Sampling
Soil samples were collected from the three alpine zones on the eastern slope of Gongga Mountain (the highest mountain in Sichuan, China,with a peak of 7556 m). The three alpine zones were dominated by alpine meadows at the high elevation zone (4200 m), bushes and pine forests at the middle elevation zone (3600 m) and alpine dark coniferous forests at the low elevation zone (3000 m) (Table S1). In each zone, we selected intact areas with flat terrain, zonal vegetation and zonal soils, and established six plots (2 m × 2 m, > 20 m intervals among the plots). We set up three sampling points (> 0.5 m intervals among sampling points) along the diagonal line of each plot. Mineral soils (a depth of 0 – 30 cm) were collected at sampling points of the plot after removal of the forest floor, and then these soils were passed through a 2 mm sieve and homogenized to obtain a representative sample for the plot. Finally, we got six soil samples from six plots at each zone. The fresh soil samples were put into ice boxes for transport back to the laboratory.
Preparation of phosphate-solubilizing microbial community inoculant.
In this study, a “phosphate-solubilizing microbial community (PSM-Community)” is defined as follows: the phosphate-solubilizing microbial community is a set of all of the microorganisms that can grow ina selective medium for the isolation of phosphate-solubilizing microorganisms. In the present experiment, Pikovskaya (PVK)(Pikovskaya, 1948) liquid mediumwas used as the selective medium for the isolation of phosphate-solubilizing microorganisms. Preparation of the PSM-Community inoculant was performed as follows. Under sterile conditions, 10 g of fresh soil was placed into a flask with 90 ml of purified water, and then the flask was sealed with a stopper. The flask was shaken for 30 minutes to form a soil suspension. Then, 0.5 ml of the suspension was sucked up with a pipette and injected into a flask with 100 ml of PVK liquid medium. After sealing with a stopper, the flask was incubated at 25 °C on an incubator shaker at 140 rpm. The incubation was stopped after three days when the concentration of microorganisms was greater than 1 × 107 CFU/ml in the PVK broth. The broth with microorganisms was the original inoculum used for the phosphate-solubilizing experiments. In the present study, the inoculantof 4200 m PSM-Community, the inoculantof 3600 m PSM-Community and the inoculantof 3000 m PSM-Community were obtained according to the soil origins.
Experimental design
We conducted a 3 × 6 shaking culture experiment in which six replicates were established for each combination of eighteen treatments, including three PSM-Community inoculum treatments (i.e., the inoculant of 4200 m PSM-Community, the inoculant of 3600 m PSM-Community and the inoculant of 3000 m PSM-Community) and six C and N addition treatments (Table S2), for a total of 108 culture flasks. The six C and N addition treatments were conducted by changing the glucose and ammonium sulfate contents in a flask with 100 ml of PVK liquid medium (Table S2). Under sterile conditions, 0.5 ml of each inoculant was sucked up with a pipette and injected into a culture flask for each experimental treatment. All of the culture flasks were incubated for 14 days at 25 °C on an incubator shaker at 140 rpm.
PLFA analysis
After the incubation ended, 1 ml of broth was sucked up from the culture flask and used for PLFA analysis. PLFAs were extracted according to the following procedures. After the 1 ml of broth was lyophilized in a tube, 1 ml of sodium hydroxide methanol solution (NaOH:CH3OH:H2O = 0.3 g:1 ml:1 ml) was added to the tube. The tube was sealed using a lid and then heated in boiling water for 30 minutes. After the tube cooled, 2 ml of hydrochloric acid-methanol solution (concentrated HCl:CH3OH:H2O = 1.5 ml:2.1 ml:1 ml) was added to the tube. The tube was shaken for 10 seconds prior to heating in hot water at 80 ± 1 °C for 10 minutes. Then, the tube was cooled in a cold water bath. Next, a 1.25 ml mixture of hexane andmethyl tertiary butyl ether (MTBE) (hexane:MTBE = 1 ml:1 ml) was added to the tube, and the tube was shaken at 250 rpm for 10 minutes. After phase separation in the tube, the water layer was removed using a pipette. Next, 3 ml of NaOH (0.3 M) and 2 drops of NaCl (saturated solution) were added to the remaining liquid in the tube, and the tube was shaken rapidly for 5 minutes. After phase separation, the upper organic phase was collected for PLFA analysis. Samples were analysed on an Agilent 6890 N Gas Chromatograph using the MIDI peak identification software (Version 4.5; MIDI Inc., Newark, DE, USA). The column was an Agilent 19091B-102 (25.0 m × 200 μm × 0.33 μm) capillary column, and H2 was used as the carrier gas. The GC temperature program was set by the MIDI software. The fatty acid 19:0 was added to the samples as an internal standard. Identification and quantification of fatty acid methyl esters were conducted automatically by the MIDI peak identification software.
Fatty acids are designated according to the nomenclature described by Petersen and Klug(Petersen & Klug 1994). The total PLFA concentration was calculated using all of the PLFAs detected (44 PLFAs). The fatty acids 14:0, 15:0, i14:0, a15:0, i15:0, i16:0, a17:0, i17:0, 10 me16:0, 16:1ω7c, 18:1ω7c, 18:0, cy17:0 and cy19:0 were used as bacterial biomarkers(DeForest et al. 2004; Thoms & Gleixner 2013). The PLFAs 18:1ω9c and 18:2ω6,9c were chosen to represent fungi(Frostegard et al. 2011).
