Characterization of broad-spectrum biocontrol efficacy of Bacillus velezensis against Fusarium oxysporum in Triticum aestivum L

Fungi are the most important phytopathogens that cause yield losses. The mycotoxins released by fungi cause spoilage of stored food consumed by humans and feed supplied to animals. Fungi-antagonistic microbes are gaining attention as potential biocontrol agents (BCAs). This study was designed to isolate bacterial isolates from different crops and evaluate their in vitro antifungal assay against three phytopathogens, plant growth promoting (PGP) characteristics, molecular identification, and in vivo efficiency against the most devastating phytopathogenic fungus Fusarium oxysporum Schltdl. In the in vitro experiment, the 3 isolates BA, GL-1, and 5a out of 360 isolates showed more than 60% inhibitory activity against the selected fungi in this study. On the basis of 16S rRNA sequencing and phylogenetic analysis, BA isolate was identified as Bacillus velezensis. All three isolates produced indole acetic acid (IAA), hydrogen cyanide (HCN), and cellulase enzymes, while the BA and GL-1 isolates also produced siderophores and the BA isolate also produced ammonia. BA was selected on basis of not only Biocontrol efficacy but also maximum PGPR activity compared to GL-1 and 5a. In vivo assay, the isolate BA showed a significant decrease in disease severity caused by Fusarium oxysporum by 64.97% after 100 days of inoculation on wheat (FD-08) seedlings in a greenhouse assay and enhanced the shoot root height, fresh and dry mass. The wide-ranging antagonistic action of Bacillus velezensis isolated from the phyllosphere of wheat crops showed promising fungicidal and plant growth-promoting capabilities, suggesting it can be used as a biofungicide.


Introduction
Phytopathogenic fungi cause diseases and catastrophic threats to plant health. Rice, wheat, maize, barley and other important crops are vulnerable to different fungal species, ultimately causing low yields, which lead to increased prices and food insecurity for humans and livestock (Mitchell et al., 2016). The widespread soilborne phytopathogen Fusarium oxysporum, a difficult fungus to control, belongs to Ascomycota, whose members infect approximately 150 different plant species and 36 hosts in Pakistan (Syed et al., 2015;Rana et al., 2017). Fusarium attacks growing seeds, juvenile plants and ruptured tissues, enters through roots and hinders the vascular system of plants, mainly causing blight and rot, and some species also produce mycotoxins (Toppo and Naik, 2015;Rana et al., 2017). Fusarium wilt in cotton, chickpea and banana (Panama disease) is associated with significant economic losses, with an estimated reduction in yield of approximately 50% (Amanda, 2015;Dita et al., 2018). Fusarium causes symptoms such as Fusarium seedling blight (FSB), root rot, and Fusarium head blight (FHB) on wheat crops (Pastuszak et al., 2021).
The soil-resident fungus Rhizoctonia solani J.G. Kuhn is a basidiomycete found in almost all geographic regions and causes disease in different important crops (Ajayi-Oyetunde and Bradley, 2018;Khan et al., 2019). On the basis of its different disease-causing mechanisms and genetically divergent groups, R. solani is called an anastomosis group (AG), and approximately 14 different AGs have been reported (Tewoldemedhin et al., 2006). Widespread sheath blight caused by R. solani in rice causes serious yield loss from 25 to 50%, and all cultivars of rice are susceptible, although the degree of susceptibility differs.
Alternaria alternata (Fr.) Keissler, a phytopathogenic fungus belonging to division Ascomycota, is the causative agent of a wide range of pre-and postharvest plant diseases, including crop, fruit, cereal and vegetable diseases, across the globe, with approximately >300 species that can spread via spores and decaying plants (Leiminger et al., 2015;Zhu et al., 2017). This fungus has seven variants called pathotypes, which produce the host-specific toxins AF toxin and AK toxin, responsible for causing black spot on strawberry and Japanese pear, respectively (Tsuge et al., 2013). Sunflower and soyabean leaf blight caused by Alternaria accounts for major yield losses, including approximately 80% seed loss and 33% oil yield loss in Pakistan and across the world (Kgatle et al., 2018).
To overcome fungi, the apparent method of choice is the use of synthetic fungicides. The main adverse effects of chemical fungicides are elevated acute and chronic mammalian nontarget and environmental toxicology and later resistance and weakening of efficacy facilitating pest reappearance (Yoon et al., 2013).
Carboxylic acid-and glucopyranosyl-containing fungicides attack DNA topoisomerase II, and amino acid formation of fungi can also affect beneficial microbes in other eukaryotic cells (Yang et al., 2011).
It is not a novel concept to use living natural systems in agriculture for plant protection and growth promotion; this approach can be traced back a long way in human history. In contrast, the need for such biocontrol agents is greater than before since concerns about the use of these compounds are persistent. The direct impact includes the uptake of insoluble nutrients, inorganic phosphate solubilization, mineralization of organic compound phytohormone production and nitrogen fixation. The indirect impacts are that biocontrol agents produce antimicrobial peptides (AMPs), metabolite production, enzyme production, antibiotics and siderophores, which can restrict the plant loss caused by plant pathogens (Velivelli et al., 2015;Lucero et al., 2021). The process of selecting microorganisms and commercialization of biocontrol products for phytopathogens is difficult because there are several standards to be analysed that are critical for the achievement of consequent steps. Mechanisms of antagonistic efficacy, growth that maintains an adequate population, and even studies of legal property rights and market penetration are vital (Fernandes et al., 2021).
The agriculture sector is Pakistan's second largest, accounting for 24% of total GDP. The productivity of major crops such as wheat, maize, rice, sugarcane, and cotton are critical to the country's economic development (Shafi et al., 2022). The purpose of this study was to evaluate broad-specturm antagonist activity of indigenous isolates against fungi and in vivo antagonistic activity against the phytopathogenic fungus Fusarium oxysporum on wheat variety FD-08 under greenhouse conditions.

