The Endophytic Bacteria Bacillus velezensis Lle-9, Isolated from Lilium leucanthum, Harbors Antifungal Activity and Plant Growth-Promoting Effects

Bacillus velezensis is an important plant growth-promoting rhizobacterium with immense potential in agriculture development. In the present study, Bacillus velezensis Lle-9 was isolated from the bulbs of Lilium leucanthum. The isolated strain showed antifungal activities against plant pathogens like Botryosphaeria dothidea, Fusarium oxysporum, Botrytis cinerea and Fusarium fujikuroi. The highest percentage of growth inhibition i.e., 68.56±2.35% was observed against Fusarium oxysporum followed by 63.12 ± 2.83%, 61.67 ± 3.39% and 55.82 ± 2.76% against Botrytis cinerea, Botryosphaeria dothidea, and Fusarium fujikuroi, respectively. The ethyl acetate fraction revealed a number of bioactive compounds and several were identified as antimicrobial agents such as diketopiperazines, cyclo-peptides, linear peptides, latrunculin A, 5α-hydroxy-6-ketocholesterol, (R)-S-lactoylglutathione, triamterene, rubiadin, moxifloxacin, 9-hydroxy-5Z,7E,11Z,14Zeicosatetraenoic acid, D-erythro-C18-Sphingosine, citrinin, and 2- arachidonoyllysophosphatidylcholine. The presence of these antimicrobial compounds in the bacterial culture might have contributed to the antifungal activities of the isolated B. velezensis Lle- 9. The strain showed plant growth-promoting traits such as production of organic acids, ACC deaminase, indole-3-acetic acid (IAA), siderophores, and nitrogen fixation and phosphate solubilization. IAA production was accelerated with application of exogenous tryptophan concentrations in the medium. Further, the lily plants upon inoculation with Lle-9 exhibited improved vegetative growth, more flowering shoots and longer roots than control plants under greenhouse condition. The isolated B. velezensis strain Lle-9 possessed broad-spectrum antifungal activities and multiple plant growth-promoting traits and thus may play an important role in promoting sustainable agriculture. This strain could be developed and applied in field experiments in order to promote plant growth and control disease pathogens.


Organic Acid Production Assay
Organic acids in the isolated strain Lle-9 were detected according to the protocol developed by Cunningham and Kuiack [30]. About 50 μl of the bacterial suspension in MgSO 4 (10 mM) was inoculated in 800 μl of Sucrose Tryptone medium (ST) containing 20 g l −1 sucrose and 5 g l −1 tryptone. The ST medium was supplemented with 10 ml of trace element solution. Samples were incubated at 30°C and 200 rpm shaking for 5 days. After incubation, organic acids in samples were detected by adding 100 μl of 0.1% alizarine red S pH indicator. After 15 min, samples with yellow color were considered as positive. Pink-colored samples indicated negative results.

Indole Acetic Acid (IAA) Detection
Indole acetic acid (IAA) production in the isolated strain Lle-9 was assayed according to the method of Gordon and Weber [31]. A bacterial suspension of 150 μl prepared in MgSO 4 (10 mM) was inoculated in 3 ml of 1/10 diluted 869-rich medium. The medium was supplemented with various tryptophan concentrations of 0 mg ml -1 , 2 mg ml -1 , 4 mg ml -1 , and 6 mg ml -1 . Samples were incubated at 30 o C for 4 days with 150-180 rpm shaking in the dark. After incubation, the cultures were centrifuged at 4,000 ×g for 20 min. About 1 ml of the supernatant was mixed with 2 ml of Salkowski's reagent (98 ml 35% HClO 4 , 2 ml 0.5M FeCl 3 ). After 20 min, change of color from yellow to pink was considered as positive for IAA production. Quantitative measurement of indole acetic acid in the samples was conducted by measuring OD at 530 nm in a spectrophotometer. The IAA quantities in samples were measured based on a standard curve of known values (Fig. S1).

