Experimental isolation procedures

Isolation tests were conducted in the Kingdom of Saudi Arabia (KSA) using soil dilutions from the rhizospheres of tomato, potato, and eggplant [28]. A single gram of soil was agitated in a sterile saline solution containing 0.85% NaCl for fourteen minutes. 200 µL of the 10^-5, 10^-6, and 10^-7 dilutions were added to the glycerol nutrient agar media and the peptone nutrient agar medium. Following a 48-hour incubation period at a temperature of 30 °C, many bacterial colonies became apparent. The detected colonies were isolated and purified using the process of isolating a single colony. The bacterial cultures were stored in a 50% glycerol broth at a temperature of -80 °C for future investigations.

Bacillus isolates antagonism test 

An in vitro antagonist test was conducted to investigate the antagonistic effect of Bacillus isolates as biocontrol agents against S. bataticola, a plant pathogenic fungus. The antagonistic bacterial isolates were lined onto potato dextrose agar (PDA) plates after being cultivated for two days. This was completed 48 hours before any fungi were introduced for testing. Located in the middle of the Petri plate was a circular area of mycelium of 5 mm in length. This area contained a vigorously growing culture of the fungus being studied. The mycelial circle was consistently positioned at a fixed distance from the border of the plate. The plate was then incubated at a temperature of 30 °C for a period of three to seven days. The percentage of inhibition was calculated according to the procedure described by Maurhofer et al. [29]. The experiment was conducted with three repetitions. The data obtained were subjected to statistical analysis using analysis of variance (ANOVA) with the use of CoStat software. Tukey's HSD test was employed to distinguish the means at a significance level of p≤0.05.

Molecular characterization of the hopeful Bacillus isolate

After inoculating 5 milliliters of Luria-Bertani broth medium with the pure colony, the mixture was stirred at a temperature of 30°C for an entire night. Using the Wizard Genomic DNA Purification Kit (Promega, USA), bacterial genomic DNA was extracted from a 1 mL bacterial pellet. The procedure was carried out following the instructions provided by the manufacturer. A polymerase chain reaction (PCR) experiment was carried out by employing certain primers of 16s rRNA (F: AGAGTGATCCTGGCTCAG, R: GGTTACCTTGTTACGACTT), following the approach that was described earlier [30]. Following that, the PCR fragments were subjected to purification and sequencing protocols. Following the uploading of the annotated sequences to GenBank, a comparison study was carried out with the isolates that had been published in the past.

Experiment on Fermentation

Bioreactor (fermentor)      

The B. amyloliquefaciens isolate KSAS6, which showed great potential, was grown and fermented in a ten-liter bench-top bioreactor (Cleaver, Saratoga, USA) equipped with four baffles and turbine impellers that had six-bladed discs. A computerized control system was employed to supervise the batch fermentation process with a working capacity of four liters. An automated method was controlled using a device equipped with a 10.4-inch color touch-screen interface. This device can store up to 59,994 distinct programs for various scenarios. The batch fermentation process was conducted at a consistent temperature of 30°C and a pH level of 7, which was carefully maintained. For pH regulation, a solution of 2 mol L^–1 NaOH and a solution of 2 mol L^–1 HCl were automatically supplied. Once the air passed through a sterile filter, it was compressed and adjusted to a flow rate of 0.5 VVM (air volume per broth volume per minute). The agitation speed was initially set at 200 rpm and then manually increased to maintain the dissolved oxygen (DO) level above 20%. The DO content and pH were measured using METTLER TOLEDO electrodes [31].

Batch fermentation process

The batch fermentation process is a method of producing a desired product by allowing microorganisms to grow and metabolize in a controlled environment for a specific time. The highly potential B. amyloliquefaciens isolate KSAS6 was inoculated as a single colony in a 500 mL^-1 Erlenmeyer flask containing 100 mL^-1 of Number 3 medium [32]. The cell broth was thereafter incubated overnight at a temperature of 30°C with continuous agitation at a speed of two hundred revolutions per minute. The batch fermentation process was initiated in a stirred tank bioreactor with an optical density (OD₅₅₀) of 0.5. The bioreactor had a working volume of 4 liters and was filled with Number 3 medium. During the fermentation process, many culture samples were collected and the quantities of biomass and glucose were measured in each sample.

