The pathogen was isolated from wheat seed coats of the infected samples. Potato dextrose agar (PDA) medium was used for the isolation and cultivation of the pathogen. Rose Bengal agar (RBA) and PDA were used for the isolation and cultivation of the antagonists obtained from soil samples. The soil samples were taken at 15-cm depth from rhizosphere to obtain a high diversity of antagonists and from 15 various areas in the province of Tekirdag (Turkey) throughout September 2018. The seeds of the susceptible wheat cultivars “Gun 91 and Sultan 95” were purchased and used to determine the antagonistic activity of the bio-agent treatments against F. graminearum. The study was conducted in vitro and in vivo assays (foliar, seed, soil and seed + soil treatments) at 5 concentrations of 1 × 105 to 1 × 109 spores/ml. In vivo assays were carried out using 30 samples of the wheat seeds per pot (22 × 15-cm diameter) containing 100 g sterile peat.
Isolation and identification of the causal pathogen and the bio-agents
The wheat seed samples were disinfected by 1% sodium hypochlorite solution (SHS) for 5 min and rinsed 3 times with sterile distilled water (SDW). After drying process on sterile filter paper, the seeds were placed onto PDA plates and incubated at 28 °C for 7 days. After incubation period, the fungal isolates were purified by a single spore technique and kept at − 10 °C throughout the study. The purified isolates were identified according to cultural, morphological (Ellis 19711976; Booth 19711977; Karaca 1974; Domsch et al. 1980; Nelson et al. 1981; Burgess et al. 1994), microscopic (Nelson et al. 1981; Toussoun and Nelson 1995), and pathological (Nelson et al. 1981; Singh et al. 1991; Toussoun and Nelson 1995) properties. The pathogenicity of 20 isolates of F. graminearum was pre-assessed on the basis of the number of dead seedlings of both wheat cultivars after 15 days under plant growth room conditions (at 25 °C, 16 h of photoperiod and 100% RH) to detect the most pathogenic fungal isolate for the next experiments. The experiment was carried out according to the same procedure as in vivo assays.
The soil samples were sieved and dried for a week at 18 °C and then kept at 10 °C. The antagonistic microbial flora was incubated for 7–9 days and isolated with serial dilution technique (Rai and Kovics 2010) on RBA (dextrose, 10 g; soy peptone, 5 g; KH3PO4, 1 g; MgSO4, 0.5 g; streptomycin sulfate salt, 1.0 g; Rose Bengal, 0.05 g; agar, 15 g; chloramphenicol, 0.1 g; distilled water, 1000 ml) for growth of antagonistic yeasts, and on PDA (agar, 15.0 g; dextrose, 20.0 g; potato extract, 4.0 g; streptomycin sulfate salt, 1.0 g; distilled water, 1000 ml) for growth of fungal antagonists. Consequently, the single spore or hyphal tip was taken from the developing fungal colonies and transferred onto PDA medium containing streptomycin sulfate salt (1.0 g/l) and incubated at room temperature (25 ± 2 °C) for a week. The selected 30 antagonistic isolates were identified according to mycological keys (Lundquist 1972; Vaughan Martini and Martini 1993 and Gams and Bissett 1998). In this study, a preliminary experiment was conducted to assess the identified isolates exhibiting the highest antagonistic activity against F. graminearum on PDA medium. The experiment was carried out according to the same procedure as in vitro assays. As a result of the assessment, the most antagonistic isolates were selected and used for the next experiments. In order to prepare the spore suspensions, the pathogen cultures were passed through 2 layers of cheesecloth, diluted with sterile distilled water to a concentration of 1 × 105 conidia/ml and centrifuged at 4.000 rpm for 5 min at 22 °C. The cultures of the bio-agents were blended in an electric blender for 2 min and prepared as a liquid suspension with sterile distilled water. All bio-agents were adjusted at 5 different concentrations from 1 × 105 to 1 × 109 spores/ml and centrifuged at 5500 rpm for 5 min for S. cerevisiae (Janson and Elshadei 2012) and at 2500 rpm for 10 min for G. roseum and S. fimicola. The obtained supernatants were filtered through grade no. 1 Whatman filter paper and used for in vitro and in vivo assays.
Determination of the antagonistic activity of the bio-agents (in vitro)
One disc (0.5-cm diameter) of 7-day-old culture of the pathogen isolate was placed to the center of the plate. The adjusted spore suspensions of G. roseum, S. cerevisiae, and S. fimicola were streaked parallel on either side of the fungal disc at a distance of 2 cm (Jalaluldeen et al. 2014). In addition, a disc (0.5 cm) of G. roseum-S. cerevisiae, G. roseum-S. fimicola and, S. cerevisiae-S. fimicola was placed at a distance of 2 cm from PDA plate edge to evaluate the antagonistic interaction between each other. The plates were incubated at 25 °C for 7–9 days. After the incubation period, the diameter of each colony was measured to evaluate the antagonistic activity as a result of the average of 5 independent replicates. The antagonist-free PDA medium, containing only SDW and a culture disk of the pathogen, was used as a control. The effect of the bio-agent treatments on mycelial growth of the pathogenic fungus was determined after 7 days and calculated using the following formula (Topps and Wain 1957).
$$ I\%=\left[\left(C-T\right)/C\right]\times 100 $$
where I % = inhibition rate, C = average diameter of mycelial growth of pathogenic fungus in control treatment, and T = average diameter of mycelial growth of pathogenic fungus after bio-agent treatments.
