Implication of plant growth-promoting rhizobacteria of Bacillus spp. as biocontrol agents against wilt disease caused by Fusarium oxysporum Schlecht. in Vicia faba L.

Isolation of Fusarium pathogen from infected faba bean

The Fusarium oxysporum was isolated from roots of naturally infected faba bean plants showing symptoms of wilt disease. The infected plants were collected from the North Sinai governorate, Egypt. The infected roots were subjected to surface sterilization by 2% sodium hypochlorite for 2 min, rinsed twice with sterilized distilled water, and kept for drying under aseptic conditions. The sterilized roots were cut into small pieces (0.5 cm) and immersed on Petri dishes containing potato dextrose agar (PDA) media (containing g L⁻¹: potato, 200; dextrose, 20; agar, 15; 1.0 L distilled H₂O) and incubated for seven days at 25±2°C. The inoculated plates were examined for pure colonies and a single spore was sub-cultured onto new PDA plates.

Identification of Fusarium spp.

The Fusarium spp. cultured were identified based on cultural followed by morphological characteristics and confirmed by macro- and micro-conidial structure [23, 24, 25].

Pathogenicity test for F. oxysporum.

The pathogenicity test of two stains of F. oxysporum was achieved at EL-Qantra Sharq Experimental Station of Desert Research Center (D.R.C.), North Sinai Governorate, Egypt (31°00’21.6” N and 32°33’48.1” E) as greenhouse experiment. The pathogenic capabilities were tested using Maryot-2 faba bean cultivar collected from Agricultural Research Center, Giza, Egypt. The Fusarium inocula were prepared by inoculation of each fungal strain into potato dextrose broth media and incubated at 25±2°C for 15 days. The sandy-loam soil used in this experiment was autoclaved, added to sterilized pots (2 kg/ pot), and mixed thoroughly with fungal inoculation (2%). The physicochemical analysis of used soil is illustrated in Table 1. The seeds of collected faba bean were surface sterilized using 2% sodium hypochlorite for 4 min., washed in distilled water. Each pot was sown with three sterilized faba bean seeds. Pots without F. oxysporum inoculation and sown with sterilized faba bean seeds were running with the experiment as a control. The experiment was achieved in triplicates. The greenhouse condition was standardized throughout the experiment at a temperature between 18 to 24°C. The plants were irrigated as necessary and observed for diseases development.

Disease severity assessment.

The wilting percentage caused by each F. oxysporum was assessed after 60 days from planting, based on leaf yellowing and/or root discoloration. The symptoms of typical wilt due to Fusarium infection are numerically assessed according to their severity in ranging between 0 to 5 as follows: (0) mean no wilt symptoms appeared; (1) mean approximately from 1 to 20% of vascular plant systems are light brown and the plant leaves appear as yellow; (2) mean approximately from 20 to 40% of vascular plant systems are light brown and the plant leaves appear as yellow; (3) mean approximately from 40 to 60% of vascular plant systems are dark brown and the plant leaves appear as yellow; (4) mean approximately from 60 to 80% of vascular plant systems are dark brown and the plant leaves appear as yellow; (5) mean approximately from 80 to 100% of the leaves are yellowish and the vascular systems are dark brown or completely dead plants.

The disease severity was calculated according to the following equation [26]:

Where: (N) means the number of plants corresponding to the symptom grade, (0), (1), (2), (3), (4), and (5); (5T) mean the total number of plants (T) multiplied by maximum symptom grade (5).

Isolation of bacterial rhizospheres.

To isolate different bacterial species from rhizosphere soil of healthy faba bean plant, approximately 10 g of collected rhizosphere soil was mixed with 100 mL sterilized distilled water and stirred for 10 min. After that, the serial dilution was made up to 10⁻⁷ and inoculate 0.1 mL of final dilution into nutrient agar media. The inoculated plates were incubated at 35 ± 2°C for 24 h. The different morphological bacterial colony was re-streaked onto new nutrient agar plates to confirm the purity. The purified isolates are inoculated onto nutrient agar slant and kept in a refrigerator at 4°C for further study.