Materials and Methods
Phytopathogenic fungi collection and maintenance The fungi Alternaria alternata, Fusarium oxysporum, and Rhizoctonia solani used in this study were kindly provided by the Agriculture Research Service, United States Department of Agriculture (ARS, USDA), and maintained on PDA (potato dextrose agar) medium at 25± 2 °C for further use.
Isolation of bacteria with antagonistic activity against phytopathogenic fungi A total of 360 samples from the phyllosphere and rhizosphere of different crops, including maize, potato, apple, pomegranate, barley, cotton, grapes, and wheat, were collected from different fields of Quetta, Baluchistan, Pakistan and processed at Applied Biotechnology Laboratory, Balochistan University of Information Technology, Engineering and Management Sciences (BUITEMS). The phyllosphere samples were processed as described by Ramzan et al. (2016), and rhizosphere samples were processed as described by Tsegaye et al. (2019). By using the spread plate method, 1 ml of each 10 -6 CFU/ml diluted sample was spread on Luria Bertani agar (LBA) media, and plates were incubated at 28±2 °C for 72 hours. The bacterial colonies were sub-cultured and assessed for antagonistic activity against phytopathogens. They were further screened for different PGP traits. The bacterial strains showing antagonistic activity against phytopathogenic fungi were kept at -80 °C in glycerol stock.

Qualitative assays
The in vitro capability of bacterial isolates to stop the growth of phytopathogens (A. alternata, R. solani and F. oxysporum) was qualitatively assessed by the agar plug method, in which 5 mm agar discs of 7-day-old fungal pathogens were separately inoculated in the middle of petri-plates containing medium (PDA), fresh bacterial isolates were inoculated on three sides of the PDA plates (at least 2.5 cm distance) that were previously inoculated with phytopathogenic fungi, and plates were kept at 27 ± 1 °C for 5-7 days (Haidar et al., 2016).

Quantitative assays
Quantitative assessment was performed by the dual culture method of Haidar et al. (2016). A 6 mm disc of 7-day-old fungi was placed in the middle of a plate containing medium (PDA), a fresh bacterial strain at 2.5 cm from the fungal disc in a straight line was co-inoculated on the same plate, and the plates were incubated for 5 days at 27± 1 °C. Agar plates inoculated only with fungi were kept as controls. The experiment was replicated in triplicate. The inhibition percentage was calculated by the formula Inhibition % = {100 × ( )} where C= circular growth of fungus (phytopathogen) on control petri plates, and T = the circular growth of fungus (phytopathogen) in the presence of antagonistic bacterial isolates (Dennis and Webster, 1971;Azeem et al., 2022).

