Management of Cumin Wilt Caused by Fusarium oxysporum Using Native Endophytic Bacteria

Abstract

Eleven endophytic bacteria were isolated from the roots of cumin, a medicinal plant grown in Assiut Province, Egypt. The strains KAU2025 and KAU2022 exhibited the highest inhibition rate against Fusarium oxysporum (FOC7) (64%) in dual culture test and were selected for further studies. KAU2022 and KAU2025 were identified as Brevibacillus parabrevis and Pseudomonas fluorescens, respectively, based on 16S rDNA sequence analysis. The isolates increased the seed germination of cumin by 46.6% compared to that in the control infected with the pathogen F. oxysporum (FOC7). Additionally, the biocontrol efficacy of B. parabrevis against Fusarium wilt in cumin was determined as 67.4% in pot experiments, and both the bioagents increased the fresh and dry weight of the cumin plant compared to the infected control. After a 24 h treatment, the peroxidase levels in the cumin plants were higher in the P. fluorescens and B. parabrevis-treated groups, and the level of polyphenol oxidase increased in both treatments compared to the infected control. The treatment of cumin with both strains increased the total phenol and flavonoid contents in the treated plants. In summary, B. parabrevis and P. fluorescens are promising agents for the biocontrol of Fusarium wilt in cumin.

1. Introduction

Cumin (Cuminum cyminum Wall 1829), which belongs to the Apiaceae family (Umbelliferae), is an essential condiment consumed in Egypt. Its essential oil is obtained by the water distillation of fruits and has antimicrobial activity. Consequently, it is widely used in the pharmaceutical and food industries [1]. Cumin wilt, caused by Fusarium oxysporum f. sp. cumini (Snyder WC, Hansen NH (1940)), is a severe disease that is limiting global cumin production. It causes high yield losses up to 100% in heavily damaged fields under favorable conditions [2]. The wilting and shriveling of the leaves, the browning of the vascular system, stunting, and dam**-off are some of the symptoms caused by the pathogen [3]. Various fungicides may provide effective control of F. oxysporum; however, the use of excessive fungicides is extremely hazardous to the environment and human health, while also posing the problem of resistance [2]. Various endophytic microorganisms with biocontrol potential have been widely used to control plant pathogens [4]. Endophytes are fungi or bacteria that colonize the internal tissues of healthy plants at all or some stages of their life cycle without causing any symptoms. They have many biological functions in plants, such as providing nutrients for the plant, participating in plant defense functions, promoting the growth of the host plant, and strengthening the plant’s resistance to biotic and abiotic stresses [4,5]. Endophytic bacteria are highly biodiverse, with more than 120 species (belonging to 54 genera) found in plants, including the common genera Bacillus, Pseudomonas, Enterobacter, and Agrobacterium [6]. The use of antagonistic microbes to control plant diseases is a highly effective, economical, and environmentally friendly alternative to synthetic fungicides [7,8].

Certain species of Bacillus and Pseudomonas can be used as plant growth-promoting bacteria and biological control agents for several plant pathogens, as they are harmless to humans and animals while also being eco-friendly [9,10]. Brevibacillus parabrevis (Migula, 1900; Shida et al., 1996) produces metabolites that inhibit fungal and bacterial activities and offers the advantages of faster growth, simple nutrition, and strong resistance to environmental conditions. Brevibacillus parabrevis is the most dominant endophytic bacterium in numerous plants [11,12,13,14,15,16], and it controls several pathogenic plant bacteria and fungi, including Colletotrichum orbiculare (Pass.) Sacc. and Roum. (as ‘lagenarium’), (1880) [11], Sclerotinia sclerotiorum (Lib.) Korf and Dumont (1972) [12], F. oxysporum [13], Ralstonia solanacearum (Smith, 1896) [14], Xanthomonas citri (Hasse, 1915) Gabriel et al., 1989 [15], and Botrytis cinerea Pers. (1794) [16]. However, there are no reports on the control, effect, and mechanism of Brevibacillus parabrevis on Fusarium oxysporum f. sp. cumini wilt in cumin.