Analytical methods

Assessment of cell biomass

After the culture's optical density (OD) at 550 nm was measured using a spectrophotometer to track the cell count. After subjecting a sample of 10 mL^-1 to centrifugation with a force of 894 g for 10 minutes, the mass of the cells was restored, purified, and then subjected to centrifugation again to determine the weight of the cells after they had been dried. Subsequently, the pellet was subjected to a drying process for the entire night at a temperature of 80°C in a dry-air oven, as described by Van Dam-Mieras et al. [33].

Measurement of glucose concentration

To determine the concentration of glucose, an enzymatic colorimetric kit was utilized. This methodology relies on the activities of peroxidase and glucose oxidase. The quinoneimine dye, which is used as a glucose concentration marker, transforms into a reddish-violet color as the final product.

Gas chromatography-mass spectroscopy (GC–MS) analysis

Through the use of GC–MS analysis, the bioactive components that were found in the cell-free supernatant of the promising B. amyloliquefaciens isolate KSAS6 were identified and described. To accomplish this, the culture filtrate was combined with ethyl acetate in a ratio of 1:1 by volume. According to Hirpara et al. [34], after the mixture had been thoroughly agitated for ten minutes and then left to sit for five minutes, two distinct layers that were not able to mix were formed. Through the utilization of a separating funnel, the top layer of the solvent, which included the biomolecules that were extracted, was separated separately. According to Sharma et al. [35], the ethyl acetate extract was further concentrated to a tiny volume by evaporating the solvent in a rotary evaporator while the vacuum was maintained. It was then that brown gum was shown after the ethyl acetate had evaporated. Following that, the crude extract was stored at a temperature of 4 degrees Celsius. An Agilent 7000D instrument (Santa Clara, California, United States) was used to perform GC–MS analysis on the residues that were produced, and the program conditions were programmed following Khamis et al. [36].

Results

Figures & Tables

Discussion

The ecological balance of soil-dwelling microbes has been upset by the widespread use of chemicals (pesticides) to manage plant diseases [37]. This has resulted in the emergence of resistant pathogen strains, contaminate groundwater, and pose clear health risks to humans [38]. The discovery of environmentally acceptable substitutes for the chemical pesticides now in use to address a variety of crop diseases is one of the major ecological concerns facing plant pathologists and microbiologists in the future [39]. Utilizing microorganisms for biological control can be a highly efficient method to reduce the negative impacts of synthetic chemical usage on the environment and decrease pollution and disturbances [40–42].

Bacillus sp. uses between 5 and 8% of its genome to synthesize bioactive secondary metabolites, which are antagonistic compounds meant to inhibit infections. Bacillus species use a broad range of antagonistic chemicals, such as polyketide compounds, bacteriocins, siderophores, and non-ribosomally produced peptides and lipopeptides, to inhibit pathogens in the root environment [43]. The most researched Bacillus species for their capacity to produce volatile organic compounds (VOCs) include B. amyloliquefaciens, B. velezensis, B. subtilis, and B. altitudinis [28,44]. B. subtilis and B. amyloliquefaciens are the producers of volatile organic compounds (VOCs), which include 2,3-butanediol. According to Saraf et al. [45], these are linked to the biocontrol of diseases and cause plants to develop induced systemic resistance (ISR). Extracellular enzymes including chitinase, β-1,3-and β-1,4-glucanases, laminarinase, oxalate oxidase, cellulases, proteases, phytases, and lipases are produced by Bacillus species. These enzymes have the ability to break down the components of fungi's cell walls, which may be a defense against fungus-related infections [46]. In this investigation, we evaluated fifteen Bacillus isolates as potential biocontrol agents against the in vitro mycelial growth of S. bataticola. When it came to S. bataticola mycelia inhibition, KSAS6 fared better than the other Bacillus isolates, with an inhibition percentage of 48.2%. The bioagent KSAS6's antagonistic effects can be attributed to its excretion of compounds and enzymes with both direct and indirect antifungal effects. Three Bacillus strains that exhibited antifungal activity against S. bataticola with an inhibition percentage of 64% were identified by Sicuia et al. [47]. This activity relates to their capacity to synthesize lipopeptides. According to Boiu-Sicuia and Cornea [48], some Bacillus strains isolated from various vegetables suppressed the fungal mycelial of S. bataticola in vitro with an inhibitory percentage equivalent to 56.7%. Additionally, the microscopic examination of S. bataticola's impeded mycelial growth demonstrated cytoplasmic content leaks, fungal perforations, and ulcerations in the cells. 