Molecular identification of strains BA, 5a and GL1
Genomic DNA from freshly cultured cells was extracted using a Thermo-ScientificTM mini kit (K0721) as stated by the manufacturer's protocol. Using the 1.3 kb universal primers U1492R (5′-GGTTACCTTACGACTT -3′) and 27F (5′-AGAGTTTGATCMTGGCTCAG -3′), bacterial isolates underwent 16S rRNA gene amplification (Awais et al., 2017). Amplifications were performed in a thermal cycler (Gradient Thermocycler, Biobase, China) in a total volume of 20 μl containing 2.5 mM MgCl2, 1 mM of each dNTP, 0.1 mM of each primer, 2.5 µl of Taq polymerase (Thermo Scientific) and 50 ng of genomic DNA. The thermal cycler protocol was as follows: initial denaturation at 95 °C for 5 minutes followed by 35 cycles of denaturation at 95 °C for 1 minute, annealing at 57 °C for 1 minute and extension at 72 °C for 1.5 minutes, with a final extension at 72 °C for 10 minutes. The amplified product was resolved in agarose gel 4 and visualized on a Gel Doc system (Biobase China). A Thermo Scientific Kit (K0691) was used to purify the amplicon according to the manufacturer's protocol. The purified amplicon was commercially sequenced at Macrogen (Korea). The inferred sequences were deposited in the GenBank database, and accession numbers were obtained. The identification of phylogenetic neighbours and calculation of highly analogous sequences were carried out by using the National Center for Biotechnology Information (NCBI); similarly, the phylogenetic tree was created in MEGA-X by using the first 50 BLAST hits (Ramzan et al., 2016).

Characteristics of plant growth promotion traits
The plant growth-promoting characteristics of the selected isolates B. velezensis, B. amyloliquefaciens, and B. subtilis were evaluated for phosphate solubilization, production of IAA, cellulose enzymes, siderophores, ammonia and HCN. All the experiments were conducted in triplicate.
Phosphate solubilization National Botanical Research Institute phosphate growth medium (NBRIP) was prepared for the assessment of phosphate solubilization (Nautiyal, 1999). The pH of the NBRIP agar was adjusted to 7.0, and the three potential antagonistic isolates were inoculated separately in the centre of plates containing NBRIP medium. NBRIP plates without bacterial inoculation served as controls. The plates were kept at 37 °C for 72 hours, and the formation of clear halo zones was observed.

Production of indole 3-acetic acid (IAA)
A single pure colony of each potential isolate was aseptically inoculated in sterile LB broth (100 ml) with the addition of 0.1 g of L-tryptophan (Sigma Aldrich) to assess IAA production (Sivasankari, 2016). The flasks were incubated at 37 °C for 48 hours with shaking at 100 rpm. Three replications were maintained, and uninoculated LB broth was kept as a control. The centrifugation of cultures was performed at 13,000 rpm (DWB-D2012plus) for twelve minutes, and the supernatants were collected. The supernatants (1 mL) were reacted with 2 mL of Salkowski reagent (1 mL of 0.5 M FeCl3 in 50 mL of 35% v/v per chloric acid) and incubated at 28 ± 2 °C for 30 minutes in the dark. The change in colour from colourless to pink-red indicated IAA production.
Screening for carboxymethyl cellulase (CMCase) CMC agar medium was used to grow the bacterial colonies for CMCase screening, and the medium contained K2HPO4 1 g/l, MgSO4·7H2O 0.5 g/l, NaCl 0.5 g/l, FeSO4.7H2O 0.01 g/l, MnSO4H2O 0.01 g/l, NH4NO3 0.3 g/l, carboxymethyl cellulose 10 g/l and agar 12 g/l. The bacterial isolates were streaked in CMC agar plates, and the plates were incubated for 96 hours at 30 °C. The plates were then flooded with gram iodine for 20 min and destained with 1 M NaCl for 20 min on a shaker at 50 rpm. A zone around the bacterial isolate after destaining was observed for cellulose production (Passari et al., 2016).
Screening for siderophore production The bacterial isolates were inoculated in broth (peptone 10.0 g/l; K2HPO4 1.5 g/l; glycerol 5 ml; MgSO4, 15.0 g/l) for 96 hours at 28 ± 2 °C. The broth cultures were centrifuged at 20,000 rpm for 7 minutes, and the cell-free portions were collected and mixed with an equal volume of chrome azurol S solution (Schwyn and Neilands, 1987). This solution was incubated in the dark for 35 min. A change in colour from blue to orange showed a positive result for siderophore production (Jacob et al., 2016). Three replications were maintained, and uninoculated broth served as a control.