Pseudomonas fluorescens (Migula, 1895) is present in the soil and water and on plant surfaces. Because of their abundant population in the soil and plant root systems and their capacity to use several plant exudates as nutrition, fluorescent pseudomonads are the best option for biological control [17]. They can adhere to soil particles and are involved in antibiotic synthesis and hydrolytic enzyme production. In addition, they possess plant growth-promoting traits, such as phosphate solubilization, nitrogen fixation, phytohormone production, and iron chelation. The varied utility of P. fluorescens allows it to be utilized as a growth promoter and disease suppressor in agriculture. It produces several substances, viz., siderophores, antibiotics, and enzymes, that induce resistance in host plants. It can also function as a competitor against pathogens for nutrition. Furthermore, the use of antibiotics, including pyoluteorin, pyrrolnitrin, 2,4, diacetylphloroglucinol (DAPG), phenazine, tensin A, cyclic lipopeptides, amphisinoomycin compound, and tropolone, as biocontrol agents has been documented previously. Additionally, plant disease stress has been shown to be dramatically reduced by adding antagonistic antimicrobial-producing bacterial strains, either individually or in combination with fungicides [17,18].

One of the most promising biocontrol strategies is the use of plant endophytic bacteria. A few studies have reported the role of endophytic bacteria isolated from the medicinal plant against Fusarium pathogen. This study focused on screening the endophytic bacteria to assess their biocontrol and growth-promoting effects on cumin pathogen F. oxysporum and explore the physiological response of cumin plants inoculation with endophytic bacteria.

2. Materials and Methods

2.1. Pathogen Isolation

F. oxysporum was isolated from naturally infected cumin seedlings showing wilt symptoms collected from different localities of Assiut Governorate, Egypt, in 2020. The roots and stems of the plants showing symptoms were surface-sterilized with 1% sodium hypochlorite for 3 min, rinsed several times in sterile water, and placed in Petri dishes containing potato dextrose agar (PDA) medium. Following a seven-day incubation period at 25 °C in the dark, the fungal growth originating from each infected root part was examined microscopically according to Pappas and Elena [3]. The hyphal tip and a single spore isolation were used to obtain pure cultures of the developed fungus. The pure cultures were isolated on PDA and stored in the refrigerator at 5 °C on PDA slants for further studies [19].

2.2. Pathogenicity Test

Nine fungus isolates were collected during the 2020 season from Baladi cumin. The pathogenic properties of the isolated fungus were determined using the Baladi cumin cultivar. The growing fungus isolates, each containing 100 mL of Czapek’s liquid medium in 250 mL conical flasks, were injected separately with 6 mm agar discs obtained from seven-day-old isolates. The flasks were incubated for 15 days at 25° C. The fungal mycelial growth medium was decanted, rinsed with distilled water, suspended in 100 mL distilled water, and blended for 5 min with a waring blender. For soil infestation, the fungal suspensions were added to 25 mL diameter pots filled with steam-sterilized sandy, loamy soil (25 CFU/g) seven days before planting. The soil was covered with polyethylene for three days after being sterilized with a 5% formaldehyde solution. Seed disinfestations were carried out by treating the seeds with 1% sodium hypochlorite solution for three mins before rinsing them three times with sterilized water. Pots containing non-infested soil were used as a control. Each pot was sown with ten seeds. Four pots were used for each isolate as replicates. The pots were kept under careful observation in greenhouse conditions and examined for the percentages of germination and wilt at 20 and 50 days, respectively, after sowing.

2.3. Molecular Identification of F. oxysporum

The highly pathogenic isolate of the pathogen, isolate No. 7, was purified from a single spore and identified by its microscopic properties [20]. To identify the ITS sequence, isolate No. 7 was sent to the Solgent Company in Daejeon, South Korea, for DNA extraction. PCR was used to amplify the ITS region covering ITS1 and ITS2 in two rounds [20]. The nucleotide sequence data of the ITS1 and ITS2 regions were subjected to pairwise alignment using the Lipman and Pearson method [21] in the “GENETYX-MAC” program (Genetyx Corp., Osaka, Japan). The sequences were further analyzed using BLAST from the National Center of Biotechnology Information (NCBI) available at: https://www.ncbi.nlm.nih.gov/ (accessed on 14 April 2022).

2.4. Effect of Culture Filtrate from Fusarium oxysporum on the Seed Germination of Cumin In Vitro

The tested isolate No. 7 from the fungus F. oxysporum was inoculated in flasks containing 100 mL of Czapek’s liquid medium. The flasks were incubated at 25 °C for three weeks. The mycelium was removed, the fungal filtrate was centrifuged for 60 min at 3000 rpm, and a Seitz filter was used to sterilize the filtrate. Then, ten cumin seeds were surface-sterilized, which included dip** the seeds in 1% sodium hypochlorite solution for three min, and then they were placed in a sterile Petri dish containing moistened filter papers with filtrate at different concentrations (1, 2, 4, 6, 8, and 10% v/v). Petri dishes containing sterilized distilled water were used as a control [22]. Four replicates were used for each treatment. Seven days later, the percentage of seed germination was monitored.