The optimal circumstances for microbe development, including agitation, aeration, pH, and temperature, can be achieved in a stirred tank bioreactor. These factors are critical for optimizing the release of secondary metabolites and the proliferation of bacterial cells. In the current study and during the exponential phase, the cell mass of B. amyloliquefaciens isolate KSAS6 expanded exponentially at a steady specific growth rate of 0.13 h^–1, which reached its maximum value of 2.1 g L^-1 at 11 hours, with a production rate of 0.17 g L^-1 h^-1.  The cell mass of B. subtilis isolate G-GANA7 increased exponentially with a constant specific growth rate of 0.3 h^-1 [31]. On the other hand, the cell mass of B. velezensis strain GB1 was cultivated in a stirred tank bioreactor with a specific growth rate of 0.1 h^-1 during a batch fermentation approach [49]. Glucose is one of the substrates that is most commonly used as a carbon source in various growing techniques. Since substrate inhibition is known to happen at specific doses. Thus, it is essential to ascertain the proper medium composition to facilitate the growth of a particular type of bacteria [50]. The glucose concentration was employed at its optimal value by the authors of this article for these reasons. After 14 hours, the glucose concentration reached 0.2 g. In this study, the biomass yield coefficient of the B. amyloliquefaciens isolate KSAS6 was 0.37 g cell/g glucose. The exponential phase of the bacterial growth curve for the B. amyloliquefaciens isolate KSAS6 revealed that the bacteria were expanding swiftly and consuming a significant amount of oxygen and carbon sources to do so, with a rate of glucose intake of 0.27 g L^-1 h^-1. Our findings indicate that during the first hour of the culture's growth, DO progressively dropped. Then, once the exponential phase began, the DO rapidly decreased, necessitating an increase in agitation speed to keep it above 20%. Stojanović et al. [51] showed that in B. subtilis cultivations, the generation of biomass and spores is significantly influenced by the agitation and aeration of the cultures, which have a direct impact on the DO concentration. B. subtilis did not sporulate when exposed to low oxygen levels, although it did continue to produce bioactive chemicals [52]. Numerous studies have found that increasing agitation and aeration rates or regulating the DO content over 20–30% can result in quicker cell development [53–55]. Nevertheless, higher agitation levels cause shear stress on cells, which can have detrimental consequences such as sporulation mechanism deactivation, uneven cell populations, smaller cells, and even cell death [56].