Siderophore Detection
The siderophore production in the strain Lle-9 was evaluated through both qualitative and quantitative tests. The bacterial cells were cultured in liquid 284 medium with chrome azurol S (CAS) shuttle solution, according to the described method [32]. About 50 μl of the bacterial suspension in 10 mM MgSO 4 was added to 800 μl of 284 medium prepared with three different iron concentrations. The iron concentrations used were: 0 μM, 0.25 μM, and 3 μM Fe(III) citrate. Samples were incubated at 30°C for 5 days with 150 rpm shaking. After incubation, 100 μl of the blue Chromium Azurol S (CAS) reagent was added to samples followed by incubation for 4 h at room temperature. After incubation, the change of color from blue to orange/yellow was considered as positive. Siderophore concentrations in all samples were further measured at 630 nm. The siderophore quantities were measured as % of siderophore units by the formula: % of siderophore units = Ar − As/Ar * 100 where, "Ar" is the absorbance of reference (CAS reagent); and "As" is the absorbance of sample at 630 nm. The ability of the strain to produce siderophores was further confirmed through qualitative test using CAS agar assay. All assays were carried out in triplicates.

Experimental Design of Greenhouse Test
The growth-promoting effects of the isolated endophytic bacterial strain Lle-9 were evaluated on the Asiatic lilium hybrids 'Tresor. ' Same-sized bulbs with normal and healthy appearance were selected from the storage house at 4 o C. For inoculation, the isolated strain Lle-9 was cultured in 5 ml LB for 10-15 h followed by further inoculation in 50 ml LB for 24 h at 30 o C with 220 rpm shaking. After incubation, the culture was re-inoculated in 400 ml LB and was kept to grow at 30 o C for 24 h. This culture was then diluted 10 times with normal water and bulbs of Tresor variety were soaked in the diluted culture for 40 min. The non-inoculated bulbs, soaked in simple LB, were used as controls. Soil pots of sizes 20 × 30 cm were prepared with soil mix of peat moss, perlite, and vermiculite in a ratio of 2:1:1. Three lily bulbs, either inoculated or non-inoculated control were sown in each soil pot. Pots were kept in a completely randomized design (CRD). Each treatment contained 5 pots. Pots were kept in plastic trays with holes in the bottom. The plastic trays were watered with equal amounts of normal tap water at regular intervals. Morphological data such as number of flowering shoots, plant height, leaf length, leaf width, bulb size and weight and root length were taken at the peak vegetative and reproductive stage.

Statistical Analysis
The data obtained were subjected to analysis of variance (ANOVA). Means were compared with Student's t-test at a probability of α = 0.05.

Isolation and Identification of B. velezensis Strain Lle-9
In our study, several bacterial endophytes were isolated from the bulb samples of Lilium leucanthum. Based on the antifungal assays, the isolated endophytic strain Lle-9 of B. velezensis was selected for further assays. The isolated strain Lle-9 formed off whitish chalky colonies with internal whitish circular lines (Fig. 1A), as a grampositive and spore-forming bacterium and exhibited small rod-shaped structures typical of the genus Bacillus as revealed by Scanning Electron Microscopic (SEM) analysis (Figs. 1B and 1C).
Molecular analysis indicated that the isolated strain Lle-9 belongs to the genus Bacillus. The BLAST results revealed that the 16S rRNA gene sequence was closely related to Bacillus velezensis. Based on the maximum likelihhod phylogenetic tree constructed with the 16S rRNA similarity (%), the Lle-9 strain revealed 99.34% similarity with Bacillus velezensis strain CR-502 (T), (AY603658) (Fig. 2). The 16S rRNA gene sequence of the isolated strain shared high similarities with other Bacillus species such as 99.25% with Bacillus siamensis, 99.25% with Bacillus nakamurai, and 99.16% with both Bacillus amyloliquefaciens and Bacillus subtillus. The 16S rRNA gene sequence of the isolated strain Lle-9 was submitted to GenBank under accession number MN461530.1.