2.5. Isolation and Purification of Endophytic Bacteria

The fresh and healthy cumin roots were cut and washed with tap water to remove the surface soil. The roots were soaked in 75% alcohol for 1 min, 2% sodium hypochlorite for 3 min, and then rinsed with sterile water three times. In addition, 200 μL of sterile water used in the last washing was spread on LB solid medium and cultured at 37 °C as a control. This showed that the disinfection was complete if no microbial growth was observed on the plate after three days. Then, 5 g of root was placed in a sterile mortar and ground into a homogenate with 2 mL of sterile water. The supernatant was diluted to one-twentieth with sterile water, and 20 μL of the diluted solution was spread on LB solid medium and cultured in an incubator at 37 °C. A single colony of each bacterial strain was selected and purified by streak plating on the medium, and the strains were preserved in a sterile tube with 30% (v/v) glycerol [22].

2.6. Assessment of the Antagonistic Activity of Endophytic Bacteria

The co-culture method was used for screening the antagonistic endophytic bacteria from cumin against F. oxysporum. The mycelial plug of the pathogen with a 0.5 cm diameter was placed at the center of a PDA plate and cultured for 24 h in an incubator at 28 °C. The endophytic bacterial strains were cultured overnight in LB broth at 37 °C. They were applied to three symmetrical locations around the mycelial plug on the PDA plate at a distance of 2.5 cm from the plate edge. The pathogen without the inoculation of the endophytic bacterial strain was used as a control. Following treatment, the cultures were incubated at 28 °C. The diameter of the inhibited and the control pathogens was checked after inoculation for four days [2]. The relative inhibition rates were determined using the following formula: (diameter of the control pathogen − diameter of the treated pathogen)/(diameter of the control pathogen) × 100%.

2.7. Endophytic Bacterial Identification by 16S rDNA Sequence and Phylogenetic Analysis

The genomic DNA of the endophytic bacteria was extracted using the CTAB method. The 16S rDNA sequences of the endophytic bacteria were amplified with PCR using the forward primer fD1 (5′-AGAGTTTGATCCTGGCTCAG-3′) and reverse primer rP1 (5′-TACCTTGTTACGACTT-3′). The reaction was carried out using 25 μL of 2× PCR mixture, 15 μL of ddH₂O, 5 μL of DNA template, and 2.5 μL of forward and reverse primers. The PCR amplifying conditions were 94 °C for 3 min, 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, and then 72 °C for 10 min. The PCR products were detected using 1% agarose gel electrophoresis. The amplification products were sequenced by Sangon Biotech (Shanghai, China) Co., Ltd. The sequence was submitted to GenBank. A homology comparison between 16S rDNA sequences was conducted using the BLAST tool, and a phylogenetic tree was constructed to analyze the taxonomic status of the strains using the MEGA 7.0 software (https://www.megasoftware.net/ (accessed on 14 April 2022)).

2.8. Biocontrol Efficacy and Growth-Promoting Effect of Brevibacillus parabrevis and Pseudomonas fluorescens on Cumin in Pot Experiments

The effect of Brevibacillus parabrevis and Pseudomonas fluorescens on dam**-off and cumin wilt was examined. Cumin seeds were soaked for 3 min in the overnight culture of each isolate. The soil was infested with F. oxysporum (25 CFU/g soil) and irrigated. Ten soaked cumin seeds were planted in the pots containing soil infected with the pathogen. Four pots were used for each particular treatment as replicates and four pots containing infested soil planted with sterilized seeds served as controls. In addition, four pots with each treatment and non-infected soil served as controls. The percentage of germination and wilt were monitored after 20 and 50 days from sowing, respectively.

The Baladi cumin cultivar seeds were selected, disinfected by soaking in 3% sodium hypochlorite for 5 min, and washed three times with sterile water. After disinfestation, the seeds were germinated in Petri dishes with wet sterile filter papers at 26 °C with 16 h of light per day. The germinated seedlings were planted in a pot containing autoclaved soil, with one plant in every pot. After ten days, 20 mL suspensions of the KENF7 strain (1.0 × 10⁸ CFU/mL) and water were poured into individual pots. After inoculation for ten days, the 20 mL spore suspensions (1.0 × 10⁸ spores/mL) of F. oxysporum were poured into each pot using the root-cutting irrigation method. Each treatment was performed in four replicates. The experiments were repeated three times.

