Isolation of actinomycetes from Norway spruce mycorrhizas
Ectomycorrhizas were collected from beneath 10-year-old Norway spruce (Picea abies) trees in a forest stand dominated by Scots pine (Pinus sylvestris) in Haigerloch, south-west Germany. Mycorrhizal rootlets from the approx. 5 cm thick organic litter layer were excised, transported on ice to the laboratory, pooled, and subsequently immersed in water to remove debris surrounding the hyphal mantle. After washing 10 times with sterile destilled water, the ectomycorrhizas were sorted and white and pale yellow mycorrhizal root tips were pooled for further study. The mycorrhizal sample was used for both bacterial isolation and the analysis of fungal populations in the mantle. First half of the pooled sample of ectomycorrhizas (0.5 g) was used for DNA extraction according to Doyle and Doyle  and sequences of fungal internal transcribed spacer regions were obtained from the ectomycorrhizas with ITS1 and ITS4 primers . The PCR products were cloned and sequenced in two directions at GeneCust (Evry, France) and compared by blastn to sequences at NCBI (http://www.ncbi.nlm.nih.gov/genbank) and at Unite (http://unite.ut.ee; ) sequence databases.
Second half of the ectomycorrhizas (0.5 g) was used for the isolation of streptomycetes. The mycorrhizal sample was added to 50 ml of HNC medium (; 6% yeast extract, 0.05% SDS, 0.05% CaCl2 pH 7.0) and incubated at 42°C with shaking for 30 min. The suspension was filtered through a fine glass mesh, and a dilution series was subsequently prepared. The filtered suspensions were plated onto ISP-2 agar , which contained 5 gL-1 cycloheximide, 2 gL-1 nalidixic acid, and 5 gL-1 nystatin. After 8 d at 27°C fifteen different actinomycete isolates could be distinguished according to their morphological appearance , and these were maintained on ISP2 agar. For 16 S rDNA gene sequencing, genomic DNA was extracted from a loopful (a few μl) of bacterial spores by GenElute bacterial genomic DNA extraction kit (Sigma, Schnelldorf, Germany). Partial 16 S rDNA sequence was amplified with the primers 27f (5-AGAGTTTGATCMTGGCTCAG-3) and 765r (5-CTGTTTGCTCCCCACGCTTTC-3) as described in Coombs and Franco . The DNA sequences were compared to NCBI’s nr database and to Greengenes database (http://greengenes.lbl.gov) by blastn to find the closest homologue for each 16 S rDNA gene fragment from taxonomically characterized homologues. Streptomyces sp. GB 4-2, isolated from Schönbuch forest near Tübingen, south-west Germany, was provided by Karl Poralla.
Fungal isolates, bacterium-fungus co-cultures
The phytopathogenic fungi, Heterobasidion abietinum 331 from Klein Kotterbachtal, Austria, H. annosum 005 from Kirkkonummi, Finland, obtained from K. Korhonen, and Fusarium oxysporum from Schönbuch forest near Tübingen, Germany, obtained from A. Honold, were maintained on 1.5% malt agar. The symbiotic fungi, Amanita muscaria strain 404, isolated from fruiting body collected from the Schönbuch forest near Tübingen, Germany, Hebeloma cylindrosporum strain H1-H7 , and Laccaria bicolor strain S238 N  were cultivated in the dark at 20 °C on MMN agar  with 10 gL-1 glucose.
The co-culture system was similar to that utilized by Maier et al. , but with some minor alterations. Actinomycetes were spread on MMN medium  so as to form a line directly in the middle of the dish, essentially dividing it in two, and were grown at 27°C for 4 days (until sporulation started). Utilizing the wide end of a Pasteur pipette to control for diameter, two plugs of the fungal inoculum were then placed inside the Petri dishes on opposite ends of the plates. Inoculi were allowed to grow for 1 week (fast growing Heterobasidion strains and F. oxysporum), for 4 weeks (H. cylindrosporum) or for 6 weeks (A. muscaria, L. bicolor and P. croceum). Thereafter the extension of fungal mycelium was recorded from the fungal inoculum to the edge of the colony.
Confrontation of mycorrhiza-derived Streptomyces strains with each other
The influence of five streptomycetes upon each other was tested pair-wise in a bioassay. Streptomyces suspension cultures were grown three days in ISP-2 medium. From the tester strain, 40 μl of this suspension culture was applied on the lower part of an agar filled Petri dish, forming a line. After the sporulation of the tester strain begun, 3 parallel lines of the receiver strain were applied perpendicularly to the tester line. For each Streptomyces pair, three tester and nine receiver lines were applied. The impact of the tester strain on the formation of receiver strain’s substrate mycelium and sporulation was recorded at the time point of the onset of sporulation in the control cultures.
Impact of Streptomyces culture filtrates and culture extracts on non-streptomycetous bacteria
Pure culture filtrates and organic extracts of streptomycetes were tested against bacteria. Streptomyces suspension cultures were grown three days in ISP-2 medium. To obtain pure culture filtrate, the cells were centrifuged (3800 rpm, 10 min), and the supernatants were filtered (0.45 μm). Organic extracts were prepared from the pure culture filtrates, which were adjusted to pH 5.0 and extracted 1:1 (vol/vol) with ethyl acetate. The organic phase was concentrated to dryness using a vacuum evaporator and re-dissolved in 1/10 of the original volume in ethanol.