Antifungal Activity Analysis
The isolated endophytic strain Lle-9 showed high potential of broad-spectrum antifungal activities against the tested phytopathogens, i.e., Botryosphaeria dothidea, Fusarium oxysporum, Fusarium fujikuroi, and Botrytis cinerea (Fig. 3A). These pathogenic strains were previously tested in an in vitro study for their pathogenicity potential and disease-causing ability in bulbs of Asiatic lilium hybrid 'Tresor. ' All pathogenic fungal strains revealed disease symptoms in bulbs of Tresor (Fig. S2). The strain Lle-9 exhibited considerable inhibition potential as revealed by zones of inhibition of the test pathogens on PDA plates. These high activities might be due to the release of some diffusible compound(s) against the test pathogens. Zones of inhibition of pathogenic fungi on PDA plates were measured as percentage values. The highest percentage of growth inhibition, i.e., 68.56 ± 2.35%, was observed against Fusarium oxysporum followed by 63.12 ± 2.83%, 61.67 ± 3.39%, and 55.82 ± 2.76% against Botrytis cinerea, Botryosphaeria dothidea, and Fusarium fujikuroi, respectively (Fig. 3B).

Plant Growth-Promoting (PGP) Assays
The plant growth-promoting effects of Lle-9 were assayed both qualitatively and quantitatively. According to our results, the endophytic bacterial strain Lle-9 showed positive results for all conducted assays.

ACC (Deaminase) and Organic Acid Detection
The ACC deaminase production was detected through a qualitative test based on change of color. The strain Lle-9 was found positive for the production of ACC deaminase (Table 1 and Fig. 4A).
The isolated strain Lle-9 was assayed for production of organic acids through a qualitative test. Strain Lle-9 showed moderate to high production of organic acids as revealed by the change of color from pink to yellow (Table 1 and Figs. 4A and 4B).

Indole Acetic Acid
Indole acetic acid (IAA) production in the isolated strain Lle-9 was detected through both qualitative and quantitative tests. Qualitative test confirmed IAA production in Lle-9 as revealed by the change of color of the culture supernatant from yellow to pink (Figs. 4A and 4C). Further, IAA was quantified in the strain at various tryptophan concentrations supplemented in the culture medium. The strain Lle-9 was able to produce IAA at different tryptophan concentrations (Table 1). Different tryptophan concentrations impacted the IAA production in the strain. The IAA content in the isolated strain increased with increasing tryptophan in the culture medium. The strain Lle-9 showed lower IAA content, i.e., 23.2 ± 1.9 μg ml -1 , at tryptophan concentration of 0 mg ml -1 . However, the IAA content increased gradually with increasing the tryptophan concentrations from 0 mg ml -1 to  6 mg ml -1 in the culture medium. The endophytic strain Lle-9 produced 79.7 ± 3.3 μg ml -1 , 117.7 ± 3.5 μg ml -1 , and 165.7 ± 5.8 μg ml -1 at tryptophan concentrations of 2 mg ml -1 , 4 mg ml -1 , and 6 mg ml -1 in the culture medium, respectively. These results suggest that the isolated strain Lle-9 has the potential to produce high content of indole acetic acid even in the absence of exogenous tryptophan. Application of exogenous tryptophan had no negative impact on IAA production; rather, it increased the production and a positive correlation was observed between the IAA production and the increasing tryptophan concentrations.

Siderophores
Production of siderophores in Lle-9 was assayed both qualitatively and quantitatively at different Fe(III) citrate concentrations added to the culture medium. The endophytic strain Lle-9 was able to produce siderophores as confirmed through a change of color from blue to orange yellow (Figs. 4A and 4D). To further test whether an iron source has any impact on the production of siderophores, the strain was cultured in the liquid 284 medium supplemented with different Fe(III) citrate concentrations ( Table 1). The strain Lle-9 showed high siderophore production when cultured in medium without Fe(III) citrate. The total siderophore quantity was reported as 51.3 ± 3.8 (psu) in the culture medium without addition of Fe(III) citrate. However, the siderophore accumulation by the strain declined as the quantities of Fe(III) citrate increased in the culture medium. Significantly greater decrease in siderophores was observed when Fe(III) citrate increased from 0 μM to 0.25 μM. About 33.3 ± 1.5 (psu) siderophores were detected at 0.25 μM Fe(III) citrate in the medium. Further reduction in siderophores was observed when Fe(III) citrate concentration was increased to 3.0 μM. However, this was not significantly different from the quantities obtained at 0.25 μM. At 3.0 μM Fe(III) citrate concentration, the strain Lle-9 accumulated 30.1 ± 1.3 (psu) siderophores. The siderophore production in the endophytic strain Lle-9 was further assayed through a qualitative test using chrome azurol S (CAS) on agar plates. An orange/yellow hallow was observed around the colonies of Lle-9 indicating the production of siderophores. The strain Lle-9 was able to quench the iron from the dye complex that resulted in a change of color from blue to orange/yellow in the form of a hallow surrounding the bacterial colony (Fig. 4B). Further, the diameter of the yellow/orange hallow produced by the strain averaged 15.32 ± 1.3 mm. This test further confirmed the high potential of the isolated endophytic strain Lle-9 to produce siderophores.