Ammonia production
The antagonistic potential bacterial isolates were inoculated in peptone broth and incubated with shaking at 120 rpm for five days at 28 ± 2 °C. After that, the culture broth was centrifuged at 10,000 rpm for 5 five minutes. Approximately 1 mL of Nessler's reagent (0.09 molL −1 K2[HgI4] with 2.5 molL −1 KOH) was mixed with 9 mL of supernatant. The change in colour from brown to yellow showed the production of ammonia, and broth without Nesseler's reagent served as a control (Jacob et al., 2016).
Production of hydrogen cyanide (HCN) HCN production by the isolates was evaluated by the Lorck method (Lorck, 1948). The isolates (BA, GL-1, 5a) were grown in bennet agar (yeast extract 1 g/l, beef extract 1 g/l, casein enzyme hydrolysate 2 g/l, dextrose 10 g/l and agar 18 g/l) supplemented with glycine (4.4 g/l). Filter paper (Whatman grade 1 filter paper) was flooded with a solution consisting of picric acid (0.5%) added to 2% sodium carbonate for one minute. The plates were sealed with parafilm and incubated at 28 ± 2 °C for five days. The alteration from yellow to orange against filter paper indicated an isolate was positive for the production of HCN. Three replications were performed for each bacterial isolate.
In vivo PGP and antagonistic assay of Bacillus velezensis on wheat The effectiveness and antagonistic effect of the isolate Bacillus velezensis, based on in vitro efficiency, was also evaluated for in vivo antagonistic and PGP assays in a greenhouse against phytopathogenic fungi (Fusarium oxysporum) on wheat (FD-08), and the indices were examined by using the process of Vasebi et al. (2013) and Passari et al. (2016) with slight modifications. Plastic pots (10 x 8 x 8 cm) were filled with pasteurized loam, perlite and peat moss (1:1:1). Wheat seeds (FD-2008) were obtained from the plant stress physiology laboratory, BUITEMS Quetta, surface sterilized for two minutes in 70% ethanol followed by 2% sodium hypochloride for five minutes and rinsed with dH2O five times. Inocula containing the phytopathogen Fusarium oxysporum was prepared by growing in PDB at 25 ± 2 °C for 8 days. The culture was filtered with 4 layers of sterile chelate cloth, and the final concentration (10 6 CFU ml −1 ) was adjusted using haemocytometer in sterile normal saline. Bacillus velezensis was grown in a 500 ml flask containing 200 ml of sterile LB broth at 30 ± 2 °C for 48 hours in a shaking incubator (Biobase, China) at 100 rpm, and a final concentration of 10 8 CFU ml −1 was maintained. The bacterial counting was done using serial dilution and subsequently dilutions were spread on LB agar medium. Seeds were soaked for 4 hours and kept on a shaker at 100 rpm in the suspension described in Table 1.
After 4 hours, the aseptic seeds were sown in seedling pots with sterile loam, perlite and peat moss. The treated seeds were sowed in the greenhouse. The trial was accomplished with 4 treatments with 3 replications each. A total of 5 mL of Bacillus velezensis at a concentration of 10 8 CFU mL −1 in the form of a soil drench was applied to T2 and T3, and Fusarium oxysporum at a concentration of 10 6 CFU ml −1 was applied to T2 to promote disease development. The bacterial and fungal inoculation treatment was repeated after seven days. Three grams of urea was given per pot two times during the experiment for each treatment. Harvesting was performed after 100 days, and root and shoot length, fresh weight and dry mass as well as root and shoot number were recorded and compared among the treatments (Passari et al., 2016).
Each plant was rated using the following scales: 0 = no symptoms, 1 = less than 25% of plants with symptoms, 2 = 26 to 50% of plants with symptoms, 3 = 51 to 75% of plants with symptoms, and 4 = 76 to 100% of plants with symptoms. Similarly, the vigour index of wheat plants in each treatment was measured by using the following formula (Ji et al., 2014):

Vigour index = (Mean shoot length + Mean root length) Germination %)
One-way ANOVA was used for statistical analysis at P < 0.05. Three replications in the greenhouse were accomplished in all trials with eleven plants per treatment.

