Phenanthrene with a purity of 98% was purchased from Sigma (USA). The rest chemicals were purchased from Dingguo (Tian jin, China).
Soil used in the study
The experiment soil, never contaminated by PAHs or heavy metals was collected from the topsoil (0-20 cm) of Hebei University of Technology, China. The soil was a kind of phaeozems (alfisol) in Hebei Province .The test soil was sieved by 2 mm sieve after dried by air. 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, 1.1% organic matter.The soil pH was 7.36 ± 0.06.
Preparation of phenanthrene contaminated soil
Phenanthrene was dissolved in acetone and added into a small part soil, one day later, after acetone volatilized, the small part soil was added into the whole sample soil and incorporated thoroughly, the final concentration of phenanthrene in soil was measured as 40+3 mg kg-1.
Preparation of cadmium contaminated soil
The aqueous solution of cadmium nitrate was added to the prepared soil, the final concentration of cadmium in soil was measured as 10+2 mg kg-1.
Preparation of phenanthrene and cadmium co-contaminated soil
The acetone stock solution containing phenanthrene was firstly added to the test soil. After acetone evaporated, cadmium nitrate aqueous solution was added to the previous soil polluted by phenanthrene. The final concentration of cadmium and phenanthrene in soil were measured as 10+2 mg kg-1 and 40+3 mg kg-1 respectively. The well prepared soil and a control soil without any pollution were shifted to boxes and aged in the dark for 15 days.
In work, the isolate of P. indica DSM11827 (German collection of microorganisms and cell cultures in Braunschweig, Germany) was applied. P. indica was supplied by Karl-Heinz-Kogel (Institute of Plant Pathology and Applied Zoology, Giessen, Germany). P. indica was maintained at 23℃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 will affect IAA production contents, 5 mg kg-1 and 20 mg kg-1 of cadmium and phenanthrene were added into the liquid cultures, respectively. CM medium was inoculated with 20 mycelium plugs from the margin of a growing colony of P. indica on CM solid medium; liquid cultures were incubated at 23℃ at 150 rpm on a rotary shaker.
Quantification of IAA in fungal growth media by HPLC
IAA production ability of P. indica under Phe, Cd and Phe+ Cd treatment was performed using Salkowski-method according to the literature . Quantification of IAA in 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 are as follows: 50% methanol: 45% water: 5% acetonitrile (v/v) was used as the mobile phase. The 0.2 mL min – 1 of flow rate was applied. The injection volume was 10 µl. Column temperature was kept at 40℃.The content of IAA was quantified by Agilent HPLC which equipped with an HCR C18 (5 µm, 4.6 x 250 mm, Agilent, USA) reversed phase chromatographic column. 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 growing on CM medium plates for 3-4 weeks, was ready for preparation of spore suspensions. To collect spores from CM agar plates, sterilized water containing 0,05% Tween-20 was added. Through gently scratching the surface of plates with a spatula, the spores were released and the suspension solution was filtered through miracloth (Calbiochem, Bad Soden, Germany) in order to remove mycelium. After that, spores were collected by centrifuging suspension solution at 3500 rpm for 7 minutes. Then, spores were washed at least 3 times with sterilized Tween-H2O. By using a hemacytometer in combination with a microscope, 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 one squared petri-dish. The seeds of M. sativa were surface sterilized in 70% alcohol for 1 min and then 3% NaClO for 15 min. After sterilized, seeds were washed by sterile deionized water repeatedly and planted into the MS medium  for germination. Seven days when the roots were presence, P. indica spores were added into the root surface. Then the seedlings contained P. indica spores were transferred into different types of soil in pot (5 kg). Three replicates were applied for each treatment.
The plants were rejuvenated for one week in shade. After then, pots were transferred to green house with natural light and watered daily to keep the soil moisture (approximate 300 mL water/ pot). Three months later, plants were harvested and soil from the rhizosphere and non- rhizosphere were collected respectively from the roots of M. sativa .