Selection of the most potent bacterial isolates

The most potent bacterial isolates were selected based on their highest efficacy to inhibit the growth of Fusarium oxysporum by using the in-vitro dual culture assay method [27]. Each rhizospheric bacterial isolate was inoculated at three equidistant points on the PDA plate and incubated at 35 ± 2°C for 24 h. At the end of the incubation period, a disk (5 mm) of a 7-days-old culture of F. oxysporum was put in the center of bacterial inoculated PDA plates. The control was designated as a PDA plate containing a disk of F. oxysporum without bacterial inoculation and running with the experiment under the same conditions. The plates were incubated at 28 ± 2°C for five days. The fungal growth inhibition percentage was calculated using the following equation:

Where R1 is the radial growth of F. oxysporum in absence of bacterial isolate (control), while R2 is the radial growth of F. oxysporum in presence of bacterial isolate. The experiment was achieved in triplicate for each bacterial isolate.

Identification of the most potent bacterial isolates.

The most potent bacterial isolates designated as Vb1, Vb3, and Vb6 were identified based on sequencing and amplification of the 16S rRNA gene. The genomic DNA was extracted according to the modified method [28]. Briefly, a separate bacterial colony was picked up by a sterile toothpick and suspended in 50 µL of sterilized deionized water. The cell suspension was put in a water bath for 10 min at 97°C, after that, the suspension was centrifuged for 10 min at 15000 rpm to recover the upper layer that contains the DNA. The intensity of DNA in the collected layer was calculated by measuring its absorbance at 260 nm by UV-spectrophotometer. A bacterial universal primers 27f (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492r (5’-GGTTACCTTGTTACGACTT-3’) were used to PCR amplification of the 16S rDNA fragment. The PCR tube containing PCR buffer (1 x), MgCl₂ (0.5 mM), Tag DNA polymerase (2.5 U, QIAGEN Inc.), Deoxynucleoside triphosphate (dNTP, 0.25 mM), universal primer (0.5 µM), and bacterial DNA (5 ng). The PCR cycling conditions were 94 °C for three minutes, followed by 30 cycles at 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 60 seconds, and final extension at 72°C for ten minutes. The forward and reverse sequencing for PCR products was achieved using Applied Biosystem’s 3730xl DNA Analyzer technology at Sigma company, Cairo, Egypt.

The obtained sequences were analyzed using BLAST as compared with those deposited in the GeneBank database. Multiple sequence alignment was performed using ClustalX 1.8 software package and a phylogenetic tree was constructed by the neighbor-joining method using MEGA (Version 6.1) software. The confidence level of each branch (1000 repeats) was tested by bootstrap analysis. The sequences obtained in the current study were deposited at GeneBank under accession numbers LC571587, LC571588, LC 571589 for strains Vb1, Vb3, and Vb6 respectably.

Characterization of the most potent rhizospheric bacterial strains as plant growth-promoting

Biocontrol of Fusarium wilt in Vicia faba plant using rhizosphere bacterial isolates

Isolation and identification of pathogenic Fusarium spp.

Faba bean (Vicia faba L.) is considered one of the most important legume crops, grown overall in the world to be used as human food and animal feed [42]. The significant losses in faba bean yield can be related to fungal infections which cause root rot, damping-off, and wilt diseases [43]. The most common fungal species that destroy faba bean growth and hence cause significant yield losses are Fusarium species [44]. Therefore, seven Fusarium spp. were isolated from the naturally infected root of faba bean collected from Sinai governorate, Egypt. The obtained Fusarium spp. were identified based on morphological and cultural characteristics as Fusarium proliferatum, Fusarium subglutinans, Rhizoctonia solani, Fusarium solani (two isolates), and Fusarium oxysporum (two isolates).