After 50 days of spore inoculation, the disease incidence, disease index values, and biocontrol efficacy were tested using the method proposed by Ghoneem et al. [23]. Disease incidence (%) = ((number of diseased plants)/total number of plants investigated)) × 100; disease index (%) = ((Σ(number of plants for each disease severity × disease rank))/(total number of plants× the highest level)) × 100. The severity of Fusarium wilt in cumin was classified into five grades (grade 0–5) according to the criteria for disease grades proposed by Ghoneem et al. [19].

2.9. Determination of Fresh and Dry Weight

Plant growth status was assessed 45 days after the sowing date. Then, ten plants were randomly selected for each treatment from the three treatment groups (KAU2025, KAU2022, and control). The plants were rooted, and their rhizosphere soil was cleaned and rinsed with flowing water and air-dried. The fresh weight (FW) of the root and foliar from each plant was recorded. The plants were then oven-dried at 80 °C for three days to measure the dry weight (DW).

2.10. Nutrient Soulubilisation, Indole Acetic Acid (IAA), and Siderophore Production

The ability of B. parabrevis and P. fluorescens to dissolve precipitated tricalcium phosphate (Ca₃(PO₄)₂) was tested on Pikovskaya (PVK) medium. The bacterial isolates were inoculation on the surface-dried plates. The plates were then incubated for seven days at 28 °C. The solubilizing index (SI) was determined based on the ratio of the total diameter (colony + halo zone) to the diameter of the colony [24].

The ability of B. parabrevis and P. fluorescens to solubilize zinc was investigated using the methods described by Saravanan et al. [25], which involved dissolving precipitated zinc oxide (ZnO) on zinc solubilizing medium. The bacterial isolates were introduced onto the surface of dried plates, and the plates were incubated at 28 °C for seven days. The ratio of the overall diameter (colony + halo zone) to the colony diameter was used to calculate the SI [24].

Individual isolates of B. parabrevis and P. fluorescens were inoculated onto Petri plates containing nutrient agar enriched with L-tryptophan and incubated for 48 h at 37 °C. The developed bacterial cultures were centrifuged at 3000 rpm for 30 min, the supernatant was transferred to a new tube, and a few drops of orthophosphoric acid, 4.0 mL of Salkowaski reagent containing 50 mL of 35% perchloric acid, and 1 mL of FeCl₃ (5 mL) solution were added, resulting in a pink color that indicated indole acetic acid production [26].

The methods reported by Schwyn and Neilands [27] were employed to encourage siderophore production by bacterial isolates using chrome azurol S (CAS) agar. Pseudomonas fluorescens and Brevibacillus parabrevis were spotted on the surface of the CAS agar medium and cultured for three days at 28 °C. The formation of an orange halo around the growth was beneficial for siderophore production.

2.11. Effect of Treatment with Brevibacillus parabrevis and Pseudomonas fluorescens on Antioxidant Enzyme and Phenol and Flavonoid Contents

2.11.1. Antioxidant Enzymes

At 0, 12, 24, 36, and 48 h of B. parabrevis and P. fluorescens treatment against F. oxysporum, antioxidant enzyme activity levels were assessed; plants inoculated only with F. oxysporum served as controls. Next, 1 g of cumin leaves were collected, washed, dried, and placed into a pre-cooled mortar, mixed with 8.0 mL of 0.05 M (pH 7.8) phosphate buffer; ground into a homogenate, and centrifuged at 4 °C and 10,000 rpm for 15 min. The supernatant was transferred to a new test tube, and the crude enzyme extract from the cumin leaves was obtained. The activity of peroxidase (POD) was measured using guaiacol colorimetry, and the amount of enzyme required for an increase in A470 of 0.01 per minute was defined as one unit of POD. The activity of polyphenol oxidase (PPO) in the cumin leaves was determined using the method described by Batra and Kuhn, [28].

2.11.2. Phenolic Compounds

The method described by Rapp and Zeigler, [29] was used to prepare the plant samples. Briefly, 1 g of cumin plant leaves (stored at −80 °C) was ground with 5 mL of 80% methanol. The material was then transferred to a 10 mL centrifuge falcon tube. The homogenate was centrifuged at 10,000 rpm for 30 min at 4 °C. The pellet was discarded, and the supernatant was transferred to new 10 mL centrifuge tubes. The tubes were stored at −20 °C for further use.

