Potential activities of Bacillus simplex as a biocontrol agent against root rot of Nigella sativa caused by Fusarium camptoceras
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This study aimed to investigate the impact of two isolates of Bacillus simplex (PHYB1 and PHYB9) for controlling the root rot disease of black cumin (Nigella sativa) caused by Fusarium camptoceras (PHYF1) under greenhouse and field conditions at Assiut Governorate, Egypt. The highest reduction percentage of infection (22.5%) was recorded by the treatment of PHYB1 as suspension than the control (60%). Both isolates of Bacillus spp. provided the root and foliar dry weight under greenhouse and seed production in the field. The results showed that the isolate PHYB1 as a formulation gave the highest impact in the root dry weight (0.28 g/plant), followed by PHYB9 (0.22 g/plant), with insignificant difference between them. PHYB9 suspension also provided the highest seed production (27.97 g/plant), whereas PHYB9 as a formulation gave the lowest (24.08 g/plant). Studies on the interaction between Bacillus spp. on F. camptoceras by scanning electron microscope (SEM) revealed that both caused a complete mycoparasitism on the fungal growth. The bacterial growth was seen to adhere and colonize the hyphae, resulting in hyphal tissue maceration. Therefore, the use of both isolates of Bacillus spp. to control root rot disease of black cumin under greenhouse and field conditions can be recommended.
KeywordsBiological control Black cumin Bacillus spp. Fusarium wilt Rhizobacteria
Black cumin (Nigella sativa L., Family: Ranunculaceae) seeds have great economic importance as they occupy an export priority in the first rank. It has especial importance among the other traditional crops in the Middle and Upper Egypt, especially in Giza, Fayoum, Beni-Suef, El-Minya, and Assiut Governorates (FAO 2016). It is an annual herb possessing a wide range of medicinal uses; the seeds are used in folk herbal medicine all over the world for the treatment and prevention of a number of diseases (Chevallier 1996). Root rot is one of the most serious diseases affecting black cumin. It is caused by Fusarium spp. (Sharma and Meena 2012). Fungal disease control is based mainly on the use of fungicides (Al-Sman et al. 2017). Fungicides may be toxic to animals, crops, and humans and could lead to the development of fungicide-tolerant pathogen strains (Abo-Elyousr et al. 2014). Biological control of soil-borne diseases, using bioagents, is an alternative method to the chemical control (Abo-Elyousr et al. 2019). It offers a way to effectively control pathogens without harmful effects on animals, humans, plants, and/or the environment (Sallam et al. 2013). In recent years, various Bacillus spp. were used as potential biocontrol agents against different Fusarium spp. The use of plant growth-promoting rhizobacteria (PGPR) has become a common practice in many countries of the world (Abo-Elyousr and Mohammed 2009 and Alamri et al. 2019).
The objective of this study was to determine the effect of two Bacillus simplex isolates, PHY1 and PHYB9 isolates, to control the root rot disease of black cumin caused by Fusarium camptoceras (PHYF1) under greenhouse and field conditions in Assiut Governorate, Egypt.
Materials and methods
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:
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).
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.
Results and discussion
Effect of B. simplex on disease severity under greenhouse conditions
Effect of B. simplex on dry weight of root and foliar dry weight under greenhouse conditions
Effect of Bacillus simplex as suspension and/or formulation on root and foliar dry weight under greenhouse conditions
Root dry weight (gm/plants)
Foliar dry weight (gm/plants)
Mean of treatments
Mean of treatments
Also, obtained results reported herein showed that bacterial suspension (PGPR) had the greatest efficacy regarding the morphological parameters of the seeds. Principally, the two treatments (formulation and suspension) were not different in most of the main studied traits of plant growth. The results agree with previous studies of Miao et al. (2018). Biocontrol agents enhanced the plant growth by production of phytohormones, which are usually believed to be involved in plant growth promotion (Glickmann and Dessaux 1995). Gopalakrishnan et al. (2012) stated that the PGPR enhanced the morphological parameters of the plant by their production of some hormone, such as indole acetic acid (IAA), siderophore production, and phosphate solubilization. IAA-producing microorganisms are known to promote root elongation and plant growth
Effect of B. simplex on disease severity and seed production under field conditions
Disease severity percentage
Effect of Bacillus simplex as suspension and/or formulation on disease severity under field conditions in 2015 and 2016 growing seasons
Disease severity percentage in field 2015
Disease severity percentage in field 2016
Mean of treatments
Mean of treatments
Effect of Bacillus simplex as suspension and/or formulation on seed production under field conditions in 2015 and 2016 growing seasons
Seed production 2015
Seed production 2016
Mean of treatments
Mean of treatments
Effect of B. simplex on volatile oils and fixed oils
Effect of Bacillus simplex as suspension and/or formulation on volatile oils and fixed oils
Volatile oils (100 gm) seeds
Fixed oils (100) gm seeds
Interaction between B. simplex strain and mycelia of F. camptoceras
The plant growth-promoting rhizobacteria (PGPR) B. simplex PHYB 1 and 9 proved significant reductions of disease severity of F. camptoceras (PHYF1), when used as a suspension or formulation. They also improved the vegetative growth and seed productivity and quantity of fixed and volatile oils in black cumin.
All authors contributed equally in the manuscript. AKM participated in the planning and implementation of the experiments, sampling, lab work, writing of the draft, and writing of the manuscript. AMK participated in the bacterial identification, implementation of the experiments, and writing of the manuscript. AE participated in the planning and implementation of the experiments, analyzing of data, and writing of the draft. AE participated in the implementation of the experiments and collection of data. All authors read and approved the final manuscript.
Ethics approval and consent to participate
This manuscript is in accordance with the guide for authors available on the journal’s website. Also, this work has not been published previously and is approved by all authors and host authorities
Consent for publication
All authors approve to publication
The authors declare that they have no competing interests.
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