Isolation of soil microorganisms
Soil samples collected from central Taiwan were taken from a depth of 0–10 cm, sifted and moistened to about 65% water-holding capacity (Tsai et al. 2012). Selective media for isolation of fungi, actinomycetes and bacteria were prepared as described previously (Ko et al. 2010b). Approximately 1.3 g soil was mixed with 100 ml sterile distilled water in an Omni mixer chamber at 5000 rpm for 30 s. The suspension was diluted to 10−4, 10−5 and 10−6 for fungi, 10−5, 10−6 and 10−7 for actinomycetes and bacteria to determine the dilution needed to obtain soil suspension essentially free of each group of microorganisms (Ko et al. 2011). A 1-ml aliquot of diluted soil suspension was mixed with 20 ml of a molten selective medium at 45°C in a Petri plate. Five plates were used for each treatment.
Vegetables including fruit of tomato (Lycopersicon esculentum), tubers of sweet potato (Ipomoea batatas), and leaves and stems of spinach (Spinacia oleracea), ong choy (Ipomoea aquatica) and common purslane (Portulaca oleracea) were purchased from local markets. For soil amendment, about 500 g soil was mixed with 4% each of chopped vegetables in a 1000-ml bottle and incubated at 24°C for at least two weeks before use. To isolate microorganisms with ability to utilize amended nutrients for multiplication, suspension of amended soil was diluted to the concentration pre-determined for each group of microorganisms and plated on the selective medium as described above. After incubation at 24°C for seven days, colonies appeared were individually transferred to 10% V-8 agar (10% V-8 juice, 0.02% CaCO3 and 2% agar) plates (Ko et al. 2010a).
Cultivation of isolated microorganisms in liquid medium
Liquid medium was prepared by grinding 4 g each of the five chopped vegetables in 100 ml water in an Omni mixer at 4000 rpm for 3 min and dispensing 50 ml broth in a 250-ml flask. After autoclaving, each flask was inoculated with two loopfuls of bacterium, or a piece (ca. 4 × 5 × 3 mm) of actinomycete or fungus agar culture. Inoculated flasks were incubated on a shaker for two weeks. After incubation, cultures were separately ground in an Omni mixer at 4000 rpm for 1 min and the mixtures were left on the bench for sedimentation. The fluid portions were used for testing their ability to control the disease.
Inoculum preparation
A. brassicicola isolate Aba-31 was grown on 10% V-8 agar at 24°C under light (Wang et al. 2010) for four to six days for the production of conidia. A conidial suspension was prepared by placing two pieces of culture blocks (ca. 5 × 5 × 3 mm) in 5 ml sterile distilled water in a test tube and by agitating the test tube for 30 s with a Vortex mixer. The concentration of conidia was adjusted to 3 conidial/μl with a Pipetman microliter pipette (West Coast Scientific, Oakland, CA) (Ann et al. 2010).
Disease control assay of culture fluids
Seeds of mustard cabbage (Brassica juncea Coss.) were grown in 8-cm pots containing a mixture of peat moss and vermiculite (9:1, v/v). Two leaves of a four-week-old plant were sprayed to run off with culture fluid of a test microorganism daily three times before inoculation on the fourth day.
Each mustard cabbage leaf was inoculated with five 2-μl drops of conidial suspension of A. brassicicola along the edge of the leaf, and a 10-μl drop of molten agar consisting of 1% agar and 1% V-8 juice at 60°C was added to each inoculum drop to fix the inoculum on the target site (Chen et al. 2011). Leaves of mustard cabbage sprayed with liquid medium were inoculated with conidia of A. brassicicola and used as a control. Inoculated plants were placed in moist chambers and kept in the greenhouse. The number and the size of lesions that developed at the inoculated sites were recorded three days after inoculation. Two leaves were used for each treatment, and all the experiments were repeated at least twice.
Extraction of the fungicidal substance
Culture fluid of E. brefeldianum isolate V3F-3 was freeze dried. One gram dry powder, obtained from approximately 50 ml of culture fluid was extracted with 25 ml of water, ethanol, methanol, acetone, ethyl acetate, or chloroform in a 250-ml flask by shaking on a shaker for 24 h (Ko et al. 2010b). The mixture was centrifuged at 1500 × g for 5 min to obtain clear extract. For bioassay and characterization of the fungistatic substance, 10 ml extract was evaporated to 2 ml followed by addition of 2 ml water and evaporation to 2 ml again. To test the ability of different solvents to extract the inhibitory substances, 10-fold concentration of the extracts was used.
Germination tests
To test the effect of the culture extract on spore germination, 10 μl of conidial suspension (2 × 104 spores/ml) of A. brassicicola was mixed with 10 μl of extract in a cavity of a sterile eight-cavity slide. Slides with spores were kept moist by placing each on a L-shaped glass rod in a 9-cm Petri plate containing 10 ml sterile distilled water. Germination was recorded after incubation at 24°C for 4 h, and 100 spores were counted in each of the three replicates. All experiments were done twice.
Characterization of the fungicidal substance
To study the effect of pH on the activity of the fungicidal substance, the pH of the culture extract of E. brefeldianum was adjusted from the original 4 to 3 with 1 N HCl, or 5 to 10 with 1 N NaOH (Ko et al. 2010b). To study the stability of the fungicidal substance under extreme pH, the pH of the extract was adjusted to 2 or 12 for 24 h and then readjusted back to 4.
For studying the effect of high temperature on the activity of the fungicidal substance, the extract was treated at 60, 80 or 100°C for 30 min or autoclaved for 15 min. For the study of the effect of the treatment of culture extract with cation exchange resins, anion exchange resins or activated charcoal on its activity against germination of A. brassicicola, 5 g of Diaion SK1B cation exchange resins (equivalent to Amberlite 1R-120), Diaion SA 12A anion exchange resins (equivalent to Amberlite 1RA-420; Tai-Young Chemical Co., Kaohsiung, Taiwan) or activated charcoal (Sigma-Aldrich) was washed with 50 ml of distilled water three times by shaking over a six-hour period to remove possible inhibitory substances. Ten milliliter extract was shaken with 1 g cation exchange resins, anion exchange resins or activated charcoal in a 150-ml flask at 100 strokes/min for 24 h and filtered through a Whatman no. 1 filter paper. The filtrate was then used for germination tests (Ko et al. 2010b). In each of the three replicates, 100 spores were counted and all experiments were done at least twice.