Fusarium oxysporum is considered the main phytopathogen that causes Fusarium wilt and can be remain to exist in the soil for several years without a main host; therefore, it is difficult to control the disease caused by Fusarium [45]. In the current study, the pathogenic severity for two F. oxysporum strains to cause wilt disease in faba bean was estimated under greenhouse experiment. Data analysis showed that the percentages of disease severity due to infection with two strains of F. oxysporum were 68% and 47% as compared with control. Therefore, we select the strain that causes the highest Fusarium wilt for further tests.

Isolation and selection of the most potent rhizospheric bacterial isolates

The interactions between rhizospheric bacterial species and the root system of plants have a positive impact on plant health, yield, and soil quality. These positive impacts can be attributed to the high efficacy of rhizospheric bacterial strains and secondary metabolites to increase the phosphate solubilization, nitrogen fixation, increase the availability of nutrients, ameliorate the biotic and abiotic stresses, biological control of phytopathogens, enhance the plant immunity through induced systematic resistance (ISR) [46]. In the current study, out of 23 bacterial species isolated from rhizosphere soil of healthy faba bean plant, seven bacterial isolates exhibited the efficacy to inhibit the growth of F. oxysporum using in-vitro dual culture assay with varying degrees ranging between 39.06 ± 0.3 and 59.1±0.2. Among seven rhizospheric bacterial isolates, three isolates designated as Vb1, Vb3, and Vb6 showed the highest efficacy to inhibit F. oxysporum growth with reduction percentages of 59.1±0.2, 46.4±0.3, and 52.5±0.3, respectively (Fig. 1). The in-vitro dual culture is a common assay method for preliminary investigation, in which the antagonistic activity was detected through measuring the inhibition percentage of radial mycelial growth towards the inoculated bacterial strains [47]. Consistent with our study, out of 35 rhizospheric bacterial isolates, five isolates exhibit F. oxysporum mycelial growth inhibition with varying percentages, and the bacterial isolate BA5 was the promising rhizospheric bacterial isolate to inhibit the mycelial growth with a percent of 58.3% [48]. Also, out of 100 microbial isolates obtained from rhizosphere soil samples, the bacterial isolate NJN-6 which was identified as Bacillus amyloliquefaciens showed the highest inhibition percentage towards F. oxysporum [49].

Figure 1

Antagonistic activity of the most potent bacterial isolates Vb1, Vb3, and Vb6 against highest pathogenic F. oxysporum strain by in-vitro dual culture assay. Bars with the different letters are significantly different (p≤ 0.05). Error bars indicate means ± SE (n = 3).

Identification of the most potent bacterial isolates

The three bacterial isolates designated as Vb1, Vb3, and Vb6 that exhibit high antagonistic activity with F. oxysporum were undergone identification based on amplification and sequencing of the 16S rRNA gene. The sequence analysis showed that the isolates Vb1 have a similarity percentage of 99.7 with Bacillus velezensis, whereas the isolates Vb3, and Vb6 have similarity percentages of 99.9 and 99.8% with two isolates of Bacillus paramycoides, respectively (Fig. 2). Similarly, the five rhizospheric bacterial isolates that showed high efficacy against Fusarium wilt, root rot, and crown diseases in tomatoes were identified using 16S rRNA as Acinetobacter calcoaceticus (AcDB3), B. amyloliquefaciens (BaMA26), B. siamensis (BsiDA2), B. subtilis (BsTA16), and B. thuringiensis (BtMB9) [50]. Accordingly, the Bacillus spp. are predominant rhizospheric bacterial species used to biocontrol of phytopathogenic fungi [47, 49, 50]. This phenomenon can be attributed to their different secondary metabolites and rapid growth which enables them to high integrate into biocontrol studies [51]. Recently, the cell immobilization of B. velezensis NH-1 was used to biocontrol of Fusarium wilt caused by F. oxysporum in cucumber [52]. Although the B. velezensis is a common bacterial species used in biocontrol, this is the first report for using it and B. paramycoides for biocontrol of Fusarium wilt caused by F. oxysporum in faba bean.