Microbial volatile organic compounds, often known as MVOCs, are a class of molecules that are generated as a result of metabolic activities performed by fungi and bacteria. There are many different chemical forms that volatile organic compounds (VOCs) can take, including ketones, alcohols, terpenoids, sulfur compounds, alkenes, and many others. Certain chemicals are shared by all of the microbes in the group, while other molecules are unique to specific strains. According to Sidorova et al. [57], a single bacterium is capable of producing up to one hundred different volatile organic compounds. According to, Bacillus species are believed to be factories of plant protective VOCs and play a variety of roles in the protection of plants against bacterial and fungal diseases [58]. Antimicrobial VOCs have been reported to be produced by B. amyloliquefaciens strains in several different studies. According to Yu et al. [59], VOCs produced by the B. amyloliquefaciens strain have the potential to impede the mycelial development of Penicillium digitatum and P. italicum. It is possible to assert that all strains of B. amyloliquefaciens are capable of producing VOCs. However, the volume, type, chemical composition, relative abundance, and spectrum of inhibition differ among the strains. Furthermore, the spectrum of inhibition was influenced by the concentration and type of nutrients present in the medium [60]. In the current study, the identification of bioactive constituents in the ethyl acetate extract of B. amyloliquefaciens KSAS6 was conducted through the application of GC–MS analysis. GC-MS chromatogram of KSAS6 revealed that twenty-one VOCs compounds, which varied from aromatic and aliphatic compounds, were produced by B. amyloliquefaciens. The major compound was the aromatic compound, diisooctyl phthalate, which constitutes 43.31% of the total area followed by the aromatic compounds, tris(2,4-di-tert-butylphenyl)phosphate and dibutyl phthalate which contributed 12.20% and 10.09%, respectively. Additionally, the aromatic compounds, 7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione, Pyrrolizin-1-one, 7-propyl-, 5-fluoro-2,2-dimethylchroman-4-one and Methanone,(1-hydroxycyclohexyl)phenyl- were detected in varying proportion. On the other hand, Dotriacontane, n-hexadecanoic acid, and Docosane as aliphatic compounds were detected, constituting 4.45%, 3.54%, and 3.41% of the total area, respectively. The primary component of Penicillium sp.'s ethyl acetate extract, diisooctyl phthalate, was shown by Lykholat et al. [61] to be the cause of the extract's antifungal action against Alternaria alternata. It can prevent the formation of germs and possesses qualities that aid in wound healing [62,63]. Diisooctyl phthalate has recently been suggested by El-Enain et al. [64] as a possible medicinal agent for the treatment of severe bacterial infections. According to Gheda and Ismail [65], cyanobacteria can produce diisooctyl phthalate, which has antibacterial properties. Dibutyl phthalate suppressed the growth of the yeast Candida albicans and the gram-positive bacterium Staphylococcus aureus, as shown by Blazević et al. [66]. Additionally, it was demonstrated that dibutyl phthalate, which is produced by B. amyloliquefaciens and B. velezensis, has antifungal properties [67]. Meloidogyne incognita J2 was inhibited by dibutyl phthalate and diisobutyl phthalate [68]; Colletotrichum fragariae spore germination and hyphal growth were significantly inhibited by dibutyl phthalate [69]; and Verticillium dahliae Kleb proliferation was significantly suppressed by 5 mmol.L-1 dibutyl phthalate in the eggplant rhizosphere [70]. Tris (2,4-di-terbutylphenyl) phosphate is an antibiotic that has biological effects. It is a member of the phenol group. According to Valinluck et al. [71], the phenolic compounds can suppress the growth of P. chrysogenum, Fusarium oxysporum, and Aspergillus niger. Furthermore, according to Abdullah et al. [72], they have the ability to suppress the growth of E. coli, S. aureus, and Pseudomonas aeruginosa. B. tequilensis generated tris (2,4-di-terbutylphenyl) phosphate, which exhibited antibacterial action [73]. According to Amborabé et al. [74], phenolic chemicals have the ability to harm the cytoplasmic membrane, raise its permeability, and cause the discharge of intracellular fluids such proteins, nucleic acids, and inorganic ions. The antifungal action of B. amyloliquefaciens KSAS6 against S. bataticola may be attributed to these volatile organic molecules.

Finally, in terms of suppressing S. bataticola's mycelial development, the B. amyloliquefaciens stain KSAS6 was more effective than other isolates. During the batch fermentation of KSAS6 in the stirred tank bioreactor, the production of secondary metabolites and culture biomass was maximized to a satisfactory degree. The highest achievable level of biomass was 2.1 g L^-1, and the yield coefficient was established at 0.37 g cells/g glucose. The GC-MS chromatogram of KSAS6 culture filtrate contained 21 distinct compounds. These compounds may be responsible for the antagonizing activity of B. amyloliquefaciens strain KSAS6 against S. bataticola. In conclusion, the B. amyloliquefaciens strain KSAS6 demonstrates significant antifungal effectiveness against S. bataticola. Consequently, it could serve as a potent biocontrol agent for managing plant fungal infections. However, additional studies and field trials are necessary to confirm results and identify potential limitations in practical agricultural applications, as well as to assess the effectiveness of this treatment on a larger scale.

The author declares no conflicts of interest.