PO43- concentration and TDP
After the incubation ended, the liquids and solids in the broth were separated by centrifugation at 10,000 rpm for 10 min and air pump filtration with a microporous membrane (0.45 μm). The liquid was composed of water and soluble substances and was used to measure the PO43- concentration. The PO43- concentration in the liquid was determined with the ascorbic acid molybdenum blue method(Murphy and Riley, 1962). The pH value of the liquid was measured with a pH meter.
After the liquid was removed by centrifugation and air pump filtration, the remaining solids were composed of microbial bodies and insoluble substances and were used to measure the TDP. To measure the remaining Ca3(PO4)2 content, the solids and 40 ml of HCl (0.1 M) were added to a centrifuge tube and shaken for 1 minute by hand. The filtrate was collected after centrifugation at 10,000 rpm for 10 min and air pump filtration with a microporous membrane (0.45 μm). The above process was repeated 5 times to dissolve all of the remaining Ca3(PO4)2 solids into the filtrates. All filtrates were placed into 500 ml volumetric flasks and brought to volume with purified water. The Ca3(PO4)2 content in the volumetric flasks was determined with the ascorbic acid molybdenum blue method. Finally, TDP was calculated by subtracting the Ca3(PO4)2 content in the volumetric flask from total amount of Ca3(PO4)2 in the medium.
Statistical analysis.
Statistical analysis was performed using SPSS 13.0. The data were subjected to the analysis of variance (ANOVA). The ANOVA test was followed by a Tukey test at the 0.05 level of significance to compare the means of the treatments.
References
DeForest, J.L., Zak, D.R., Pregitzer, K.S., and Burton, A.J. (2004). Atmospheric nitrate deposition, microbial community composition, and enzyme activity in northern hardwood forests. Soil Sci Soc Am J 68, 132-138.
Frostegard, A., Tunlid, A., and Baath, E. (2011). Use and misuse of PLFA measurements in soils. Soil Biol Biochem 43, 1621-1625.
Murphy, J., and Riley, JP. (1962). A modified single solution method for the determination of phosphate in natural waters. Analytica chimica acta 27, 31-36.
Pikovskaya, R.I. (1948). Mobilization of phosphorus in soil in connection with the vital activity of some microbial species. Mikrobiologiya 17, 362-370.
Petersen, S.O., and Klug, M.J. (1994). Effects of sieving, storage, and incubation-temperature on the phospholipid fatty-acid profile of a soil microbial community. Appl Environ Microb 60, 2421-2430.
Thoms, C., and Gleixner, G. (2013). Seasonal differences in tree species' influence on soil microbial communities. Soil Biol Biochem 66, 239-248.
Table 1S Site information and soil properties.
Elevation / Coordinates / Vegetation / pH / Soil moisture / SOC / TN / TPm asl / (WGS 84) / % / mg g-1 / mg g-1 / mg g-1
4200 / N29° 32' 38"
E101° 57' 23" / Alpine meadow (Kobresia
pygmaea;
Allium prattii) / 4.9(0.1) / 44.9(3.4) / 34.7(6.8) / 1.5(0.3) / 1.7(0.1)
3600 / N29° 33' 00"
E101° 58' 06" / Bushes and pine forest (Abies
fabri;Rhododendron
sp.) / 3.8(0.1) / 69.1(1.6) / 32.8(3.0) / 2.0(0.1) / 1.1(0.02)
3000 / N29° 34' 24"
E101° 59' 21" / Alpine dark coniferous forest (Abiesfabri) / 3.8(0.2) / 62.9(1.7) / 36.0(4.3) / 2.0(0.2) / 1.2(0.05)
Values in parentheses represent the standard error.
Table S2 Phosphate-solubilizing experiments design.
Level of C and N / Group / C / N / P / Initial pH / C/N ratiomg C / 100 ml / mg N / 100 ml / mg P / 100 ml
Low levelgroups
1 / 65.5 / 10.6 / 100 / 7.0±0.2 / 6.2 (low)
2 / 364 / 10.6 / 100 / 7.0±0.2 / 34 (middle)
3 / 1455 / 10.6 / 100 / 7.0±0.2 / 137 (high)
High level groups
4 / 364 / 58.8 / 100 / 7.0±0.2 / 6.2 (low)
5 / 1455 / 42.5 / 100 / 7.0±0.2 / 34 (middle)
6 / 364 / 2.65 / 100 / 7.0±0.2 / 137 (high)
C, N and P addition by glucose, (NH4)2SO4 and Ca3(PO4)2 (an insoluble phosphorus). The word “(low)” denotes a low C/N ratio; The word “(middle)” denotes a middle C/N ratio; The word “(high)” denotes a high C/N ratio.
Figure S1 Phosphate-solubilizing efficiency under different C and N levels when C/N = 6.2. Low C, N level: C = 65.6 mg and N = 10.6 mg; High C, N level: C = 364 mg and N = 58.8 mg. 4200 m: the 4200 m PSM-Community; 3600 m: the 3600 m PSM-Community; 3000 m: the 3000 m PSM-Community; CK: a control check group without treatments.
Figure S2 Phosphate-solubilizing efficiency under different C and N levels when C/N ratio = 137. Low C, N level: C = 364 mg and N = 2.65 mg; High C, N level: C = 1455 mg and N = 10.6 mg.
Figure S3 The pH values with the different treatments of C and N.
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