Figure 2

Phylogenetic analysis of 16sr RNA sequences of the bacterial isolates with the sequences retrieved from NCBI. The analysis was conducted with MEGA 7 using the neighbor-joining method with bootstrap value (1000 replicates).

Characterization of the rhizospheric bacterial strains as plant growth-promoting

The antagonistic activity of rhizospheric bacterial species towards phytopathogenic fungi can be attributed to their plant growth-promoting traits. Among these traits, the efficacy of rhizospheric bacteria to producing hydrolytic enzymes, nutrient competitions, nitrogen fixations, siderophores production, phosphate solubilization, phytohormones production, and ammonia production [53]. The preliminary screening of three selected rhizospheric bacteria showed their efficacy to fixed nitrogen by producing blue color around the bacterial colony grown on nitrogen-free malate medium. The quantification nitrogen-fixing assay was achieved through the acetylene reduction method. Data on solid media (qualitative assay) are compatible with those recorded from liquid media (quantitative assay) after being analyzed by gas chromatography. Data analysis showed that the highest nitrogen-fixing was recorded for bacterial isolate Vb1 with the ethylene production value of 391.4±1.5 n mole C₂H₄/ml/24h, followed by Vb3 and Vb6 with values of 351.8±1.2 and 258.7±1.01 n mole C₂H₄/ml/24h (Table 2). Similarly, the nitrogen-fixing efficacy of rhizospheric bacterial species associated with Calligonum polygonoides and Lasiurus sindicus was assessed qualitatively via color change and quantitatively via acetylene reduction assay [29].

Another mechanisms utilized by rhizospheric microbes to enhance plant growth and then compete with phytopathogens are phosphate solubilization and ammonia production [54]. Among macronutrients required by a high amount for enhancement of plant growth is phosphorus ion, which exists in most cases as an insoluble form. Rhizospheric bacteria have the ability to convert phosphorus from insoluble to soluble form through different mechanisms such as the production of enzymes and/or organic acids [55]. In the current study, the appearance of a clear zone around the bacterial growth on Pikovskaya agar media indicates their success in phosphate solubilizing. The diameter of clear zones was 1.3±0.02, 9.4±0.05, and 9.9±0.12 mm for Vb1, Vb3, and Vb6, respectively (Table 2) which correlated with the amount of liberated phosphate. The quantitative assay is depending on the amount of liberated organic acid detected by the phosphomolybdate method. The results showed that the highest phosphate solubilizing was achieved by bacterial isolate Vb6 followed by Vb3 and Vb1. Also, the difference between the amount of liberated phosphate recorded by bacterial isolates Vb6 and Vb3 are non-significant which recorded 91.5±1.2 µg ml⁻¹ and 91.1±1.3 µg ml⁻¹ respectively at 2^nd days and reached 152.2±1.3 µg ml⁻¹ and 151.8±1.3 µg ml⁻¹ at tenth days (Fig. 3). The lowest amount of phosphate solubilization is recorded by bacterial strain Vb1 which was 41.82±0.75 µg ml⁻¹ after 2^nd days and decreased to 37.73±0.62 µg ml⁻¹ after tenth days as compared with control which recorded 25.64±0.59 µg ml⁻¹ and 27.31±0.49 µg ml⁻¹ after second and tenth days, respectively (Fig. 3). According to the obtained data, the quantitative assay is compatible with those reported by the qualitative assay (formed clear zone). The production of low molecular weight organic acid is considered the main mechanism for phosphate solubilization by different bacterial species [56].

Figure 3

The amount of P-liberated by rhizospheric bacterial strains Vb1, Vb3, and Vb6 in liquid media.

The obtained data highlighted that all rhizospheric bacterial isolates have the ability to produce ammonia with varying degrees according to color intensity after adding Nessler’s reagent (Table 2). The highest intensity for brownish color was reported for isolate Vb3 followed by Vb1 and Vb6. Ammonia production enhanced plant growth by the synthesis of biomolecules containing nitrogen. Also, ammonia can increase the root and shoot length, enhance the fresh weight of the plants, and increase the efficacy of the plant against phytopathogens attacks [57].

