Medicago sativa seeds were provided by Professor Diter Von Wettstein, Department of Crop and Soil Sciences, Washington State University.
Phenanthrene with a purity of 98% was purchased from Sigma (USA). The rest of the chemicals were purchased from Dingguo (Tian Jin, China).
Soil used in the study
The experimental soil had never been contaminated by PAHs or heavy metals and was collected from the topsoil (0-20 cm) at Hebei University of Technology, China. The soil is a phaeozem (alfisol) from Hebei Province . The test soil was sieved with a 2 mm sieve after being air-dried. The soil samples were measured using standard methods  before phytoremediation. The composition of the sample soil was physico-chemically characterized: 55.6 ± 2.1% silt, 30.8 ± 1.8% sand and 13.6 ± 1.5% clay; 0.06% total N, 8 mg/kg available P, 40 mg/kg available K, and 1.1% organic matter. The soil pH was 7.36±0.06.
Preparation of phenanthrene-contaminated soil
Phenanthrene was dissolved in acetone and added to a small part of the soil. One day later, after the acetone had volatilized, a small part of the soil was added to the whole sample soil and incorporated thoroughly. The final concentration of phenanthrene in the soil was measured as 40±3 mg kg-1.
Preparation of cadmium-contaminated soil
An aqueous solution of cadmium nitrate was added to the prepared soil, and the final concentration of cadmium in the soil was measured as 10±2 mg kg-1.
Preparation of phenanthrene and cadmium co-contaminated soil
The acetone stock solution containing phenanthrene was first added to the test soil. After the acetone evaporated, the cadmium nitrate aqueous solution was added to the soil previously polluted by phenanthrene. The final concentrations of cadmium and phenanthrene in the soil were measured as 10±2 mg kg-1 and 40±3 mg kg-1, respectively. The prepared soil and a control soil without any pollution were moved into boxes and aged in the dark for 15 days.
In this work, the P. indica isolate DSM11827 (German collection of microorganisms and cell cultures in Braunschweig, Germany) was applied. The P. indica was supplied by Karl-Heinz-Kogel (Institute of Plant Pathology and Applied Zoology, Giessen, Germany). The P. indica was maintained at 23°C on CM medium . For solid medium, 14 g L-1 agar was added; for liquid cultures, 100 mL medium was inoculated in a 300-mL Erlenmeyer flask. To test whether Cd and Phe would affect IAA production, 5 mg kg-1 and 20 mg kg-1 cadmium and phenanthrene, respectively, were added to the liquid cultures. The CM medium was inoculated with 20 mycelium plugs from the margin of a growing colony of P. indica on CM solid medium. The liquid cultures were incubated at 23°C at 150 rpm on a rotary shaker.
Quantification of IAA in fungal growth media by HPLC
The IAA production ability of P. indica under the Phe, Cd and Phe+ Cd treatments was measured using the Salkowski method according to the literature . The quantification of IAA in the fungal growth media was performed by HPLC. Fungal culture filtrates were harvested, acidified and extracted twice with ethyl acetate as described in . The parameters used for HPLC were as follows: 50% methanol: 45% water: 5% acetonitrile (v/v) was used as the mobile phase. A flow rate of 0.2 mL min–1 was applied. The injection volume was 10 µl. The column temperature was kept at 40°C.The IAA content was quantified by Agilent HPLC equipped with an HCR C18 (5 µm, 4.6 x 250 mm, Agilent, USA) reverse-phase chromatographic column. The IAA concentrations were always determined in parallel in medium in which no fungus had been cultured but which had been incubated under the same conditions.
M. sativa treatment and P. indica inoculation
P. indica grown on CM medium plates for 3-4 weeks was used for the preparation of the spore suspensions. To collect spores from the CM agar plates, sterilized water containing 0.05% Tween-20 was added. The spores were released by gently scratching the surface of plates with a spatula, and the suspension solution was filtered through Miracloth (Calbiochem, Bad Soden, Germany) to remove the mycelium. After that, spores were collected by centrifuging the suspension solution at 3500 rpm for 7 min. Then, the spores were washed at least 3 times with sterilized Tween-H2O. By using a hemacytometer in combination with a microscope, the spore densities were determined. The spore concentration was adjusted to 500,000 spores/mL with sterilized Tween-H2O. For inoculation, three mL spore suspension was pipetted on top of plant roots in a square Petri dish. The seeds of M. sativa were surface-sterilized in 70% alcohol for 1 min and then in 3% NaClO for 15 min. After sterilization, seeds were repeatedly washed with sterile deionized water and planted in the MS medium  for germination. After seven days, when roots had grown, P. indica spores were added to the root surface. Then, the seedlings containing P. indica spores were transferred into the different types of soil in pots (5 kg). Three replicates were applied for each treatment.
The plants were rejuvenated in the shade for one week. Then, the pots were transferred to a greenhouse with natural light and watered daily to maintain soil moisture (approximately 300 mL water/pot). Three months later, the plants were harvested, and soil from the rhizosphere and nonrhizosphere of M. sativa was collected.