2.11.3. Total Phenol Content

The total phenol content in the leaf samples was determined according to the method described by Şahin et al. [30]. Briefly, 0.02 mL of reaction mixture sample (previously prepared and stored at −20 °C) was placed in the spectrophotometer cuvette. Then, 0.5 mL of Folin reagent (Folin–Ciocalteu reagent/Folin–Denis reagent) was added to the reaction mixture. Aliquots of 0.75 mL of 20% sodium carbonate (Na₂CO₃) solution and 8 mL of sterile distilled water were added. The cuvette containing the reaction mixture was incubated at 37 °C in a water bath for 60 min. An additional reaction mixture lacking the sample was treated as a control. After 1 h, the total phenol content was assayed using a spectrophotometer at 767 nm. The reaction readings were observed thrice at an interval of 30 min and three replicates were observed for each sample. The total phenol contents were expressed as mg/g plant FW using gallic acid (0–5 mg) as a standard. Total phenol was determined as mg gallic acid/g plant material.

2.11.4. Total Flavonoid Content

The total flavonoids in the cumin leaves were determined using a colorimetric assay according to the method described by Fattahi et al. [31]. Briefly, 100 µL of the leaf sample was added to 4 mL of sterilized double distilled water. Then, 300 µL of 5% sodium nitrite (NaNO₂) was added; after 5 min, 300 µL of 10% aluminum chloride (AlCl₃) was added. Next, 2 mL of 1 M sodium hydroxide (NaOH) was added to the mixture. After that, the mixture was diluted by adding 3.3 mL of sterilized double distilled water and the sample was thoroughly mixed. The reaction cuvettes were left for 5 min at room temperature before the readings were determined at 510 nm. Cuvettes without the reaction mixture sample were used as blanks. The flavonoid content was expressed as mg/g of plant material. The total flavonoids were determined as = mg/g of plant material.

2.12. Statistical Analysis

All the in vitro and greenhouse experiments were conducted in four replicates. The greenhouse experiments were performed using a complete randomized design, and all the collected data were analyzed using the Statistix ver. 8.1 (Analytical software, statistix; Tallahassee, FL, USA, 1985–2003) software. The data collected for disease severity were transformed into arcsine values and a one-way analysis of variance (ANOVA) was performed. The means of the replicates for all treatments were compared using Fisher’s least significant difference test at p = 0.05 [32].

3. Results

3.1. Isolation and Pathogenicity Tests of the Causal Pathogen

The data shown in

Table 1. Pathogenicity tests of nine tested fungi isolates on the Baladi cumin variety under greenhouse conditions during the 2020 season.

Isolate No.	Germination (%)	Wilt Severity (%)
1	30.0 ± 3.1 d	31.21 ± 2.1 d
2	30.0 ± 3.2 d	30.00 ± 2.0 d
3	40.0 ± 2.4 c	33.33 ± 2.1 d
4	40.0 ± 2.45 c	53.33 ± 1.5 b
5	40.0 ± 2.44 c	40.00 ± 1.7 c
6	60.0 ± 2.1 b	43.33 ± 1.8 c
KENF7 (7)	20.0± 3.7 e	66.67± 1.1 a
8	60.0 ± 2.1 b	36.67 ± 1.9 d
9	30.0 ± 3.1 d	26.67± 2.3 e
Control (0)	100.0 ± 1.1 a	0.0 ± 0.0 f

The same letters within each treatment represent values that were not significantly different at the p < 0.05 level of confidence according to the LSD multiple range test.

indicate that all nine fugal isolates could infect cumin plants and cause the typical symptoms of Fusarium wilt, including reduced plant height and foliar symptoms, with different degrees of disease severity. The foliar symptoms developed throughout the experiment; the expression was highest 60–80 days after the sowing date. The results indicated that the tested isolates were able to infect cumin plants. In general, isolate KENF7 (No. 7) caused the highest percentage of infection and reduced plant height compared to the control at 32.22 and 31.11%, respectively, followed by isolates No. 1, 6, 8, and 9. However, isolates No. 2, 3, 4, and 5 gave the lowest infection percentage.

3.2. Identification of No. 7 (KENF7) by ITS Sequencing

The ITS sequence was used to characterize the FOL isolate No. 7 as KMOAMN.FCumn (ITS1 and ITS2). A BLAST search of the NCBI data libraries for ITS sequence similarities yielded the highest results with 100% sequence similarity to the F. oxysporum isolate FC 1 (GenBank accession No. KY046315.1). The ITS sequence for the KMOAMN.FCumn isolate was deposited in the GenBank sequence database under the accession number (OM918708.1).