The extracellular hydrolytic enzymes secreted by rhizospheric microbes are important for the decomposition of different polymers such as chitin, cellulase, starch, and lignin which ultimately to mineralization to beneficial minerals such as P, N, and S [58]. The secretion of these hydrolytic enzymes by different rhizosphere microbes has a critical role in the suppression of phytopathogenic fungi through the degradation of their cell wall [59, 60]. For example, hydrolytic amylase, lipase, cellulase, pectinase, and protease enzyme are released by rhizospheric bacterial species such as Bacillus, Pseudomonas, and Enterobacter are showed inhibitory action against phytopathogenic fungi [61]. In the current study, the rhizosphere bacterial strains B. velezensis Vb1, B. paramycoides Vb3, and B. paramycoides Vb6 possess the efficacy to secret amylase, cellulase, protease, and catalase except the bacterial strains Vb3 and Vb1 lack the efficacy to secrete amylase and catalase (Table 2). Data analysis showed that the highest clear zone formed around bacterial strain was recorded for cellulase enzymes with values of 36±0.0, 28.3±3.8, and 30±1.53 mm. Catalase enzymes promote plant growth by the indirect mechanism through scavenging the free radicals released because of biotic and abiotic stresses [36].

Siderophores have a critical role in the biocontrol of soilborne fungi as reported previously [62]. The inhibitory effect of siderophores against phytopathogenic fungi due to converting Fe³⁺ (inorganic form) to Fe²⁺ (organic form), and hence making it available for plant and unavailable for phytopathogens [63]. The selected bacterial strains Vb1, Vb3, and Vb6 are recorded as siderophores producers through the appearance of an orange halo around the bacterial growth. Similarly, Bacillus siamensis, B. subtilis, and B. amyloliquefaciens are characterized by their efficacy in producing siderophores which help in the biocontrol of Fusarium wilt caused by F. oxysporum [50]. Also, the efficacy of Pseudomonas aeruginosa JAS-25 to colonizing the roots, secretion of antibiotics, and antagonistic activity against pathogenic fungi can be related to their potentiality to siderophores production [64]. Hydrogen cyanide (HCN) is defined as a volatile secondary metabolite secreted by various rhizosphere bacterial species. It is characterized by its efficacy in suppression of soilborne pathogenic fungi through blocking the electron transport system and hence disrupting the supply of the cell by energy which ultimately to cell death [65]. Data showed that all selected rhizosphere bacterial strains have the ability to produce HCN with different degrees due to the color change of filter paper from deep yellow to reddish-brown (Table 2). Recently, various rhizosphere bacterial species are recorded as positive for HCN production such as Aeromonas, Pseudomonas, Bacillus, Alcaligenes, and Rhizobium [66].

The ability of rhizosphere bacterial strains to produce auxin and cytokinin is a promising tool to study the profound effect of these strains on plant growth [67]. Indole-3-acetic acid (IAA) is considered the main auxins synthesized by plants and has a profound role in leaf formation, root initiation, embryo development, phototropism, fruit development, geotropism, abscission. It has a positive impact on the root length, increases root branches, root laterals, and root hair that facilitate the nutrient uptake from the surrounding environment [68]. The efficacy of three rhizospheric bacterial strains Vb1, Vb3, and Vb6 to produce IAA were screened qualitatively at broth media in the absence and presence of L-tryptophan (0, 1, 2, and 5 mg ml⁻¹) as a precursor for IAA after 15 days. Data represented in Fig. (4A) showed that the maximum IAA production in absence of L-tryptophan was achieved for bacterial isolate Vb6 with a value of 5.2±1.03 µg ml⁻¹ and this concentration was increased up to 67.9±3.3 µg ml⁻¹ at 5 mg ml⁻¹ L-tryptophan. Moreover, the IAA production by bacterial strains Vb1 and Vb3 was increased from 2.1±0.2 and 2.8±0.4 µg ml⁻¹ in absence of L-tryptophan to 81.3±1.9 and 58.2±4.3 µg ml⁻¹ in presence of 5 mg ml⁻¹ tryptophan respectively (Fig. 4A). Compatible with our study, the IAA production by B. siamensis was increased in liquid media supplemented with L-tryptophan as compared with those without L-tryptophan, and the highest IAA production was achieved at 250 µg ml⁻¹ [69].