Measurement of chlorophyll contents and chlorophyll fluorescence
Chlorophyll content in the youngest fully expanded leaves (0.1 g) was extracted by 80% acetone and centrifuged at 4000 rpm for 20 min; the optical density of the supernatant was read at 663 and 645 nm wavelengths for Chl a and Chl b, respectively . The following parameters of chlorophyll fluorescence were measured by analyzing the first fully grown leaves of M. sativa using a portable fluorometer (Hansatech, Instruments LTD, UK): F0 (minimal fluorescence level in the dark-adapted state), Fm (maximal fluorescence level in the dark-adapted state), Fv/Fm (maximum quantum efficiency of PSII photochemistry) and ETR (the relative PSII electron transport rate). M. sativa plants were dark-adapted for 30 min to measure the influence of the experimental factors on photosystem II (PSII) efficiency.
Measure of soil microorganism activity
Soil microorganism activity was measured according to methods in the literature . First, the soil was freeze-dried by a freeze-drying machine (Alpha 1-2 L D plus, Germany). Five grams of freeze-dried soil was dissolved in 15 mL of phosphate buffer solution (NaCl–8.5 g, Na2HPO4–2.2 g, NaH2PO4–0.1 g, pH 7.6) at room temperature. The turbid liquid was shaken at 180 rpm for 30 min, and then 0.5 mL of fluorescein diacetate (FDA) (2 g L−1, in acetone) solution was added into the mixture. The absorbance value was recorded at OD490nm.
Analysis of phenanthrene in the rhizosphere and nonrhizosphere
A three-step sequential extraction method was used to detect the concentrations of phenanthrene in different chemical speciations in the soil . The ultrasonic extraction and high-performance liquid chromatography (HPLC) ultraviolet detection method was used. Ten grams of freeze-dried soil sample was dissolved in 50 mL of acetone-hexane (1:1, v/v) mixed extraction solvent. The extraction process was performed for 1 h in the ultrasonic cleaner. The extracted liquid was poured into a filter funnel containing 10 g anhydrous Na2SO4. The extracted liquid was concentrated to 5 mL by a rotary evaporation instrument in a 60°C water bath. Then, 5 mL concentrated extracted liquid was transferred to a silica gel-alumina column for chromatography and eluted with a methylene chloride-hexane (1:1, v/v) mixture. The condensed elution was nearly dried, diluted to a final volume of 1 mL and used for HPLC determination. The parameters used for HPLC were as follows: methanol and water (87:13, v/v) were used as the mobile phase. A flow rate of 1 mL min – 1 and detection wavelength of 254 nm were applied. The content of phenanthrene was quantified by Agilent HPLC equipped with an HCR C18 (5 µm, 4.6 x 250 mm, Agilent, USA) reverse-phase chromatographic column. The phenanthrene recovery from soil was 97 ± 3%.
Analysis of phenanthrene in plants
The phenanthrene in plants was extracted by acetone and dichloromethane (v/v, 1:1). After centrifugation and rotary evaporation, the concentrated phenanthrene was exchanged with 1 mL hexane for analysis. The content of phenanthrene was quantified by HPLC equipped with an HCR C18 (5 µm, 4.6 x 250 mm, Agilent, USA) reverse-phase chromatographic column. The oven temperature was first maintained at 100°C for 2 min, then increased to 300°C at a rate 10°C min−1, and finally kept at 300°C for 10 min. A phenanthrene standard was added to the uncontaminated plants and soil to measure phenanthrene recovery. A procedural blank as well as a spiked blank and duplicate samples were included every batch of ten samples in the analysis. The phenanthrene recovery from plants was 99 ± 5%.
Analysis of cadmium in soil and plants
Cadmium was determined according to methods in the references [40, 49]. Soil from the rhizosphere and nonrhizosphere of M. sativa was extracted by mixing 0.5 g soil with 10 mL HCl solution and then heating for 3 h (45°C). After cooling, cadmium was extracted by mixing the soil with HNO3 and HClO4 (v/v, 4:1) for digestion (220°C, 1 h) and then adding HF and HClO4 (v/v, 5:1) for further digestion (220°C, 2 h). The same method was used to extract cadmium from the plants. At the end of the extraction step, the supernatant was harvested by centrifuging at 6000 rpm for 20 min. Then, the supernatant was filtered through a 0.45 µm microfiltration membrane and quantified by inductively coupled plasma optical emission spectrometry (ICPOES).
Analysis of enzyme activity in the rhizosphere and nonrhizosphere
The enzyme activity of polyphenol oxidase was determined according to the soil enzyme analytical methods manual . Ten grams of freeze-dried soil was dissolved in 10 mL pyrogallol (1%) and shaken at 150 rpm min-1 for 1 min. Then, the sample was kept in the dark at 30°C for 2 h. Four milliliters of citric acid-phosphate buffer (disodium hydrogen phosphate-35.61 g L-1, citric acid-21.01 g L-1, pH 4.5) was added to the sample. Finally, 35 mL ether was added and shaken for 2 min. The absorbance value at 430 nm was recorded after 30 min of extraction.
In this study, all data are expressed as the means ± SE and represent at least three independent biological experiments. The significance of differences was analyzed by using one-way analysis of variance (ANOVA) with Duncan’s multiple range test.