References

1. El-Kazzaz MK, Ghoneim KE, Agha MKM, Helmy A, Behiry SI, Abdelkhalek A, et al. Suppression of Pepper Root Rot and Wilt Diseases Caused by Rhizoctonia solani and Fusarium oxysporum. Life, (2022); 12(4): 587.
2. Jain A, Sarsaiya S, Wu Q, Lu Y, Shi J. A review of plant leaf fungal diseases and its environment speciation. Bioengineered, (2019); 10(1): 409–424.
3. Wolfgang A, Taffner J, Guimarães RA, Coyne D, Berg G. Novel strategies for soil-borne diseases: Exploiting the microbiome and volatile-based mechanisms toward controlling Meloidogyne-based disease complexes. Frontiers in Microbiology, (2019); 10(JUN): 1296.
4. Lodha S, Mawar R. Population dynamics of Macrophomina phaseolina in relation to disease management: A review. Journal of Phytopathology, (2020); 168(1): 1–17.
5. Abdelkhalek A, Aseel DG, Király L, Künstler A, Moawad H, Al-Askar AA. Induction of Systemic Resistance to Tobacco mosaic virus in Tomato through Foliar Application of Bacillus amyloliquefaciens Strain TBorg1 Culture Filtrate. Viruses, (2022); 14(8): 1830.
6. Yadav RC, Sharma SK, Varma A, Singh UB, Kumar A, Bhupenchandra I, et al. Zinc-solubilizing Bacillus spp. in conjunction with chemical fertilizers enhance growth, yield, nutrient content, and zinc biofortification in wheat crop. Frontiers in Microbiology, (2023); 14: 1210938.
7. Kloepper JW, Lifshitz R, Zablotowicz RM. Free-living bacterial inocula for enhancing crop productivity. Trends in biotechnology, (1989); 7(2): 39–44.
8. Shao J, Xu Z, Zhang N, Shen Q, Zhang R. Contribution of indole-3-acetic acid in the plant growth promotion by the rhizospheric strain Bacillus amyloliquefaciens SQR9. Biology and Fertility of Soils, (2015); 51: 321–330.
9. Raguchander T, Jayashree K, Samiyappan R. Management of Fusarium Wilt of Banana using Antagonistic Microorganisms. Journal of Biological Control, (1997); 11: 101–105.
10. Foysal MJ, Lisa AK. Isolation and characterization of Bacillus sp. strain BC01 from soil displaying potent antagonistic activity against plant and fish pathogenic fungi and bacteria. Journal of Genetic Engineering and Biotechnology, (2018); 16(2): 387–392.
11. Fendrihan S, Constantinescu F, Sicuia OA, Dinu S. Beneficial Bacillus strains improve plant resistance to phytopathogens: a review. International Journal of Environment, Agriculture and Biotechnology, (2016); 1(2): 238512.
12. Khan N, Martínez-Hidalgo P, Ice TA, Maymon M, Humm EA, Nejat N, et al. Antifungal activity of Bacillus species against Fusarium and analysis of the potential mechanisms used in biocontrol. Frontiers in microbiology, (2018); 9: 2363.
13. Arguelles-Arias A, Ongena M, Halimi B, Lara Y, Brans A, Joris B, et al. Bacillus amyloliquefaciens GA1 as a source of potent antibiotics and other secondary metabolites for biocontrol of plant pathogens. Microbial cell factories, (2009); 8: 1–12.
14. Kiesewalter HT, Lozano-Andrade CN, Wibowo M, Strube ML, Maróti G, Snyder D, et al. Genomic and chemical diversity of Bacillus subtilis secondary metabolites against plant pathogenic fungi. Msystems, (2021); 6(1): 10–1128.
15. Amanullah A, Otero JM, Mikola M, Hsu A, Zhang J, Aunins J, et al. Novel micro‐bioreactor high throughput technology for cell culture process development: Reproducibility and scalability assessment of fed‐batch CHO cultures. Biotechnology and bioengineering, (2010); 106(1): 57–67.
16. Bareither R, Bargh N, Oakeshott R, Watts K, Pollard D. Automated disposable small scale reactor for high throughput bioprocess development: a proof of concept study. Biotechnology and bioengineering, (2013); 110(12): 3126–3138.
17. Krishna C, Nokes SE. Predicting vegetative inoculum performance to maximize phytase production in solid-state fermentation using response surface methodology. Journal of Industrial Microbiology and Biotechnology, (2001); 26(3): 161–170.
