Growth of endophytic fungus - Penicillium resedanum LK6
Approximately, 200 root pieces were collected from C. annuum plants growing in water deficient conditions (soil water potential 41.23 hPa). The root pieces were surface sterilized with 2.5% sodium hypochlorite (30 min in shaking incubator at 120 rpm) and washed with autoclaved distilled water (DW) to remove the contaminants, rhizobacteria and superficial fungi. The pepper root pieces (about 0.5 cm) were kept in petri-plates containing Hagem medium (0.05% NH4Cl, 0.1% FeCl3, 0.05% KH2PO4, 0.5% glucose, 0.05% MgSO4.7H2O, 1.5% agar and 80 ppm streptomycin; pH 5.6 ± 0.1). The sterilized roots pieces were imprinted to ensure the effectiveness of sterilization process Redman et al. . The emerging fungal spots from the root pieces were isolated and transferred to Potato Dextrose Agar (PDA) medium under aseptic conditions. Among isolated endophytes, a bioactive strain was selected through screening bioassays using dwarf mutant and normal cultivars of Oryza sativa. The endophyte was identified by DNA extraction, PCR techniques, sequencing and phylogenetic analysis of Internal Transcribed Spacer [ITS-1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS-4 (5′-TCC TCC GCT TAT TGA TAT GC-3′)] with the method previously described by Redman et al.  and Khan et al. . The sequence of the endophyte (P. resedanum) was submitted to GenBank and was given accession no. JX111908. The endophyte was inoculated in Czapek broth (1% peptone, 1% glucose, 0.001% FeSO4.7H2O, 0.05% MgSO4.7H2O, 0.05% KCl; pH 7.3 ± 0.2) and incubated for 10 days at 28°C under shaking (150 rpm) conditions to undertake further experiments [17, 18].
C. annuum growth with endophyte
The C. annuum seeds were sterilized with 2.5% sodium hypochlorite for 30 min, and rinsed with autoclaved DW. Seeds were incubated in darkness for 24 h to obtain equally germination. The pre-germinated seeds were cultivated in autoclaved pots (121°C for 15 min; two times; 10 × 5 cm) with substrate (peat: perlite: vermiculite – 1:1:1 by volume). The endophyte was cultured in Czapek broth containing conidia (20 ml with 25 propagules/pot) and added to substrate as described previsouly [16–18]. The control plants only received 20 ml/pot of endophyte-free Czapek broth. Thus, pre-germinated pepper seeds and endophyte were grown together for three weeks in the growth chamber (day/night cycle: 14 h; 28°C/10 h; 25°C; relative humidity 60–70%; light intensity 1000 μEm-2-s Natrium lamps) irrigated with distilled water.
Drought stress, endophyte association and SA treatments
The experiment was conducted with a completely randomized block design. Salicylic acid (SA-10-6 M) was exogenously applied to pepper plants. The treatments included (i) control, (ii) control plants under drought stress, (iii) plants with endophyte (EA), (iv) EA plants under stress, (v) SA-treated plants, (vi) SA-treated plants under stress, (vii) SA and endophyte-infected plants and (viii) SA and endophyte-associated plants under stress (SA+EA). Each treatment contained 18 plants and the experiment was repeated three times. Drought stress was initiated by exposing plants to 15% polyethylene glycol (PEG 10,000 MW; -3.02 MPa osmotic potential) for 2, 4 and 8 days. The growth parameters i.e. shoot length and fresh weights were measured at harvest while chlorophyll content of leaves was measured by chlorophyll meter (SPAD-502 Minolta, Japan). All readings were taken in triplicate.
The effect on the plant biomass was measured after endophyte and SA treatments under different stress regimes [18
]. The biomass gained/lost in endophyte-inoculated and non-inoculated plants were compared by using this formula:
DW is the dry weight while E+ and E- are plants with or without endophyte infestation respectively.
Determination of electrolytic leakage
Electrolytic leakage was determined according to the method of Liu et al. . Briefly, fresh leaf samples (200 mg) were cut into 5 mm small pieces length and placed in test tubes containing 10 ml DW. The preliminary electrical conductivity (EC1) was measured after the tubes were kept in water bath at 25°C for one hour. The samples were autoclaved at 121°C for 20 min to completely kill the tissues and release all electrolytes from leaf tissues. When the samples were cooled down to 25°C, final electrical conductivity (EC2) was measured. The electrolyte leakage (EL) was estimated using formula: EL = EC1/EC2.
Microscopic analysis and colonization
Plant roots infected with fungal endophyte were sectioned and treated with sodium hypochlorite (2.5%) for 10 min for clarification. Latter, it was treated with KOH (20%) for 24 h which was extensively rinsed with autoclaved DW. The root pieces were acidified with HCl (10%); stained for 24 h using tryptophan blue (0.8%) and lactic acid (95%). At the end, the root pieces were distained in lactic acid for 24 h. The endophytic colonization in roots pieces was assessed through light microscope (Stemi SV 11 Apo, Carl Zeiss). The rate of colonization was determined according to the method of Kumar and Hyde .