Measurement of chlorophyll contents and chlorophyll fluorescence
Chlorophyll content in the youngest fully expanded leaves (0.1 g) was extracted by 80% acetone, centrifuged at 4000 rpm for 20 min, and then 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 factors on photosystem II (PSII) efficiency.
Measure of soil microorganism activity
Soil microorganism activity was measured referenced to the literature . Firstly, the soil was freeze-dried by Freeze drying machine (Alpha 1-2 L D plus, Germany ). The 5 g freeze-dried soil was dissolved in 15 mL 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 shaked 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 non- rhizosphere
Similar 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 chromatograph (HPLC) ultraviolet detector detection method was used. Ten gram freeze-dried soil samples was dissolved in 50 mL of acetone- hexane (1:1, v/v) mixed extraction solvent. The progress of extraction was performed for 1 h in the ultrasonic cleaners. Extracted liquid was filled with filter funnel containing 10 g anhydrous Na2SO4. The extracted liquid was concentrated to 5 mL by rotary evaporation instrument in 60℃water bath. Then 5 mL concentrated extracted liquid was transferred to silica gel-Alumina column chromatography and washed with methylene chloride-hexane (1:1, v/v) mixture elution. The condensed elution nearly drying was diluted to a final volume of 1mL and used for HPLC determination. The parameters used for HPLC are as follows: methanol and water (87:13, v/v) was used as the mobile phase. The 1 mL min – 1 of flow rate and 254 nm of detection wave length was applied. The content of phenanthrene was quantified by Agilent HPLC which equipped with an HCR C18 (5 µm, 4.6 x 250 mm, Agilent, USA) reversed phase chromatographic column. The recoveries for 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 to 1 mL hexane to be analyzed. The content of phenanthrene was quantified by HPLC which equipped with a HCR C18 (5 µm, 4.6 x 250 mm, Agilent, USA) reversed phase chromatographic column. The oven temperature was firstly maintained at 100℃for 2 min, then increased to 300℃ at a rate 10℃ min−1, and finally kept at 300℃for 10 min. Phenanthrene standard was added to the uncontaminated plants and soil to measure the phenanthrene recovery. Procedural blank together with spiked blank and duplicate samples were included with every batch of ten samples in the analysis. The recoveries for plants was 99 ± 5%.
Analysis of cadmium in soil and plant
Cadmium was determined according to the references [40, 49]. Soil about the rhizosphere and non- rhizosphere of M. sativa was extracted by mixing 0.5 g soil with 10 mL HCl solution and then heating for 3 h (45℃). After cool down, Cadmium was extracted by mixing the soil with HNO3 and HClO4 (v/v, 4:1) for digestion (220℃, 1 h) and then adding HF and HClO4 (v/v, 5:1) for further digestion (220℃, 2 h). The same method was used for cadmium extraction from plants. At the end of extraction step, the supernatant was harvested by centrifuging at 6000 rpm, 20 min. Then the supernatant was filtered through 0.45 µm microfiltration membrane and quantified by Inductively Coupled Plasma Optical Emission Spectrometry (ICPOES).
Analysis of enzyme activity in the rhizosphere and non- rhizosphere
Enzyme activity of polyphenol oxidase was determined according to the protocol book of soil enzymes and analytical methods . Ten gram freeze–dried soil was dissolved in 10 mL pyrogallol (1%) and shaked with 150 r. min-1 for 1 min. Then the sample was kept in dark at 30℃ for 2 h. Four milliliter citric acid - phosphate buffer ( disodium hydrogen phosphate-35.61g/ L, citric acid-21.01g/ L, pH4.5) was added into the sample. Finally, 35 mL ether was added and shaked for 2 min. The absorbance value at 430 nm was recorded after 30 min extraction.
In this study, all data are expressed as means ±SE and represent at least three independent biological experiments. The significant differences were analyzed by using a one-way analysis of variance (ANOVA), which referred to Duncan’s multiple range test.