuang town, Sanya, Hainan Province, China (18.305 N, 109.463 E), with a pH of 5.24 (soil:water =1:2.5, w/v), organic matter of 18.65 g·kg, total N of 1.16 g·kg, NH4 -N of 6.2 mg·kg, and NO3 -N of 17.4 mg·kg. Composite samples were taken at 0–10 cm of depth. Moist field soil samples were sieved through 2 mm to remove stone and plant roots and then stored at –10 C to inhibit biological activity. The original UA and NP of the moist soil were 32.4 mg UreaN per kg·h and 0.925 mg NO2 -N per kg·h, respectively, measured with the Received for publication 18 Feb. 2015. Accepted for publication 18 Feb. 2015. This study was supported by the Natural Science Foundation of Hainan Province (312100), the National Natural Science Foundation of China (41301324), the National Science and Technology Support Program of China (2011BAD11B04), and the Program for Hainan cultivated land improvement (HNGDg121501). We gratefully acknowledge Zhunian Wang from Chinese Academy of Tropical Agricultural Sciences for the assistance in plant species identification. To whom reprint requests should be addressed; e-mail shiyunfeng8189@sina.com. 744 HORTSCIENCE VOL. 50(5) MAY 2015 same methods as described in the following short-term incubational experiments. Tropical medicinal plant materials and their extracts. A total of 24 species of tropical medicinal plants were collected from Danzhou Tropical Medicinal Plant Germplasm Resource Nursery, Chinese Academy of Tropical Agricultural Sciences during Sept. and Dec. 2013. The selection of the plant materials in the present study was based on their potential medicinal uses and their distribution in sampling locations. The Latin names of the plant species, used parts, and labels in this article were shown in Table 1. Plant samples were brought back to the laboratory, washed, air-dried, crushed into powder with a particle size of less than 0.37 mm, and preserved under dark refrigerated condition. The powdered plant samples were dissolved in water or ethanol at the ratio of 1:10 (solid:liquid, w/v) and ultrasonically extracted for 20 min. The extracts were centrifuged at the speed of 12,000 rpm for 10 min. The residues were extracted twice with the same fresh solvent, and all the extracts combined. Water and organic extracts were concentrated in a rotary evaporator, and then the extracts were freeze-dried. After that, the solid matters were weighted and the total solid yields were calculated and expressed in terms of milligrams of extracts per gram of dry weight material (Table 1). The phenols content of plant extracts was determined by the spectrophotometric method using Folin–Ciocalteu reagent, with gallic acid (GA) as the standard (Singleton et al., 1999). The results were expressed in terms of milligrams of GA equivalents per gram of extract (mg GAE/g extract) (Table 1). Short-term incubation experiments. The effects of the 48 tropical medicinal plant extracts obtained with water and ethanol on soil UA and NP were assayed by the following short-term incubation experiments. The soil UA in short-term incubation was determined with urea-N residual method (Tabatabai, 1994). Briefly, a 5 mL of urea solution (2 mg urea-N/mL) containing 0.25 mg of plant extract was added to 5 g of soil, incubating at 37 C for 5 h. After that, the soil was extractedwith a 50mL of KCl (2 mol·L)-phenyl mercuric acetate (5 mg·L) solution, and urea-N content was estimated. The results were expressed in Table 1. Total solid yields and phenols contents of waterand ethanol-extracted plant extracts and urease activity (UA) and nitrification potential (NP) in the plant extract-treated soil under 5-h incubation. The data were shown as means of triplicated measurements with SD. The plant extracts for further study are also

Inhibitory Effects of Tropical Medicinal Plant Extracts on Urea Hydrolysis and Nitrification in Soil: A Preliminary Study