Figure 4

Phytohormones production by three rhizospheric bacterial species Vb1, Vb3, and Vb6. A) denote the qualitative production of IAA at different concentrations of tryptophan (0, 1, 2, and 5 mg ml⁻¹); B) denote the quantitative production of IAA at different interval times in presence of 5 mg ml⁻¹ tryptophan, C) denote the production of ABA, Benzyl, Kinten, and Ziaten phytohormones detected by HPLC analysis, D) denote the production of GA₃ detected by HPLC. Bars with the different letters are significantly different (p≤ 0.05). Error bars indicate means ± SE (n = 3).

According to qualitative assay, the concentration of 5 mg ml⁻¹ L-tryptophan is the best for higher IAA production and hence it was selected to investigate the quantitative IAA assay. Analysis of variance showed that the maximum IAA concentration was recorded at 10, 14, and 14 days for rhizospheric bacterial strains Vb1, Vb3, and Vb6 with values of 61.4±3.7, 25.9±1.4, and 59.6±0.5 µg ml⁻¹, respectively.

Furthermore, the rhizospheric bacterial strains were quantitative screening for their efficacy to produce various phytohormones using HPLC analysis. Data analysis showed that the successful rhizospheric bacterial species Vb1, Vb3, and Vb6 to produce ABA, Benzyl, Kinten, Ziaten, and GA₃ with values of (0.04±0.01, 0.03±0.01, and 0.02±0.01 mg/100 ml), (1.53±0.1, 0.23±0.05, and 0.22±0.03 mg/100 ml), (0.34±0.01, 0.96±0.01, and 0.27±0.01 mg/100 ml), (0.54±0.01, 0.67±0.01, and 0.38±0.01 mg/100 ml), and (14.45±1.6, 13.88±0.9, and 7.80±0.8 mg/100 ml), respectively (Fig. 4 B and C). The ABA has an important role in improving plant adaptation under various stresses and regulating plant growth [70]. Also, Gibberellic acid is another phytohormone that has a critical role in ameliorating different stress, directly affects plant growth, plant yield, nitrogen metabolism, and mineral nutrition [71]. The efficacy of rhizosphere bacteria strains to secrete different phytohormones are mentioned in different published studies [70, 72, 73]. Based on the above plant growth-promoting traits of three rhizosphere bacterial strains can be use of these strains as bioinoculant to increase plant growth and hence increase the tolerance of a plant to phytopathogens infection.

Biocontrol of Fusarium wilt in Vicia faba L. plant using rhizosphere bacterial isolates.

Field experiment

Data represented in Table 4 showed the potency of B. velezensis Vb1, B. paramycoides Vb3, and B. paramycoides Vb6 to improve F. oxysporum-infected faba bean plants either separately or in a consortium. Inoculation of faba bean seeds with rhizospheric bacterial strains and planting into F. oxysporum-infected soil causes a significant increase in plant heights, the number of pods/plant, and fresh and dry weight as compared with those planting into infected soil without bacterial inoculation. For instance, inoculation with separate bacterial species enhance plant height with percentages of 21.3% – 24.3%, whereas the bacterial consortium highly improves faba bean plant height with percentages of 28.3% – 48.3% and the highest significant value was recorded for V-mix treatment (Vb1+Vb3+Vb6) as compared with the infected plant (plant sowing in infected soil in absence of bacterial strains). Moreover, the number of pods/plants were highly reduced in the infected plant (12.3±1.5) as compared with a healthy plant (21.0±1.0). The presence of plant growth-promoting rhizobacterial strains causes a significant increase in the number of pods as compared with healthy plants and the highest number was recorded for the bacterial consortium.