3.3. Effect of Fusarium oxysporum KENF7 Culture Filtrate on the Seed Germination of Baladi Cumin In Vitro

Surface-sterilized cumin seeds in sterile Petri dishes containing filter papers were saturated with different concentrations of F. oxysporum culture filtrate. The data shown in

Table 2. Effect of Fusarium oxysporum KENF7 culture filtrate (different concentration 1–10%) on the seed germination of Baladi cumin in vitro.

Filtrate Concentration (%)	Seed Germination (%)
1	80 ± 3.1 b
2	70 ± 3.7 c
4	40 ± 4.1 d
6	40 ± 4.2d
8	30 ± 5.1 e
10	10 ± 5.5 f
Control (0)	100 ± 0.0 a

The same letters within each treatment represent values that were not significantly different at the p < 0.05 level of confidence according to a least significant difference (LSD) test.

demonstrate that all treatments significantly decreased the percentage of seed germination. Seed germination decreased with an increase in the concentration of the culture filtrate in the medium. In addition, higher seed germination was found at fungus culture filtrate concentrations of 1 and 2% (80 and 70%, respectively). The lowest seed germination percentage was detected at culture filtrate concentrations of 8 and 10% (30% and 10%, respectively).

3.4. Isolation of Endophytic Bacteria and Screening of Antagonistic Strains against Pathogenic Fungi

Eleven strains of endophytic bacteria antagonistic to F. oxysporum were isolated from the roots of cumin. They were screened using the panel confrontation method for an inhibitory effect on F. oxysporum. Among the 11 strains, KAU2025 and KAU2022 had the highest inhibition rate against F. oxysporum and they were selected for further experiments (

Table 3. In vitro screening of different bacterial isolates against highly virulent isolate of Fusarium oxysporum.

Isolates No.	Inhibition Zone (mm)
1	6.2 ± 4.5 b
KAU2022 3	7.9 ± 3.3 a
4	6.4 ± 4.5 b
5	6.3 ± 4.2 b
6	6.5 ± 4.1 b
7	4.4 ± 4.9 c
8	6.4 ± 4.2 b
9	6.3 ± 4.0 b
KAU2025	6.2 ± 4.1 b
10	7.6 ± 3.2 a
11	6.5 ± 4.3 b
Control (0)	0.0 ± 0.0 d

The same letters within each treatment represent values that were not significantly different at the p < 0.05 level of confidence according to a least significant difference (LSD) test.

).

3.5. 16s rDNA Sequencing and Phylogenetic Analysis of Endophytic Strains KAU2025 and KAU2022

The genomic DNA of the endophytic bacterial strains KAU2025 and KAU2022 was extracted and amplified using PCR. Based on physiological and molecular the isolates, No. 2, KAU2022 and No. 10, KAU2025 were identified as B. parabrevis and P. fluorescens, respectively (Tables S1 and S2).

3.6. PGPR Evaluation

The screening of the two isolates of Brevibacillus parabrevis and Pseudomonas fluorescens for zinc and phosphate solubilization was carried out in vitro. The two isolates were found to be capable of solubilizing zinc and phosphorus (

Table 4. Detection of zinc and phosphate solubilization and siderophore and indole acetic acid production by Brevibacillus parabrevis and Pseudomonas fluorescens isolates.

Isolates	Zinc Solubilization	Phosphate Solubilization	Siderophore Production	Indole Acetic Acid Production
Brevibacillus parabrevis	+++	++	+	++
Pseudomonas fluorescens	+++	++	+	++

(+++) strong; (++) moderate; (+) weak.

). The data presented in Table 4 also show that both isolates of B. parabrevis and P. fluorescens were able to solubizatize zinc. B. parabrevis was graded as highly (+++), whereas P. fluorescens had positive effects and was scored as (+). Pseudomonas fluorescens positively affected phosphate solubilization and was graded as moderate (++), while B. parabrevis had negative effects and was scored as nil (−).

The data presented in Table 4 show the production of siderophores and IAAs by the Brevibacillus parabrevis and Pseudomonas fluorescens isolates. According to these results, the isolates could produce IAA at varying different degrees. The P. fluorescens isolate produced more than B. parabrevis. In addition, the B. parabrevis isolate showed improved siderophore production compared to that of the P. fluorescens isolate.

