Source of microorganisms
Pathogenic fungi isolate Fusarium camptoceras (PHYF1), the causal agent of root rot disease, was isolated from black cumin (Al-Sman et al. 2017). The isolate was grown in Petri dishes (9 cm in diameter) which contained potato dextrose agar medium (PDA) (20 g dextrose, 15–20 g agar, 200 g potato, and 1 L water). Petri dishes were incubated for 7 days at 27 °C. Two isolates of Bacillus simplex (PHYB1 and PHYB9), isolated from the rhizosphere of black cumin, at Assiut Governorate, Egypt, were used. The isolates were identified according to their morphological and physiological characteristics (Dye 1968; Schaad 1988, and Holt et al. 1994).
Inoculum preparation and soil infestation with the pathogenic fungi
Inoculum of pathogenic isolate were prepared by inoculating of discs 0.5 cm in diameter of 5-day-old cultures of PHYF1 in bottles which contained the autoclaved barley medium (100 dried coarse sand, 75 g dried barley grains, 75 ml tap water). Bottles were incubated for 15 days at 25 ± 1 °C (Abo-Elyousr et al. 2009). After this period, the contents were mixed together and used as a source of inoculum. Pots (30 cm diameter) filled with 5 kg soil were infested by mixing about 150 g of the inoculums with the soil and then irrigated directly. For control treatments, sterilized uninoculated soil were used (Gabr et al. 1998). Ten disinfested seeds of black cumin were seeded in each pot, 7 days after soil infestation with the pathogen (Hilal et al. 2000). Pots were irrigated directly after sowing and subsequently as needed for 8 weeks.
Inoculum preparation of suspension antagonistic of Bacillus simplex
The antagonistic bacterial isolates PHYB1 and PHYB9 were used in this study. Inoculum of each bacterial isolate was prepared by growing it in a nutrient yeast extract broth, incubated at 25 °C on an orbital shaker at 200 rpm for 24 h. Bacteria were afterward polluted by centrifugation at 15,000 rpm for 5 min and washed in distilled water and the concentration of the bacteria was adjusted to 108 cfu.
Preparation of formulation antagonistic Bacillus simplex
To prepare the Bacillus spp. formulation, the method of Abo-Elyousr and El-Hendawy (2008) was followed. In brief, each isolate was grown separately at 20 °C in 250-ml flask each containing 100 ml of tryptic soya broth liquid medium and shaking at 1000 rpm for 48 h. A 400-ml aliquot of the bacterial suspension containing (108 cfu ml−1) was mixed by 1 kg of the talc powder (dry-sterilized at 105 °C for 12 h), 15 g CaCO3 (to adjust the pH to 7), and 10 g carboxymethylcellulose.
Effect of Bacillus simplex as suspension/or formulation on disease severity under greenhouse conditions
Inoculums and pot infestation of the tested pathogenic isolate PHYF1 were prepared as previously mentioned. Each pot was planted by ten sterilized seeds of black cumin after they were soaked in the antagonistic bacterial suspension for 20 min and was also treated with the formulation bacteria; they were left to dry then grown directly. Two controls were used in this experiment: one was healthy control (untreated with bioagents or pathogens) and the second was infected control (untreated with bioagents but infected with the pathogen). The treatments were arranged in a randomized complete block design with five replicates. Results were recorded as disease severity percentage after 80 days of planting. The root rot was scored on 0–3 scale, where:
0 = healthy, creamy white on discoloration of sub crown internode and crown roots, 1 = light brown discoloration of sub crown internode and crown roots, 2 = brown discoloration of sub crown internode and crown roots, and 3 = dark brown to black discoloration of sub crown internode and crown roots and/or roots mostly decayed (Tinline et al. 1975). The disease severity (disease index) was estimated according to the following equation (Seleim et al. 2011):
$$ \mathrm{Root}\ \mathrm{rot}\kern0.37em \mathrm{index}=\frac{\left(n\times 1\right)+\left(n\times 2\right)+\left(n\times 3\right)\;}{T\times 3}\times 100. $$
n = number of plants in each scale of disease plants (1, 2, 3)
T = total number of plants
At the end of the experiment, plants from various treatments were removed, washed thoroughly with running water, then oven dried at 65 °C for 72 h for dry weight. The experiment was repeated twice.
Control of the disease using Bacillus simplex under field conditions
The field trials were conducted at the Experimental Farm of Plant Pathology Department, Faculty of Agriculture, Assiut University, Egypt, during 2014/2015 and 2015/2016 growing seasons. The plots were divided into two rows: length/row 2 m and five holes/row; three plots were used for each treatment. The distance between holes is 30 cm and the infestation was carried out by adding 10 g substrate containing inocula of the pathogen PHYF1. Seeds were treated by bioagents as in greenhouse experiment. Disease severity percentage was recorded after 80 days from planting, and seed production (g/plants) was recorded at the end of experiment.
Scanning electron microscopic (SEM) examination and photography
The method of Zhou et al. (2011) for preparing the samples was followed. After growing the bacterial suspensions of PHYB1 and PHYB9 with the pathogen PHYF1 in Petri dishes, 2–3 samples, size 3–5 mm, from the direct interactions were cut and fixed in 5% cold buffered glutaraldehyde for 2 min. The samples were scanned at 15 kV, using a JEOL JSM 5300 Lv scanning electron microscope, and photographed.
Evaluation of fixed and volatile oils
Distillery fixed oils
Soxhlet extractor was used to get fixed oils of black cumin seeds, according to Salea et al. (2013).
Distillery volatile oils
Clevenger type device was used to get an oil pilot from black cumin seeds. In this method, steam was passed through the seeds containing the volatile oils; 100 g of the seeds were placed in flasks with 500 ml water and left for 3 h for the seeds boiling. The water evaporated carrying the volatile oil and dried over anhydrous sodium sulphate (Khalid and Shedeed 2016).
Statistical analysis
Analyses of variance were carried out, using MSTAT-C program version 2.10 (1991) and least significant difference (LSD) at P ≤ 0.05 was employed to test significant difference between treatments (Gomez and Gomez 1984). All experiments were performed twice.