18. Gangadharan D, Sivaramakrishnan S, Nampoothiri KM, Pandey A. Solid culturing of bacillus amyloliquefaciens for alpha amylase production. Food Technology and Biotechnology, (2006); 44(2): 269–274.
19. Hewitt CJ, Nienow AW. The scale‐up of microbial batch and fed‐batch fermentation processes. Advances in applied microbiology, (2007); 62: 105–135.
20. Ruiz‐Sanchez J, Flores‐Bustamante ZR, Dendooven L, Favela‐Torres E, Soca‐Chafre G, Galindez‐Mayer J, et al. A comparative study of taxol production in liquid and solid‐state fermentation with Nigrospora sp. a fungus isolated from Taxus globosa. Journal of applied microbiology, (2010); 109(6): 2144–2150.
21. Kumar S, Sharma N, Pathania S. Optimization of process parameters and scale-up of xylanase production from Bacillus Amyloliquifaciens Sh8 in a stirred tank bioreactor. Cellulose Chemistry and Technology, (2017); 51(5–6): 403–415.
22. Allman T. Bioreactors: design, operation, and applications. In: Fermentation Microbiology and Biotechnology, Fourth Edition, CRC Press; 2018. p. 283–308.
23. Kiebus-Rpaa A, Karcz J. Mass Transfer in Multiphase Mechanically Agitated Systems. In: El-Amin M (ed.) Mass Transfer in Multiphase Systems and its Applications, London, UK: InTech; 2011. p. 93–116.
24. Liu S. Bioprocess engineering: kinetics, sustainability, and reactor design. Amsterdam, The Netherlands: Elsevier; 2020.
25. Glick BR. Some Techniques to Elaborate Plant–Microbe Interactions: Beneficial Plant-Bacterial Interactions, Springer, Cham 2015, p 97-122.
26. Hathi Z, Mettu S, Priya A, Athukoralalage S, Lam TN, Choudhury NR, et al. Methodological advances and challenges in probiotic bacteria production: Ongoing strategies and future perspectives. Biochemical Engineering Journal, (2021); 176: 108199.
27. Sarmiento-López LG, López-Meyer M, Maldonado-Mendoza IE, Quiroz-Figueroa FR, Sepúlveda-Jiménez G, Rodríguez-Monroy M. Production of indole-3-acetic acid by Bacillus circulans E9 in a low-cost medium in a bioreactor. Journal of bioscience and bioengineering, (2022); 134(1): 21–28.
28. Abdelkhalek A, Al-Askar AA, Elbeaino T, Moawad H, El-Gendi H. Protective and Curative Activities of Paenibacillus polymyxa against Zucchini yellow mosaic virus Infestation in Squash Plants. Biology, (2022); 11(8): 1150.
29. Maurhofer M, Keel C, Haas D, Défago G. Influence of plant species on disease suppression by Pseudomonas fluorescens strain CHAO with enhanced antibiotic production. Plant pathology, (1995); 44(1): 40–50.
30. Yassin Y, Aseel D, Khalil A, Abdel-Megeed A, Al-Askar A, Elbeaino T, et al. Foliar Application of Rhizobium leguminosarum bv. viciae Strain 33504-Borg201 Promotes Faba Bean Growth and Enhances Systemic Resistance Against Bean Yellow Mosaic Virus Infection. Current Microbiology, (2024); 81(8): 220.
31. Moustafa HE, Abd-Elsalam HE, Abo-Zaid GA, Serour EA. as Biocontrol Agent, II. Biotechnology, (2009); 8(1): 35–43.
32. Asaka O, Shoda M. Biocontrol of Rhizoctonia solani damping-off of tomato with Bacillus subtilis RB14. Applied and environmental microbiology, (1996); 62(11): 4081–4085.
33. Van Dam-Mieras MCE, Jeu WH, Vries J, Currell BR, James JW, Leach CK, et al. Techniques used in bioproduct analysis. 1992.
34. Hirpara DG, Gajera HP, Bhimani RD, Golakiya BA. The SRAP based molecular diversity related to antifungal and antioxidant bioactive constituents for biocontrol potentials of Trichoderma against Sclerotium rolfsii Scc. Current genetics, (2016); 62: 619–641.
35. Sharma D, Pramanik A, Agrawal PK. Evaluation of bioactive secondary metabolites from endophytic fungus Pestalotiopsis neglecta BAB-5510 isolated from leaves of Cupressus torulosa D. Don. 3 Biotech, (2016); 6(2): 210.
36. Khamis WM, Heflish AA, El-Messeiry S, Behiry SI, Al-Askar AA, Su Y, et al. Swietenia mahagoni Leaves Extract: Antifungal, Insecticidal, and Phytochemical Analysis. Separations, (2023); 10(5): 301.