3.7. Brevibacillus parabrevis and Pseudomonas fluorescens Biocontrol Ability in Pot Experiments

The cumin plants inoculated with B. parabrevis KAU2025 and P. fluorescens KAU2022 showed milder symptoms than the control plants. In cumin plants inoculated with B. parabrevis, the disease incidence of Fusarium wilt decreased by 28.33%, the disease index decreased by 46.50%, and the biocontrol efficacy reached 58.55% compared to that of the control (

Table 5. Biocontrol efficacy of Brevibacillus parabrevis and Pseudomonas fluorescens against Fusarium oxysporum f in pot experiments.

Treatment	Disease Incidence (%)	Germination (%)	Biocontrol Efficacy (%)
Brevibacillus parabrevis	42.6 ± 2.4 b	70.0 ± 1.17 a	54.19
Pseudomonas fluorescens	52.0 ± 2.4 b	60.0 ± 1.32 b	44.1
Control	93.0 ± 2.8 a	5.0 ± 1.74 c	-

±SE—standard error of the mean. Data are the means of three replicates of 20 plants each. Identical letters within the same treatment condition indicate a lack of significant difference at the p < 0.05 threshold, as determined via a least significant difference (LSD) test.

). All treatments containing biocontrol agents significantly reduced the percentage of wilt disease compared to the control (untreated seeds with bioagents and untreated soil with bioagents).

3.8. Growth-Promoting Effect of Endophytic Bacteria Pseudomonas fluorescens KAU2025 and Brevibacillus parabrevis; KAU2022on Cumin Plants

The fresh weight (FW) and dry weight (DW) of the cumin plants increased significantly in the P. fluorescens and B. parabrevis treatment groups compared with the control group (

Table 6. Effect of Brevibacillus parabrevis and Pseudomonas fluorescens on fresh and dry weight of cumin plants after inoculation with Fusarium oxysporum.

Treatments	Fresh Weight (g)	Dry Weight (g)	Root Weight (g)	Root Dry Weight (g)
Brevibacillus parabrevis	2.45 ± 0.16 b	0.29 ± 0.01 b	0.25 ± 0.01 a	0.13 ± 0.01 a
Pseudomonas fluorescens	1.95 ± 0.16 c	0.12 ± 0.01 c	0.19 ± 0.03 b	0.11 ± 0.03 a
Healthy control	3.30 ± 0.20 a	0.35 ± 0.02 a	0.29 ± 0.01 a	0.14 ± 0.01 a
Infected control	1.25 ± 0.20 d	0.09 ± 0.02 d	0.12 ± 0.01 c	0.03 ± 0.01 b

±SE—standard error of means. The same letters within each treatment represent values that were not significantly different at the p < 0.05 level of confidence according to a least significant difference (LSD) test.

). The plant height, root length, and stem of the cumin plants increased significantly in the P. fluorescens and B. parabrevis treatment groups compared with the control pathogens (Table 6).

3.9. Effect of Pseudomonas fluorescens KAU2025 and Brevibacillus parabrevis; KAU2022 on the Phenol, Flavonoid, and Antioxidant Enzyme Activities of Cumin Plants

The P. fluorescens KAU2025 and B. parabrevis KAU2022 strains could promote the expression of antioxidant enzymes in cumin plants to a certain extent, and various antioxidant enzymes were affected to different degrees. The PO and PPO activity showed a trend of increasing first and then decreasing, reaching a peak at 48 h after Fusarium oxysporum inoculation. The same trend was observed with phenol and flavonoid content; both increased after treatment with P. fluorescens KAU2025 and B. parabrevis KAU2022, reaching a maximum level at 36 h and then decreasing (Figure 1A–D).

4. Discussion

Under greenhouse conditions, the pathogenicity tests of nine isolates from the Baladi cumin variety revealed that all isolates were able to infect the cumin plants. They caused different degrees of wilt and dam**-off diseases. This result confirms the findings of Satish–Lodha and Ritu-Mawar [33,34,35].

The seed germination percentage was decreased when the filtrate concentration was increased from 1% to 10%. Higher seed germination was observed at culture filtrate concentrations of 1% and 2–80% and 70%, respectively, while the lowest seed germination was detected at concentrations of 8% and 10% (30% and 10%, respectively). This showed that the F. oxysporum fungus produced a phytotoxic compound that had a negative effect on the percentage of seed germination [36,37]. ** et al. [38] found that fusaric acid (5-n-butylpicolinic acid), produced by Fusarium spp., was associated with wilt in banana, cotton, pea, tomato, and other plants. In addition, when enniatin was added as a solution to germinating wheat seeds, decreased seedling growth was directly related to increased enniatin concentrations.