Results
Selection of bacteria with antagonistic activity Twenty bacterial isolates showed positive antifungal activity against all three fungi (Alternaria alternata, Rhizoctonia solani and Fusarium oxysporum) of the 360 isolated bacteria. Table 2 shows the colony characteristics of the twenty antagonistic bacterial isolates.  The isolates that showed positive antifungal activity were quantified using the dual culture method. All 20 isolates showed antagonistic activity ranging from 24-79%, as shown in Figure 1. Among 20 isolates, BA, GLI and 5a showed very strong antagonistic activity against the three tested phytopathogenic pathogens such as A. alternata, F. oxysporum, and R. solani as shown in Figures 1 and 2. All three isolates were gram-positive; BA and 5a were spore-bearing, while Gl-1 was nonspore-forming. BA, Gl-1 and 5a were characterized for molecular identification and for in vitro plant growth-promoting traits.

Molecular identification of the biocontrol bacterial isolates
BLAST examination using the 16S rRNA gene sequence showed that the BA, GL-1 and 5a isolates were aligned (98-99.11%) to Bacillus isolates. Using the neighbour-joining method, the evolutionary history was inferred (Saitou and Nei, 1987), and the optimal tree of the current study is shown in Figure 5. The replicate tree percentage is shown next to the branches where the associated taxa clustered together in the bootstrap test (1000 replicates) (Shimodaira and Hasegawa, 2001). Using the maximum composite likelihood method, the evolutionary distances were computed in units of the number of base substitutions per site. This analysis involved 50 nucleotide sequences. The ambiguous sites were detached for each sequence pair through the pairwise deletion option. Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018). Using the neighbour-joining dendrogram, phylogenetic analysis indicated that BA is closely related to B. velezensis. Gl-1 and 5a are related to B. amyloliquefaciens and B. subtilis, respectively.

Characteristics of plant growth promoting traits
The bacterial isolates BA, GL-1 and 5a, which showed very strong antifungal activity against the tested phytopathogenic fungi, were selected for characterization of in vitro PGP (plant growth-promoting) traits, as summarized in Table 4. 8

Indole-3 acetic acid (IAA) production and phosphate solubilization
The hormone indole-3 acetic acid (IAA), which is a vital hormone for plant growth promotion, tested positive in all three selected isolates by using Salkowski's reagent supplemented with tryptophan. Using the tryptophan-dependent pathway method, qualitative estimation of the results showed IAA production in the presence of tryptophan. None of the isolates solubilized phosphate.   Cellulase (CMcase) and ammonia production All three isolates showed cellulase production on CMC agar by forming a clear zone around the bacterial colonies. Among the isolates, GL-1 showed the largest zone (30 mm), followed by BA (19 mm) and 5a (16 mm). For ammonia screening, all the selected isolates (BA, GL-1 and 5a) were tested qualitatively for ammonia production. The isolate BA showed a positive result for ammonia production. The medium colour changed to brownish, indicating a positive result for ammonia production.
Hydrogen cyanide (HCN) and siderophore production All antagonistic isolates (BA, GL-1 and 5a) tested positive for hydrogen cyanide production. The result was based on colour intensity developed by soaking filter papers. On CAS agar medium, siderophore production was detected in all tested isolates (BA, GL-1 and 5a) by forming an orange zone around the colonies. Isolate BA produced the largest zone (13 mm), followed by 5a (11 mm) and GL1 (9 mm).
In vivo antagonistic assay of a selected isolate The bacterial isolate BA (Bacillus velezensis) was selected for the greenhouse experiment because it showed very strong antifungal activity in vitro against the tested fungi but also has better plant growthpromoting activity than other isolates and is a spore former. The phytopathogen Fusarium oxysporum was chosen for this experiment, as this fungus is relatively more lethal to wheat than the other fungi mentioned earlier. The in vivo biocontrol capability of Bacillus velezensis against Fusarium oxysporum showed that the isolate Bacillus velezensis reduced the disease rate significantly (64.97%) compared to pathogen treatment only, as shown in Figure 3. The symptoms such as necrosis, yellowing and wilting was reduced significantly by Bacillus velezensis compared to control. A significant rise in root length, shoot length, fresh weight and dry mass after 100 days of inoculation (DOI) was observed, as shown in Figure 4. The bacterial strain BA displayed significant PGP (plant growthpromoting) activity in terms of shoots and root length (p < 0.05) when compared with the pathogen-inoculated control (T4) and comparable to the uninoculated negative control (T1). In addition, the BA isolates were competent to produce IAA, ammonia, HCN, and siderophores and showed higher antifungal activity against all tested phytopathogens, including Fusarium oxysporum in vitro.