On the other hand, the fresh weight and dry weight of healthy and infected faba bean plants after 60 days of sowing were (51.7±1.2 g and 18.0±2.6 g) and (38.7+2.1 g and 13.3+1.5 g), respectively. The presence of F. oxysporum in absence of bacterial rhizospheric strains has a negative impact on all morphological characteristics of faba bean. Inoculation of plants with different bacterial strains Vb1, Vb3, and Vb6 has been significantly improving fresh and dry weight in presence of fungus. For instance, the presence of B. velezensis Vb1 improves the fresh weight and dry weight with values of 18.6% and 8.7% respectively, whereas the bacterial consortium (V-mix) increases the fresh weight and dry weight with percentages of 37% and 20.4% respectively in presence of F. oxysporum strain (Table 4). The obtained data are in harmony with those reported that the rhizobacterial strains Rhizobium sp., Bacillus pumilus, and Pseudomonas alcaligenes have the efficacy to improve the number of pods, dry weight, fresh weight, and plant height of lentil in presence of Fusarium oxysporum causing wilt disease [79]. Similarly, rhizobacterial strain Bacillus sp. SJ-5 was used to biocontrol disease caused by Fusarium oxysporum & Rhizoctonia solani in soybean plants [80].

The current study showed that the three rhizospheric Bacillus spp. have the potency to improve the growth performance of the F. oxysporum-infected faba bean plant. This phenomenon can be attributed to antagonistic activity between rhizospheric bacteria and pathogens, producing lytic enzymes, secretion of bioactive inhibitory compounds, or competition between bacteria and pathogenic fungi on essential nutrients [81, 82]. Bacillus spp. are considered the most common rhizospheric bacterial strains used to decrease the wilting disease caused by F. oxysporum in various plants [83, 84]. The various published studies reported that the Bacillus spp. have various suppressive mechanisms including producing volatile substances, induction of systematic resistance, stimulating plant growth, and aggressive plant roots colonization [13, 79].

The planting of faba bean in infected soil with F. oxysporum caused 71.7 ± 2.1 wilt severity after 60 days of planting (Table 4). The inoculation of faba bean seeds with plant growth-promoting rhizospheres causes a significant decrease in wilt disease. For example, the presence of bacteria consortium V-mix (Vb1+Vb3+Vb6) has the efficacy to protect faba bean plant from wilt with percentages of 83.3%, followed by bacterial treatment Vb1+Vb6 that reduce the wilt with a percentage of 73.7%. Not only rhizobacterial consortium can reduce the wilt disease, but also separate species have the ability. For instance, the presence of B. velezensis Vb1 can reduce the wilt disease with a percentage of 67.7% as compared with infected plants followed by B. paramycoides Vb6 and B. paramycoides Vb3 (Table 4). Un-compatible with our study, Akköprü and Demir reported that the treatment with single rhizobacterial species was more effective than Glomus intraradices as mycorrhizal fungi or consortium between mycorrhizae and rhizosphere bacteria to biocontrol of F. oxysporum f. sp. lycopersici in tomato plant [85]. The obtained results are in harmony with those recorded by Khalil and co-authors, who reported that the rhizospheric bacterial strains B. siamensis BsiDA2 and B. subtilis BsTA16 have significantly reduced wilt disease in tomatoes caused by Fusarium oxysporum spp. lycopersici [50]. The rhizospheric Bacillus spp. are used by various researchers to biocontrol wilt disease caused by F. oxysporum in various plants under greenhouse and field conditions [74, 81]. In the current study, the activity of Bacillus spp. to reduce the wilt disease can be attributed to producing siderophores, HCN, hydrolytic enzymes, and ammonia which help in suppressive phytopathogens.