37. Muhammad M, Ahmad MW, Basit A, Ullah S, Mohamed HI, Nisar N, et al. Plant growth-promoting rhizobacteria and their applications and role in the management of soilborne diseases. In: Bacterial secondary metabolites, Elsevier; 2024. p. 59–82.
38. Moawad H, Abd El-Rahim WM. Bioremediation of irrigation water contaminated with textile dyes. Fresenius Environmental Bulletin, (2003); 12(7): 786–792.
39. Mnif I, Grau-Campistany A, Coronel-León J, Hammami I, Triki MA, Manresa A, et al. Purification and identification of Bacillus subtilis SPB1 lipopeptide biosurfactant exhibiting antifungal activity against Rhizoctonia bataticola and Rhizoctonia solani. Environmental Science and Pollution Research, (2016); 23: 6690–6699.
40. Usta C. Microorganisms in biological pest control—a review (bacterial toxin application and effect of environmental factors). Current progress in biological research, (2013); 13: 287–317.
41. Abdelkhalek A, El-Gendi H, Al-Askar AA, Maresca V, Moawad H, Elsharkawy MM, et al. Enhancing systemic resistance in faba bean (Vicia faba L.) to Bean yellow mosaic virus via soil application and foliar spray of nitrogen-fixing Rhizobium leguminosarum bv. viciae strain 33504-Alex1. Frontiers in Plant Science, (2022); 13: 933498.
42. Elsharkawy MM, Sakran RM, Ahmad AA, Behiry SI, Abdelkhalek A, Hassan MM, et al. Induction of systemic resistance against sheath blight in rice by different Pseudomonas isolates. Life, (2022); 12(3): 349.
43. Fira D, Dimkić I, Berić T, Lozo J, Stanković S. Biological control of plant pathogens by Bacillus species. Journal of biotechnology, (2018); 285: 44–55.
44. Grahovac J, Pajčin I, Vlajkov V. Bacillus VOCs in the context of biological control. Antibiotics, (2023); 12(3): 581.
45. Saraf M, Pandya U, Thakkar A. Role of allelochemicals in plant growth promoting rhizobacteria for biocontrol of phytopathogens. Microbiological research, (2014); 169(1): 18–29.
46. Kumar P, Dubey RC, Maheshwari DK. Bacillus strains isolated from rhizosphere showed plant growth promoting and antagonistic activity against phytopathogens. Microbiological research, (2012); 167(8): 493–499.
47. Sicuia OA, Olteanu V, Ciuca M, Cîrstea DM, Cornea CP. Characterization of new Bacillus spp. isolates for antifungal properties and biosynthesis of lipopeptides. (2011); 54: 482–491.
48. Boiu-Sicuia OA, Cornea CP. Bacterial strains involved in soilborne phytopathogens inhibition. (2021); 64: 641–646.
49. Soliman A, Matar S, Abo-Zaid G. Production of Bacillus velezensis Strain GB1 as a biocontrol agent and its impact on Bemisia tabaci by inducing systemic resistance in a squash plant. Horticulturae, (2022); 8(6): 511.
50. Ghasemi S, Ahmadzadeh M. Optimisation of a cost-effective culture medium for the large-scale production of Bacillus subtilis UTB96. Archives of Phytopathology and Plant Protection, (2013); 46(13): 1552–1563.
51. Stamenković-Stojanović S, Karabegović I, Beškoski V, Nikolić N, Lazić M. Bacillus based microbial formulations: Optimization of the production process. Hemijska industrija, (2019); 73(3): 169–182.
52. Carvalho ALU de, Oliveira FHPC de, Mariano R de LR, Gouveia ER, Souto-Maior AM. Growth, sporulation and production of bioactive compounds by Bacillus subtilis R14. Brazilian archives of biology and technology, (2010); 53: 643–652.
53. Monteiro SM, Clemente JJ, Henriques AO, Gomes RJ, Carrondo MJ, Cunha AE. A procedure for high‐yield spore production by Bacillus s ubtilis. Biotechnology Progress, (2005); 21(4): 1026–1031.
54. Cho JH, Kim YB, Kim EK. Optimization of culture media for Bacillus species by statistical experimental design methods. Korean Journal of Chemical Engineering, (2009); 26: 754–759.
55. Posada-Uribe LF, Romero-Tabarez M, Villegas-Escobar V. Effect of medium components and culture conditions in Bacillus subtilis EA-CB0575 spore production. Bioprocess and biosystems engineering, (2015); 38: 1879–1888.
56. Sahoo S, Rao KK, Suraishkumar GK. Reactive oxygen species induced by shear stress mediate cell death in Bacillus subtilis. Biotechnology and bioengineering, (2006); 94(1): 118–127.