Discussion
Pathogens in soil, such as bacteria, fungi, viruses and nematodes, cause major economic losses to crop. To overcome this constraint, synthetic chemical compounds are routinely applied to crops, which not only causes environmental contamination but also health problems for humans consuming chemically affected crops. The practice of applying insecticides or pesticides is undeniably a current exercise to control phytopathogens. However, it is no longer a defensible approach for the control of pathogens, as they are emerging with resistance to fungicides and pesticides. This has encouraged researchers around the world to have increasing interest in biocontrol agents. Presently, there is no target site for the isolation of potential bacteria that could provide resistance against plant diseases, although the suitable location for finding such a bacterial strain is analogous to that for the target pathogen. It has been reported that synergic factors are required for biocontrol efficiency, such as the characteristics of biocontrol agents, the epidemiology of the target pathogen and the environmental conditions.
The goal of this research was to characterize the efficacy of bacterial isolates from the rhizosphere and phyllosphere that would provide resistance against phytopathogenic fungi in vitro and in vivo and promote the growth of crops.
In in vitro assay, out of 360 bacterial isolates, 20 were screened and found to possess antagonistic efficiency at some level against all three fungi (Alternaria alternata, Fusarium oxysporum, and Rhizoctonia solani) used in this study. Qualitative antagonistic activity through agar plug method was the first line of screening, and all isolates that had no antagonistic activity against phytopathogens were eliminated.
Furthermore, three bacterial isolates (B. velezensis, B. amyloliquefaciens, and B. subtilis) were subjected to quantification assays against the fungi used in this study. The three bacterial isolates were selected based on very strong antifungal inhibition against all three fungi. The inhibition zone was determined in an in vitro study, which may suggest the release of fungicidal metabolites by potential bacterial isolates.
The 16S rRNA gene sequences of isolates BA, GL-1 and 5a displayed 98-99% homology with reference sequences in GenBank of NCBI. The BA isolate is gram-positive and spore-bearing; despite the fact that all three bacterial isolates have strong antagonistic efficiencies against the tested phytopathogens, BA (Bacillus velezensis) was chosen for in vivo study based on PGP activity of Ammonia production, which was not observed in GL-1 and 5a (Bacillus amyloliquefaciens and Bacillus subtilus). The same type of bacterium with PGP and biocontrol activity was isolated from the rhizosphere of rice soil by Madhaiyan et al. (2010). Studies have shown that Bacillus strains can be used as biocontrol agents for plant disease (Gajbhiye et al., 2009), and our bacterial isolate BA showed 98.66% similarity to Bacillus velezensis.
For plant growth and development, phytohormone production is a significant element of plant growth and development of biocontrol isolates. IAA production by isolates allows plants to develop a well-grown root system, which raises the plants' nutrient uptake. In our study, the three isolates (BA, GL-1 and 5a) tested positive to produce IAA using l-tryptophan, indicating their genetic makeup is responsible for the variation production of IAA by multiple pathways. Previous studies found the same results and reported that bacterial isolates belonging to the same genus can produce different amounts of IAA (Gilbert et al., 2018). In this study, none of the three tested isolates showed phosphate-solubilizing activity on NBRIP agar medium by establishing clear halo zones around the bacterial colony. Similar results were obtained by Jacob et al. (2016), who showed promising findings from a GH (greenhouse) study, but the isolate was unable to demonstrate phosphate solubilizing activity.
Ammonia production by bacteria can act as an activating factor by suppressing plant pathogens and is responsible for indirect plant growth through the accumulation and supply of nitrogen to the plant. Consequently, root and shoot elongation are stimulated, which eventually increases plant biomass. The isolate BA produces ammonia. These findings are consistent with those of Joseph et al. (2012), who reported ammonia production in 95.0% Bacillus spp.

