Survey and isolation of the pathogens
The survey was performed and samples were collected from the turfgrass areas in İstanbul, Antalya, Ankara, İzmir, Kayseri, Bursa, Aydın, and Muğla Provinces in 2015. Segments of leaves and roots were sterilized for 1 min, in 1% sodium hypochlorite (NaOCl) solution, then washed with sterile water and air dried in a laminar flow cabinet before culturing on potato dextrose agar (PDA, Difco, USA) containing 50 mg/l streptomycin sulfate. Isolates were incubated under the light and dark regimes, respectively on 28 ± 1 °C for 7 days.
Bacterial isolates
Five antagonistic domestic bacterial strains (215b, 44bac, 88cfp, 166fp, and 88bfp) used in this study were isolated from the tomato and cucumber rhizospheres in a previous study, where isolates 166fp and 88cfp managed with Pythium deliense, Sclerotinia minor, and Alternaria solani on tomato were detected (Aşkın 2008). Also, isolate 44bac managed downy mildew on cucumber (Aşkın and Ozan 2013) under field conditions. Molecular identification of the 5 antagonistic bacterial isolates, used in this study, was first determined in this study.
Molecular identifications of fungal and bacterial isolates
Isolation of fungal DNA was carried out by Blood and Tissue Kit (QIAGEN Inc. Valencia, CA), as specified by the manufacturer. The PCR reaction mixture and conditions were made by modifying according to Mahadevakumar et al. (2016). DNA amplification was performed, using the optimized cycles optimized with Techne TC-5000 thermal cycler. Primers ITS-1 (5 ′TCC GTA GGT GAA CCT GCGG 3′) and ITS-4 (5 ′TCC TCC GCT TAT TGA TATGC 3′) were used for amplification of ITS regions (White et al. 1990). The polymerase chain reaction (PCR) was performed in a 50-μl reaction mixture containing 1 μl template DNA, 1 μl forward primer (10 mM), 1 μl reverse primer (10 mM), 5 μl reaction buffer (10×), 4 μl dNTP (each 2.5 mM), 0.5 μl Taq DNA Polymerase (5 U/μl), and 37.5 μl sterile double-distilled water. The PCR cycling protocol consisted of initial denaturation at 94 °C for 4 min, followed by 30 cycles of 94 °C for 45 s, 55 °C for 45 s, 72 °C for 2 min, and a final elongation step of 72 °C for 10 min. As a negative control, the template DNA was replaced by sterile double-distilled water.
Molecular definition of bacteria was made according to the protocol of DNA isolation from Blood and Tissue Kit (QIAGEN Inc. Valencia, CA). The 16S rDNA gene fragments were amplified by PCR using the universal primers 27F 5′AGAGTTTGATCMTGGCTCAG3′ and 1492R 5′TACGGYTACCTTGTTACGACTT3′ (Lane, 1991). The PCR reaction mixture and conditions were modified to carry out the PCR reaction. DNA replications were performed in the ABI Veriti (Applied Biosystem) thermal cycler using the following cycles:
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1.
The initial denaturation consist of 5 min at 94 °C, 35 cycles of amplification step consisting of denaturation of 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 120 s, and final extension of 10 min at 72 °C (Lane 1991).
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2.
The PCR product was directly subjected to Sanger sequence treatment in a special Arge Laboratory (BM Gene Research and Biotechnology Company, Ankara, Turkey).
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Bipartite raw sequence electropherograms were compared to the isolate sequences in Gen Bank after BLAST screening in NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
Determination of mycelial compatibility groups (MCGs) of S. rolfsii isolates
In order to determine the mycelial compatibility among 32 isolates, obtained from different areas grown with turfgrass areas, mating each isolate with themselves and with all other isolates was carried out (Punja and Grogan 1983). Mating studies were performed on PDA medium with 0.25% food coloring (Ponceau 4R, Turkey). Mycelial discs of two isolates were reciprocatively plated on PDA medium with a distance of 3–4 cm. Cultures were incubated at 25 ± 1 °C and colony growth was observed after 7–14 days (Kohn et al. 1991). Hyphal interaction among the isolates mate was observed at the end of 7 days after culturing. Compatibility between each of the two groups was evaluated according to a red line with separation in the region, where the hyphae collided. When the red line was seen, it was accepted as incompatible, otherwise not (Punja and Grogan 1983).
Fungal inoculums
S. rolfsii was grown on wheat bran medium in bottles of 500 ml, sterilized in an autoclave for 20 min. at 121 °C, for 15 days at 28 ± 1 °C (Aşkın 2008).