Gram-positive bacteria (Bacillus subtilis DSM 10, Staphylococcus aureus DSM 20231, Mycobacterium phlei DSM 750) and Gram-negative bacteria (Escherichia coli K12 (W1130), Pseudomonas fluorescens DSM 50090) were tested. Bacillus subtilis DSM 10 was initially cultured in DSMZ 1 medium at 37°C and tested on DSMZ 1 and MM 1 agar media. Staphylococcus aureus DSM 20231 was initially cultured in KM 1 medium at 37°C and tested on KM 1 agar medium. Mycobacterium phlei DSM 750 was initially cultured in KM 1 medium at 27°C and tested on KM 1 agar medium. Escherichia coli K12 (W1130) was initially cultured in KM 1 medium at 37°C and tested on KM 1 and MM 1 agar media. Pseudomonas fluorescens DSM 50090 was initially cultured in KM 1 medium at 27°C and tested on KM 1 and MM 1 agar media. KM 1 medium consisted of 8 g Difco nutrient broth, 5 g NaCl, 20 g agar per 1 liter of de-ionized water. The pH was adjusted to pH 7.2 prior to sterilization. KM 5 medium consisted of 4 g yeast extract, 10 g malt extract, 4 g glucose, 20 g agar per liter un-distilled water. The pH was adjusted to pH 5.5 prior to sterilization. DSMZ1-medium consisted of 5 g Bacto peptone, 3 g malt extract, 10 mg MnSO4 x H2O and 20 g agar per liter of un-distilled water. The pH was adjusted to 5.5 prior to sterilization. MM1 medium  consisted of 5 g glucose, 0,5 g tri-sodium-citrate x 2 H2O, 3 g KH2PO4, 7 g K2HPO4, 0.1 g MgSO4 x 7 H2O, 1 g (NH4)2SO4 and 15 g Bacto agar.
The bacteria were cultivated for a period of 24 h in 100 ml in respective liquid media in 500 ml Erlenmeyer flasks with one baffle at 27°C or 37°C on a rotary shaker at120 rpm. The cultures were centrifuged, re-suspended in saline, and set to achieve an optical density of 1.3 at a wavelength of 546 nm. In the case of minimal medium (MM1), cultures were washed one time with saline to get rid of complex media used for inoculation. Two hundred ml of complex medium (DSMZ 1, KM 1, and KM 5) containing agar were inoculated with 2 ml of this defined suspension of organisms (OD = 1.3). Ten ml of inoculated agar were poured into each Petri dish. Streptomyces pure culture filtrate (10 μl) or organic extract (10 μl) was applied on paper discs (diameter: 6 mm) and air dried. The paper discs were then placed on the previously prepared agar media. After 24 h, microbial growth inhibition was recorded by measuring the diameter of the inhibition zone.
Fermentation of streptomycetes for the analysis of secondary metabolites
The strains AcM9, AcM11, AcM20, AcM29 and AcM30 were cultivated in 100 ml ISP-2-medium at 120 rpm and 27 °C for 3 days. Of these cultures, four ml were used to inoculate 100 ml SGG, OM and MMN medium in 500 ml-Erlenmeyer flasks with one baffle. SGG-medium consisted of 10 g soluble starch, 10 g glucose, 10 g glycerol, 2.5 g cornsteep powder (Marcor, Hartge Ingredients, Hamburg), 5 g Bacto peptone (Difco), 2 g yeast extract (Ohly Kat, Deutsche Hefewerke, Hamburg), 1 g NaCl and 3 g CaCO3 per liter of tap water. The pH was adjusted to pH 7.3 prior to sterilization. OM medium consisted of 20 g oat meal (Holo Hafergold, Neuform, Zarrentin) and 5 ml of the following micronutrient solution: 3 g CaCl2x2 H2O, 1 g iron-III-citrat, 200 mg MnSO4 x 1 H2O, 100 mg ZnCl2, 25 mg CuSO4 x 5H2O, 20 mg Na2B4O7 x 10 H2O, 4 mg CoCl2 x 6H2O, and 10 mg Na2MoO4 x 2 H2O per liter of deionized water. The pH was adjusted to pH 7.3 prior to sterilization. Modified MMN medium was prepared according to Molina and Palmer . Fermentations were carried out on a rotary shaker at 120 rpm and 27°C. After 2, 4 and 6 days (24, 48 and 72 hours) 10 ml of bacterial culture were centrifuged (3800 rpm, 10 min) and bacterial biomass was determined (volume percent). The culture filtrate - separated from the bacterial mycelium by centrifugation - was used for further analyses of secreted bacterial metabolites.