Determination of in vivo antagonistic activity of the bio-agents on leaf infection
The wheat seed samples were disinfected by 1% SHS for 5 min and rinsed 3 times with SDW before bio-agent treatments. The seeds of wheat cultivars “Gun 91and Sultan 95” were planted in experimental pots (22 × 15-cm diameter) containing a sterile peat and were grown in the plant growth room conditions. When plants reached the two-leaf stage in 2 weeks, G. roseum, S. cerevisiae, S. fimicola, and a mixture of the bio-agents (G. roseum + S. cerevisiae, G. roseum + S. fimicola, S. cerevisiae + S. fimicola) were sprayed as a foliar treatment separately at concentrations of 1 × 105 to 1 × 109 spores/ml to upper and lower surfaces of wheat leaves with a dose-adjusted spray at the rate of 0.2 ml per leaf. After the inocula were allowed to dry on the leaves (approximately 1–2 h), the pathogen inoculum at a concentration of 1 × 105 conidia/ml was applied to wheat leaves in the same way. Following the inoculation process, the plants were kept in a plant growth room to provide optimum conditions for disease development for 72 h. Afterwards, the seedlings were kept at room temperature for 24 h (Kawasaki 2008). Consequently, the leaves were detached separately from the plants, and the disease incidence was determined according to visually necrotic lesion and/or sporulation area of the disease. The lesion area was measured as the average of 5 independent replicates and compared with the control pots sprayed with a spore suspension of the fungal pathogen only. The effect of the bio-agent treatments on leaf infection was calculated using the formula of Topps and Wain (1957).
$$ I\%=\left[\left(C-T\right)/C\right]\times 100 $$
where I % = inhibition rate, C = average lesion length in control treatment, and T = average lesion length after bio-agent treatments.
Determination of in vivo antagonistic activity of the bio-agents on damping-off
To evaluate the antagonistic activity of the bio-agents against F. graminearum, different concentrations of each of G. roseum, S. cerevisiae, S. fimicola, and their mixtures (G. roseum + S. cerevisiae, G. roseum + S. fimicola, S. cerevisiae + S. fimicola) were examined as the seed, soil, and seed + soil treatments.
To determine the antagonistic activity of the seed treatments against damping-off in wheat cultivars, the pathogen inoculum at the rate of 5% w/v (Hussein 1973) was transferred to pots containing an autoclaved sterile peat, mixed thoroughly, and kept by moisturizing for 7 days. After the incubation period, the bio-agent suspensions were applied at different concentrations to the disinfected wheat seeds by spraying with a dose-adjusted spray to cover the seed surface homogenously at the rate of 20 ml/kg of seeds before sowing in pots.
To determine the antagonistic activity of the soil treatments, the pathogen inoculum at the rate of 5% w/v (Hussein 1973) and the spore suspensions of tested bio-agents (1 × 105, 1 × 106, 1 × 107, 1 × 108, 1 × 109 spores/ml) were added as a soil drench at the rate of 30 ml per each pot, mixed thoroughly, and kept by moisturizing for 7 days. After a week, the wheat seeds were sown in pots following disinfection with SHS.
For the seed + soil treatments, the pathogen inoculum and the spore suspensions of tested bio-agents were added to pots as a soil drench, mixed thoroughly, and kept by moisturizing for 7 days. At the end of a week, disinfected seeds were sprayed by spore suspensions of the tested bio-agents and were sown in pots. The experiment was attempted with an average of 5 independent replicates, compared with the control pots inoculated with a spore suspension of the fungal pathogen only. The study was conducted in a plant growth room under a 16 h photoperiod cycle at 25 °C. The number of dead seedlings was determined after 15 days, and the effect of the bio-agent treatments on damping-off was calculated using the following formula (Topps and Wain 1957).
$$ I\%=\left[\left(C-T/C\right)\right]\times 100 $$
where I % = inhibition rate, C = average of non-emerged seedlings in control treatment, and T = average of non-emerged seedlings after bio-agent treatments.
Statistical analysis of data was subjected to ANOVA (one-way analysis of variance). Significant differences (p < 0.05) were tested by the general linear model (GLM) procedure, using the Duncan’s multiple range test (DMRT) for disease incidence, mycelial growth, and lesion length of the pathogenic fungus after bio-agent treatments.