Determination of antioxidants
To determine reduced glutathione (GSH), leaves tissues (100 mg) of all the treated pepper plant samples were ground in 3 ml 5% (v/v) trichloroacetic acid using chilled mortar and pestle. The homogenate was obtained through centrifugation (at 15000 rpm for 15 min at 4°C). The homogenate obtained was analysed for reduced glutathione (GSH) activity as described by Ellman . The reaction mixture comprised of sample supernatant (0.1 ml), monosodium phosphate (3.0 ml; 150 mM NaH2PO4; pH 7.4) and Ellman’s reagent (0.5 ml). The mixture was incubated at 30°C for 5 min. Absorbance was determined at 412 nm and the GSH activity was calculated by a standard curve.
Total polyphenol content was determined by the Folin-Ciocalteau method as mentioned by Kumazawa et al. . Plant tissues (100 mg) were ground with 80% ethanol and the resultant extracts (0.5 ml) were mixed with Folin-Ciocalteau reagent (0.5 ml) and 10% Na2CO3 (0.5 ml). The absorbance of the reaction mixture was measured at 760 nm after 1 h incubation at room temperature. Total polyphenol content was expressed as micro g/mg (gallic acid equivalents).
The detection of superoxide anion (O2
-) was based on its ability to reduce nitro blue tetrazolium (NBT) as performed by Doke . Treated plant tissues (100 mg) were cut into 1 mm2 pieces and immediately immersed in 10 mM phosphate buffer (pH 7.8), containing NBT (0.05% (w/v)) and 10 mM NaN3. The reaction mixture was left for incubation till one hour at room temperature. The reaction mixture was heated at 85 ± 2°C for 15 min and cooled quickly to 0°C. The absorbance was measured at 580 nm. The O2
- content was expressed as an increase of absorbance / 0.1 g dry weight.
The extent of lipid peroxidation was determined by the method of Ohkawa et al. . The optical density of the resulting light pink colour was recorded at 532 nm. Tetramethoxypropane was used as an external standard. The level of lipid peroxides was expressed as micro moles of malondialdehyde (MDA) formed/g tissue weight.
All treated plant’s leaves (200 mg) were homogenized in 50 mM Tris–HCl buffer (pH 7.0) composed of 3 mM MgCl2, 1 mM EDTA and 1.0% PVP and then centrifuged (15,000 rpm for 15 min at 2°C). The supernatant was used for enzymatic analysis. All parameters were expressed as activity per mg protein. Total proteins were determined according to Bradford  method.
Catalase (EC 188.8.131.52) activity was measured as describe by Aebi . The crude enzyme supernatant was treated with 0.2 M H2O2 (0.5 ml) in 10 mM phosphate buffer (pH 7.0). The enzymatic activity was determined by the decrease in absorbance of H2O2 at 240 nm. The one unit of catalase is given as μg of H2O2 released mg protein min-1. Peroxidase (EC 184.108.40.206) and polyphenol oxidase (EC 220.127.116.11) activities were measured as described by Kar and Mishra et al.  with a little modification. The pepper leaf samples (200 mg) were homogenized with phosphate buffer pH 6.8 (0.1 M) and centrifuged (2°C for 15 min at 17,000 rpm). The clear supernatant was obtained which was analysed for enzymatic activity. The reaction mixture of peroxidase activity composed of 0.1 M phosphate buffer (pH 6.8), pyrogallol (50 μl), H2O2 (50 μl) and enzyme extract (0.1 ml). After incubation (5 min at 25°C), the reaction was stopped by adding 5% (v/v) H2SO4 (0.5 ml). The amount of purpurogallin synthesized during the reaction was measured by the absorbance at 420 nm. The same assay mixture, used for peroxidase (without H2O2), was measured for the activity of polyphenol oxidase. The absorbance of purpurogallin formed was read at 420 nm. One unit of peroxidase and polyphenol oxidase was defined as an increase of 0.1 units of absorbance.
Endogenous salicylic acid analysis
SA was extracted and quantified as described previously by Seskar et al. . The freeze-dried leaf tissues (0.4 g) of all treated samples were grinded to powder. The powder was sequentially extracted with 90 and 100% methanol by centrifuging (at 15,000 rpm and 4°C). Both the extraction steps were repeated four times until the sample decoloured. The combined methanol extracts were vacuum-dried. Dry pellets were re-suspended in 5% trichloroacetic acid (2.5 ml) while the supernatant was partitioned with ethyl acetate: cyclopentane: isopropanol (100:99:1, v/v). The organic layer containing free SA was transferred to a 4 ml vial and dried with nitrogen gas. The dry SA was rigorously suspended in 1 ml of 70% methanol. High Performance Liquid Chromatography (HPLC) analysis were carried out on Shimadzu coupled with fluorescence detector (Shimdzu RF-10AXL, excitation and emission 305-365 nm respectively) fitted with C18 reverse-phase HPLC column (HP hypersil ODS, particle size 5 μm, pore size 120Å Waters) (Additional file 1: Table S1). The flow rate was 1.0 ml/min. The experiment was repeated three times.
The eight different treatments comprised of eighteen plants per treatment while the experiments were performed in triplicate. The mean, standard error and the graphical representation was done using Graph Pad Prism software (version 5.0, San Diego, California USA). To identify significant effects between the treatments and control with or without stress conditions and endophyte, we used Duncan’s multiple range tests (DMRT) on Statistic Analysis System (SAS 9.1, USA).