Pathogenicity tests
Pathogenicity tests of S. rolfsii isolates were conducted under greenhouse conditions. The fungus inoculums grown on wheat bran (4 g inoculums/kg soil) was added to the sterilized garden soil, fine sand, and burnt fertilizer mixed (2:1:1) and then distributed in the pots (10 cm in diameter). Control pots contained the sterilized garden soil, fine sand, and burnt fertilizer mixed (2:1:1) free from the inoculum. Three pots were replicated for each treatment. All pots were covered by a sanitized polyethylene nylon and incubated for 3 days. At the end of the duration, 30 seeds of turfgrass (cv. Festuca arundinacea) were placed on the soil surface, covered with 1 cm of sterile natural soil, and watered with 9–10 ml of water. The infected plants were counted 3 weeks later (Zhang et al. 2014) and recorded. Evaluation was made according to a scale of 0 to 5: 0 = no disease symptoms, 1 = 1–10% hypocotyl infecting and/or shortening, 2 = 11–30% hypocotyl infecting and/or shortening, 3 = 31–50% hypocotyl infecting and/or shortening, 4 = 51–80% hypocotyl infecting and/or shortening, and 5 = entire hypocotyl infecting and/or shortening (Ichielevich Auster et al. 1985). Disease severity was calculated according to the Townsend–Heuberger formula (Townsend and Heuberger 1943):
$$ \mathrm{Disease}\ \mathrm{severity}\%\kern0.5em =\kern0.5em \Sigma \kern0.5em \left(N\kern0.5em \times \kern0.5em V\right)/Z\kern0.5em \times \kern0.5em N\kern0.5em \times 100 $$
(N is the number of samples in the scale with different disease grades, V is the scale value, Z is the highest scale value, and N is the total number of samples observed)
Bacterial inoculums
Bacterial isolates were cultured in potato dextrose broth. After 24 h, the bacterial concentration was verified through spectrophotometry at a wavelength (λ) of 600 nm seeking for an absorbance between 0.9 and 1 equivalent to a concentration of 1 × 108 c/ml and by counting the colony forming units (cfu) per milliliter through the total viable count. Surface-disinfected seeds of turfgrass were inoculated with bacterial solutions by soaking with agitation for 12 h. Rhizobacterial stock cultures were maintained in nutrient agar medium amended with 15% glycerol and stored at − 80 °C. Before being used in the bioassays, stock cultures were streaked onto nutrient agar plates and incubated at 28 ± 1 °C for 48 h.
Biocontrol assays
This study was carried out using turfgrass seeds mixture containing 4 cvs: Festuca rubra, Lolium perenne, Poa pratensis, Festuca arundinacea, and the most virulent S. rolfsii isolate (Sr34-10). The soil used in the experiment was prepared in the form of a mixture of 2:1:1 garden soil to stream sand to burnt fertilizer. Soil mix was sterilized in an autoclave at 121 °C for 45 min. The inoculum of S. rolfsii was developed on wheat bran. Antagonistic bacteria were applied by coating to the seeds. Experiments were carried out in both sterilized and non-sterilized soils, where three treatments were performed: (1) negative control by sowing uncoated turfgrass seeds in non-infested soils, (2) positive control by sowing uncoated turfgrass seeds in infested soils to evaluate the varietal sensitivity, and (3) sowing coated turfgrass seeds in infested soils to evaluate the biocontrol efficacy of each antagonistic isolate against S. rolfsii. The mixture of inoculum and soil (5 g to 1 kg of soil) were filled in sterile plastic pots (10 cm in diameter). After 4–5 days, 30 coated and uncoated turfgrass seeds were sown at a depth of 1 cm per pot. The plants were grown in a plant growth medium containing 12 h of light, 12 h of darkness, and 25 ± 1 °C temperature. The experiments were carried out in 3 replicates, according to randomized plot design. After the inoculation, observations were made at intervals of 10 days and a 0–5 scale was used 30 days after sowing, and disease severity was calculated as mentioned before.
Statistical analysis
Variance analyses were carried out, using the SPSS GLM statistical program, to determine the differences among virulence levels of isolates and disease rates in biocontrol assay.
Disease assessment
Disease ratios were estimated according to the devised scale by Townsend-Heuberger formula (Townsend and Heuberger 1943). The calculated disease severity and the activity of bacterial isolates using the Abbott formula was determined from the disease severity values. Disease severity was compared by Tukey’s multiple comparison test on these ratios.