57. Sidorova DE, Plyuta VA, Padiy DA, Kupriyanova E V, Roshina N V, Koksharova OA, et al. The effect of volatile organic compounds on different organisms: Agrobacteria, plants and insects. Microorganisms, (2021); 10(1): 69.
58. Poulaki EG, Tjamos SE. Bacillus species: factories of plant protective volatile organic compounds. Journal of Applied Microbiology, (2023); 134(3): lxad037.
59. Yu SM, Oh BT, Lee YH. Biocontrol of green and blue molds in postharvest satsuma mandarin using Bacillus amyloliquefaciens JBC36. Biocontrol Science and Technology, (2012); 22(10): 1181–1197.
60. Raza W, Wei Z, Ling N, Huang Q, Shen Q. Effect of organic fertilizers prepared from organic waste materials on the production of antibacterial volatile organic compounds by two biocontrol Bacillus amyloliquefaciens strains. Journal of Biotechnology, (2016); 227: 43–53.
61. Lykholat YV, Khromykh NO, Didur OO, Drehval OA, Sklyar T V, Anishchenko AO. Chaenomeles speciosa fruit endophytic fungi isolation and characterization of their antimicrobial activity and the secondary metabolites composition. Beni-Suef University Journal of Basic and Applied Sciences, (2021); 10: 1–10.
62. Amer MS, Barakat KM, Hassanein AEA. Phthalate derivatives from marine Penicillium decumbens and its synergetic effect against sepsis bacteria. Biointerface Res. Appl. Chem, (2019); 9(4): 4070–4076.
63. Huang L, Zhu X, Zhou S, Cheng Z, Shi K, Zhang C, et al. Phthalic acid esters: Natural sources and biological activities. Toxins, (2021); 13(7): 495.
64. El-Enain A, Zeatar A, Zayed A, Elkhawaga M, Mahmoud Y. Diisooctyl Phthalate as A Secondary Metabolite from Actinomycete Inhabit Animal’s Dung with Promising Antimicrobial Activity. Egyptian Journal of Chemistry, (2023); 66(12): 261–277.
65. Gheda SF, Ismail GA. Natural products from some soil cyanobacterial extracts with potent antimicrobial, antioxidant and cytotoxic activities. Anais da Academia Brasileira de Ciências, (2020); 92: e20190934.
66. Blažević I, Radonić A, Mastelić J, Zekić M, Skočibušić M, Maravić A. Hedge mustard (Sisymbrium officinale): Chemical diversity of volatiles and their antimicrobial activity. Chemistry & biodiversity, (2010); 7(8): 2023–2034.
67. Soliman SA, Khaleil MM, Metwally RA. Evaluation of the antifungal activity of Bacillus amyloliquefaciens and B. velezensis and characterization of the bioactive secondary metabolites produced against plant pathogenic fungi. Biology, (2022); 11(10): 1390.
68. Yang G, Zhou B, Zhang X, Zhang Z, Wu Y, Zhang Y, et al. Effects of tomato root exudates on Meloidogyne incognita. PLoS One, (2016); 11(4): e0154675.
69. Li X, Jing T, Zhou D, Zhang M, Qi D, Zang X, et al. Biocontrol efficacy and possible mechanism of Streptomyces sp. H4 against postharvest anthracnose caused by Colletotrichum fragariae on strawberry fruit. Postharvest Biology and Technology, (2021); 175: 111401.
70. Yin YL, Zhou BL, Tang YP. Effects of three allelochemicals on Verticillium dahliae antagonistic microbes in eggplant rhizosphere. Allelopathy Journal, (2015); 35(1): 23–34.
71. Varsha KK, Devendra L, Shilpa G, Priya S, Pandey A, Nampoothiri KM. 2, 4-Di-tert-butyl phenol as the antifungal, antioxidant bioactive purified from a newly isolated Lactococcus sp. International journal of food microbiology, (2015); 211: 44–50.
72. Abdullah ASH, Mirghani MES, Jamal P. Antibacterial activity of Malaysian mango kernel. African Journal of Biotechnology, (2011); 10(81): 18739–18748.
73. Murniasih T, Masteria Yunovilsa P, Untari F. Antibacterial activity and GC–MS based metabolite profiles of Indonesian marine Bacillus. Indones J Pharm, (2022); 33: 475–483.
74. Amborabé BE, Fleurat-Lessard P, Chollet JF, Roblin G. Antifungal effects of salicylic acid and other benzoic acid derivatives towards Eutypa lata: structure–activity relationship. Plant Physiology and Biochemistry, (2002); 40(12): 1051–1060.