Extraction and HPLC-UV-visible spectral analysis of Streptomyces secondary metabolites
Culture filtrates (5 ml) of AcM 9, AcM11, AcM20, AcM29 and AcM30 were adjusted to pH 5 and extracted with 5 ml ethyl acetate for 30 min under shaking conditions. The organic extracts were concentrated to dryness using vacuum evaporator and resuspended in 0.5 ml of methanol. The 10-fold concentrated extracts were centrifuged (3 min, 13 000 rpm) and 5 μl of each sample was subjected to HPLC on a 5 μm Nucleosil C18-column (Maisch, Ammerbuch, Germany, 125 mm x 3 mm, fitted with a guard-column: 20 mm x 3 mm) with 0.1% -o-phosphoric acid as solvent A and acetonitrile as solvent B at a linear gradient (from 4.5 to 100% B in 15 min and a 3-min hold at 100% B) at a flow rate of 0.85 ml/min. The chromatographic system consisted of a 1090 M liquid chromatograph (Hewlett Packard, Waldbronn, Germany) equipped with a diode array detector and a Kayak XM 600 ChemStation (Agilent Technologies, Waldbronn, Germany). Multiple wavelengths monitoring was performed at 210, 230, 260, 280, 310, 360, 435 and 500 nm and UV-visible spectra were measured from 200 to 600 nm.
HPLC-ESI-MS analysis of Streptomyces secondary metabolites
HPLC-DAD-ESI-MS analysis was carried out with an Agilent 1200 HPLC series equipped with a binary HPLC pump, autosampler and diode array detector, and an Agilent LC/MSD Ultra Trap System XCT 6330 (Agilent, Waldbronn, Germany). The Samples (2.5 μL) were separated on a 3 μm Nucleosil C18-column (Maisch, Ammerbuch, Germany, 100 mm x 2 mm with a precolumn 10 mm x 2 mm) and separated by linear gradient elution from 10% eluent B to 100% eluent B in 15 minutes (0.1% formic acid as eluent A, 0.06% formic acid in acetonitrile as eluent B) at a flow rate of 400 μl/min. Wavelength monitoring was performed at 230 nm, 260 nm, 280 nm, 360 nm and 435 nm. MS Instrument settings were as follows: Ionization: ESI (positive and negative, alternating); Mode: Ultra Scan; Capillary voltage: 3.5 kV; Temperature: 350°C; Tuning mass: m/z 400. The production levels of the following metabolites were quantified based on the comparison of their peak area with that obtained by HPLC analysis of known amount of pure substance: Acta 2930 B1, actiphenol, cycloheximide, ferulic acid.
Inoculation of Arabidopsis thaliana with streptomycetes and with Alternaria brassicicola, chlorophyll fluorescence and disease index measurements
Sterile Arabidopsis thaliana Col-0 seeds were placed on half strength MS  medium containing 1% glucose and 0.8% agar for germination. After 7 days, seedlings were transferred to ½ MS with 2% agar. To grow seedlings in an upright position with leaves free from contact with the agar surface, the top third of solid medium was removed from the Petri dish. Seedlings were placed with roots on the agar and leaves in the airspace. Petri dishes were then stored in a vertical position to allow root growth on the agar surface. Plants were cultivated at 22°C, 200μE/m2s with a light/dark cycle of 8/16 h.
After 7 days, roots were inoculated with AcM 9, AcM11, AcM29, AcM29, AcM30 and positive control Streptomyces GB 4-2 . Bacterial cultures grown in ISP-2 medium for 4 to 5 days were separated from growth medium by centrifugation, washed three times in sterile water and diluted to an OD of 0.3. Fourteen μl were applied to each root. Control plants (no bacterial inoculation) received 14 μl of sterile water. This time point was referred to as “d-7” and was the first time point of measurement of maximal photosystem II efficiency (Fv/Fm), which was measured using Imaging-PAM fluorometer (Walz, Effeltrich, Germany) in the following manner: seedlings were subjected to a saturated light impulse of 3000 μE/m2s and 0.7 sec duration to establish maximal fluorescence and basic fluorescence, from which maximal Fv/Fm was calculated. Results were based on two values of 10 plants per each time point. Each treatment contained in total 30 plants in three independent repetitions. Standard deviation was calculated based on mean values of those repetitions. Seven days after bacterial inoculation of roots (referred to as “d0”), 2 to 3 leaves of each seedling were infected with 1 μl each of a 5x105 spores/ml suspension of Alternaria brassicicola (kindly donated by Birgit Kemmerling, ZMBP, University of Tuebingen).
Disease index was determined regularly from day 3 post Alternaria brassicicola infection (d3) based on Epple et al. . The spread of fungal infection on each leaf was assessed at d3, d5, d7, d11, and d14 post Alternaria brassicicola inoculation, and quantified in classes 1 to 6: class 1: no infection, class 2: infection restricted to site of inoculation, class 3: symmetric spread of infection around inoculation site, class 4: asymmetric spread of infection around inoculation site, class 5: beginning sporulation of pathogen, and class 6: >50% of leave surface infected. Disease index (DI) was calculated as DI = ∑ i x l/n where i is infection class, l number of leaves in the respective class and n is total number of infected leaves. Results were calculated as mean values of three independent repetitions each containing 20 infected leaves of 10 plants per treatment. Standard deviations were calculated from mean values of independent repetitions.