Biological control is considered as a potential and sustainable method for control due to the decreased chance of environmental pollution and low health risk. Brevibacillus parabrevis, with its high antagonistic activity toward Fusarium oxysporum, can be successfully employed as a potential biocontrol agent to for various crop diseases, including alfalfa anthracnose, caused by Colletotrichum truncatum [39], ginseng gray mold, caused by B. cinerea [40], tomato bacterial wilt, caused by Ralstonia solanacearum [41], and potato common scab, caused by Streptomyces scabies [42]. Brevibacillus parabrevis has also been reported as the most dominant endophytic bacterium in multiple plants and controls several pathogenic plant fungi and bacteria [13,16,43].

The present study revealed that Brevibacillus parabrevis KAU2025 and Pseudomonas fluorescens KAU2022 inhibited the growth of F. oxysporum. The in vitro hyphal growth inhibition rate was 67.50%, and the biocontrol efficacy of KENF7 was 58.55% and 11.33%. B. parabrevis is one of the most frequently studied biological control agents.

From a previous study of Bishi and Vakharia, [44], they concluded that lytic enzymes such as β-1, 3 glucanase, chitinase, and protease are associated with the ability of Pseudomonas sp. to control plant pathogens [44].

In addition, Pseudomonas fluorescens KAU2022 inoculation promoted cumin growth, with a significant increase in fresh weight (FW) and dry weight (DW) compared to that of the control. These results indicated that the antagonistic bacteria Brevibacillus parabrevis KAU2025 and P. fluorescens KAU2022 could serve as plant growth stimulators.

The results of our study also showed that the bioagents could produce siderophores and indole acetic acid in different degrees. These results are in accordance with those of Singh et al. [45], who tested 26 isolates of Pseudomonas fluorescens for their ability to produce IAA and siderophores and solubilize phosphorous in in vitro conditions and found that all isolates showed plant growth promoting abilities. Wakatsuki [46] mentioned that microbes were a potential alternative that could help fulfill plant zinc requirements by solubilizing complex zinc in soil. Several genera of rhizobacteria belonging to Pseudomonas spp. and Bacillus spp. have been confirmed to solubilize zinc.

B. parabrevis is known to have biocontrol efficacy against plant diseases via multiple mechanisms, including the secretion of antimicrobial substances, competition for ecological niches and nutrients, and the induction of host systemic resistance, growth promotion, and colonization ability [47,48]. The antimicrobial mechanism of P. fluorescens KAU2022 may involve the secretion of active protease enzymes. Our results showed that P. fluorescens KAU2022 triggered resistance to Fusarium wilt in cumin plants by increasing the levels of defense-related antioxidant enzymes. The PO and PPO activities and the phenol content significantly increased in the plants treated with the P. fluorescens KAU2022 bacterial strain.

Our results showed that the plants treated with both bioagents had increased phenol and flavonoid contents compared to the untreated plants. These results are in agreement with those of many other researchers, e.g., Abo-Elyousr et al. [49], who reported that the treatment of tomato plants with P. fluorescens increased phenolic compounds and decreased the disease severity of bacterial wilt caused by Ralstonia solanacearum. In addition, the treatment increased the peroxidase (PO) and polyphenol oxidase (PPO) activity in the host tissues; the role of both enzymes is essential for quinones, which are more toxic to pathogens than their non-oxidized forms [50]. Moreover, they change the pH of the plant cell cytoplasm, leading to an increase in phenolic content and the inhibition of pathogen development [51].

Lanubile et al. [52] showed that an increase in the activity of the POD and PPO enzymes was helpful in the treatment of various fungal diseases. An increase in plant resistance to a pathogen can be attributed to an increase in peroxidase activity, as it is known that increased PO leads to an increase in plants’ ability to resist diseases [53,54].

5. Conclusions

Brevibacillus parabrevis KAU2022 and Pseudomonas fluorescens KAU2025 isolates were first selected from cumin for their antagonistic activity against Fusarium oxysporum—they acted with a relatively broad target spectrum by damaging the mycelial structure. The B. parabrevis and P. fluorescens isolates significantly decreased the disease incidence of Fusarium wilt and increased PO and PPO activities in cumin in the pot experiments. In addition, the B. parabrevis and P. fluorescens strains promoted cumin seedling growth. These findings suggest that the B. parabrevis and P. fluorescens strains can promote plant growth and enhance plant stress resistance.