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The production of hydrogen cyanide (HCN) is another vital attribute of PGP, which plays a substantial role in combatting disease. The host plant is usually not affected by HCN generated by bacteria. It can operate as a biological control agent, whereas cyanide acts as a metabolic inhibitor to discourage competition predation or act as a safeguard from phytopathogenic fungi. All isolates in this research study (BA, GL-1, 5a) tested positive for HCN production. These findings are similar to Ghodsalavi et al. (2013), who found that approximately 90% of Bacillus spp. Are capable of producing HCN.
The role of siderophores in plant-bacteria association is to mobilize various metal ions, such as Fe, Cu and Zn (Tabli et al., 2018). Siderophores may excite the biosynthesis of other antimicrobial compounds directly by enriching the accessibility of minerals to bacteria that may stop the development of phytopathogenic fungi as well as other pathogenic microorganisms. The present study showed that the isolates BA and GL-1 were positive for siderophore production. The isolate BA (Bacillus velezensis) produced siderophores that are in agreement with Pereira Castro-Silva (2010), who reported that Bacillus subtilis (BS-8), isolated from rhizospheric soil, exhibited a high capacity for siderophore production.
Cellulase play a significant role in disease suppression but also helps in plant growth elevation and organic matter decomposition. In the current work, all three isolates (BA, GL-1 and 5a) exhibited positive cellulase production by forming a clear halo zone around the bacterial colonies on CMC agar, verifying the studies of Vipul et al. (2012) and Das et al. (2010), which showed that all Bacillus spp. In the experiment were positive for cellulase production.
In case of in vivo experiment, the isolate B. velezensisreduced Fusarium oxysporum disease symptoms by 64.97% in a greenhouse environment based on biocontrol observations and a significant vigour index compared to the control. According to Wang et al. (2020b), Bacillus velezensis BM21 showed antifungal activity against F. graminearum. Similarly, Jiang et al. (2019) used Bacillus velezensis F21 to inhibit Fusarium oxysporum in watermelon under greenhouse conditions. Analogous outcomes described by Yigit Dikilitas (2007) and Passari et al. (2016) reported that Pseudomonas fluorescence and Bacillus Sp. repressed Fusarium oxysporum growth in tomato plants under greenhouse conditions. According to Wang et al. (2020a), Bacillus velezensis FKM10 not only inhibited F. verticillioides but also enhanced the plant growth of Malus hupehensis R. Biocontrol bacteria displayed activity on a widespread range of agricultural crops, facilitating growth development and increasing crop yield, disease control, and stress tolerance. Research was performed to assess the competence of isolates and to screen the plant growth stimulating factors of Bacillus spp. using an in vitro antagonistic approach. The isolate (BA) involved in the elevation of growth in a greenhouse setting showed a significant antagonistic effect and an increase in root growth, shoot length and dry mass of wheat (FD-08) seedlings compared to the control after 100 days of seedling growth. The current outcome was significant (p < 0.05) when related to the control. According to Jasrotia et al. (2021), Bacillus spp. not only inhibited Magnaporthe oryzae, causing blast of rice but also significantly enhanced the shoot and root length of basmati rice. Several studies have also shown that endophytic bacteria can increase the root and shoot length in various plants, and such a significant effect on the growth and development of plants may compensate the host plant with respect to overall growth (Eid et al., 20021).

Conclusions
The study's goal was to characterise the efficacy and plant growth promoting activity of indigenous biocontrol bacterial isolates with broad antifungal activity in vitro. Furthermore, the selected biocontrol isolate will be tested in greenhouse conditions against Fusarium oxysporum on wheat crop. We believe that Bacillus velezensis (BA) could be useful as a biocontrol agent, liquid culture or efficient carrier material, and plant growth promoting inoculum in the production of agricultural crops.