Sampling and fungal isolation
Root samples were collected from some diseased cucumber plants showing typical symptoms of damping off, growing in some plastic tunnels located in the desert farms in Najaf province, Iraq. The samples were transferred to the laboratory of plant diseases at the Faculty of Agriculture, Kufa University, Iraq, for isolation of fungal pathogens. Collected roots were washed by tap water, cut into small pieces, sterilized with NaOCl (1%) solution for 2 min, and washed with sterile distilled water to remove any residues of NaOCl. Root pieces were then dried using filter papers to remove any excess water and transferred to Petri dishes containing potato dextrose agar (PDA) media, supplemented with chloramphenicol antibiotic at a concentration of 200 mg/L. All Petri dishes were incubated at a temperature of 25 ± 2 °C for about 4 days.
Morphological identification
The appeared fungi were purified and maintained on the same medium (PDA) and were used for morphological identification by microscopic examination.
Molecular characterization
The isolated fungi were molecularly identified using PCR technique and determining the nucleotide sequences as follows:
DNA extraction
From each fungal isolate, 50–100 mg of fresh 5-day-old colonies were taken by a sterile scalpel and transferred into an Eppendorf tube for DNA extraction using a specific extraction kit (Zymo Research, Cat. No. D6005), following the manufacturer’s instructions. The quality and quantity of DNA extracted from each isolate were measured by a UV spectrophotometer (Thermo Scientific, Germany). DNA was then stored at − 20 until use.
PCR amplification and DNA sequencing of rDNA-ITS region
The internal transcribed spacer (ITS) region of R. solani isolates were amplified, using the universal primers ITS1 (TCCGTTGGTGAACCAGCGG) and ITS4 (TCCTCCGC TTATGATATGC) (White et al. 1990) using Taq DNA polymerase (Roche, Cat. No. 11146 173 001). The final volume of each PCR reaction mixture (sample) was 20 μl containing; 2 μ1 10 × PCR buffer, 1 μl of each primer (10 pmol), 2 μl dNTPs (2 mM), 3 μl template DNA (30 ng/μ1), 1 unit Taq polymerase, then completed to 20 μl by adding nuclease-free sterile distilled water. PCR amplification was performed using the following conditions: initial denaturation at 94 °C for 1 min followed by 35 cycles each consisting of final denaturation at 94 °C for 30 s, annealing temperature at 55 °C for 30 s, initial extension for 1 min, and final extension at 72 °C for 5 min (White et al. 1990. PCR-amplified products were electrophoretically separated on a 1% agarose gel for 140 min at 80 V and 400 mA and visualized with ethidium bromide under UV illumination, and images were captured using Vilber Lourmat, Taiwan, gel documentation system.
For DNA sequencing, the PCR-amplified products were gel-purified using the FavorPrep PCR Purification Kit (Cat. No. FAGCK 001, Favorgen, Taiwan) and sent along with the primer pair (ITS1 and ITS4) to the Macrogen DNA sequencing service in Korea. PCR products were directly sequenced in both directions using the respective forward and reverse primers. The obtained nucleotide sequences were aligned and compared with the sequences belonged to the R. solani isolates in the NCBI database using the Basic Local Alignment Search Tool (BLAST) (Zhang et al. 2012). Using the MEGA6 software, multiple alignments of the nucleotide sequences and construction of phylogenetic trees were performed using the neighbor-joining method (Tamura et al. 2013).
Pathogenicity of F. solani and R. solani to cucumber
Sterilized soil (1 kg/pot) was distributed in 14-cm diameter pots and F. solani and R. solani isolates separately grown on millet grains were added into the potting soil at 1% (W:W). The pots were watered and kept for 4 days before sowing. Seeds of cucumber were surface sterilized in 1% sodium hypochlorite solution for 2 min, then were rinsed in sterile distilled water, and sown in the pots (5 seeds/pot). Seeds were also sown in non-infested soil to serve as a control. Four replicates (pots) were established for each treatment, and the pots were randomly distributed in the greenhouse, where they were watered and fertilized as needed. The percentages of pre- and post-emergence damping off were determined after 20 days of sowing. The percentages of seed germination were calculated according to the following formula: seed germination (%) = (number of seeds germinated/total number of seeds) × 100.
Efficacy of P. fluorescens and B. subtilis as bio-control agents against R. solani and F. solani on cucumber
Preparation of fungal inoculums
Clean 250-ml flasks were filled with millet grains and autoclaved at 121 °C for 1 h for two successive days. Five-millimeter diameter discs from the margins of the fungal colonies (R. solani or F. solani) were added to the flasks. Flasks were incubated at 25 ± 2 °C for 2 weeks and were shaken every 2 days to ensure uniform colonization of the fungus.
Bacterial isolation
Soil samples were collected from the rhizosphere of cucumber non-symptomized plants growing adjacent to the plants that are showing damping-off or wilt symptoms. One gram of each collected sample was suspended in 9 ml of sterile distilled water and serially diluted until getting the dilution of 10−7. One milliliter of each sample was spread on a Petri dish plate containing nutrient agar medium, and all plates were incubated at 37 °C for 24 h. Individual bacterial colonies were picked up using a sterilized loop, transferred to nutrient agar plates, and incubated at 37 °C for 24 h. The developed single colonies were transferred to nutrient agar medium slants and pure cultures were stored in a refrigerator at 4 °C.
The isolated bacterial isolates were identified, using morphological (staining and motility), cultural (Nutrient agar, Cetrimide agar), and biochemical tests (IMIC test, triple sugar iron test, nitrate reduction test, catalase test, casein hydrolysis, oxidase test, starch hydrolysis, lipid hydrolysis, gelatin liquefaction, and carbohydrate tests).
In vitro evaluation of antifungal activity of the isolated bacterial isolates
The antagonistic effects of P. fluorescens and B. subtilis against R. solani and F. solani were evaluated in vitro. A streak of either P. fluorescens or B. subtilis was placed on PDA plates at 28 °C for 1 day; then a mycelial disc (0.5 cm) of either R. solani or F. solani was placed onto the center of each PDA plate. All plates were incubated at 28 °C until the fungal growth of the control plates reached the edge of the plate. The reduction of the fungal mycelial growths was calculated according to Fokemma (1973). Radial growth of R. solani and F. solani was recorded and inhibition percent of the growth was calculated according to the following formula: growth reduction (%) = [(growth in control − growth in treatment)/growth in control] × 100
Effect of B. subtilis and P. fluorescens on cucumber seed germination and damping-off disease in pots
A pot experiment was designed using small pots containing reasonable weight (300 g.) of sterilized soil (sandy loam). R. solani and F. solani grown on millet grains were mixed with the potting soil at 1% W:W. After 3 days, bacterial suspensions containing 1 × 109 CFU/ml of the tested bacterial isolates were also added, and the infested pots were irrigated for 5 days before sowing. Ten cucumber seeds were sown in each pot with 3 replicates (pots) for each treatment in completely randomized design (CRD). The experiment included 10 treatments namely non-infested soil (control), soil treated with R. solani only, soil treated with F. solani only, soil treated with B. subtilis only, soil treated with P. fluorescens only, soil treated with R. solani + B. subtilis, soil treated with R. solani + P. fluorescens, soil treated with F. solani + B. subtilis, and soil treated with F. solani + P. fluorescens. Pots were kept under greenhouse conditions till the end of the experiment (3 weeks of sowing). The percentage of seed germination and damping off of cucumber seedlings were determined at the end of the experiment.