Potential for Nitrogen Fixation and Phosphate Solubilization
The nitrogen-fixing potential of the isolated B. velezensis Lle-9 was assessed by its ability to grow on nitrogenfree minimal medium (NFM). The Escherichia coli strain DH5α, which is unable to grow on nitrogen-free medium was used as a negative control. Both strains were cultured on nitrogen-free medium and medium supplemented with 5 mM NH 4 Cl, which is a preferred source of nitrogen. Results revealed that the growth of B. velezensis Lle-9 was clearly visible on the NFM, while the Escherichia coli DH5α, which does not fix nitrogen, grew only on medium supplemented with reactive nitrogen (Fig. 4C). These results demonstrate the potential of the isolated endophytic B. velezensis Lle-9 to fix nitrogen.
The phosphate solubilization potential of the endophytic B. velezensis strain Lle-9 was assayed on solid NBRIP medium. In this medium, Ca 3 (PO 4 ) 2 was provided as the sole source of inorganic phosphate. The endophytic strain Lle-9 was able to grow on the medium for longer incubation time and it solubilized the inorganic phosphate as indicated by the clearing zone surrounding bacterial colonies (Fig. 4D).

Plant Growth Promotion
The plant growth-promoting potential of the isolated B. velezensis Lle-9 was further assessed on the vegetative growth and bulbs production of the Asiatic lilium hybrids 'Tresor' under greenhouse conditions. Bulbs of 'Tresor' were inoculated with the isolate strain before cultivation in soil pots. Upon completion of vegetative growth, a number of plant growth parameters like plant height, number of flowering shoots, leaf length, leaf width, stem diameter and weight of bulbs were measured between the inoculated and un-inoculated control plants. Our results revealed that inoculation of bulbs with B. velezensis Lle-9 resulted in significant increase in overall plant growth relative to control plants ( Table 2). Inoculation of bulbs led to a significant increase in most of the tested growth parameters. The inoculated plants showed significantly high (p ≤ 0.05) increase in the number of flowering shoots, i.e. 3.47 ± 0.30, as compared to 2.73 ± 0.45 in un-inoculated control plants. Likewise, significant differences were observed in plant height between inoculated and un-inoculated plants (Fig. 5A, Table 2 (Fig. 5B). Overall, these results revealed that the B. velezensis Lle-9 was able to improve the vegetative and reproductive growth of lily plants upon inoculation.

Discussion
In the present study, a new endophytic bacterial strain Lle-9 of Bacillus velezensis was isolated from the bulbs of Lilium leucanthum. The isolated strain was assessed for its potential of antifungal activities against several fungal pathogens and it showed antimicrobial activity and resisted the growth and proliferation of these pathogens. The strain was identified to be B. velezensis through morphological and molecular analysis. Several plant growth promotion assays were conducted to confirm whether the strain could promote growth of the associated plants through several underlined mechanisms and whether it could also provide protection against disease-causing agents. Previous studies indicated that several B. velezensis strains isolated from various hosts, showed broadspectrum antimicrobial activities and plant growth promotion effects. Bacillus amyloliquefaciens FZB42, now recognized as a B. velezensis strain was reported in 2007 as the first gram-positive biocontrol bacteria to have its genome sequenced [36]. This isolate is now used as a model strain to promote plant growth and to confer disease resistance against broad-spectrum phytopathogens [37]. In addition, several other strains of B. velezensis have been used as antagonists of plant pathogens and as plant growth promoters in sustainable agriculture [12,36,38]. Further, a Bacillus velezensis strain CC09, isolated from healthy leaves of Cinnamomum camphora, showed immense potential as a new biocontrol agent, in control of many phytopathogenic diseases including wheat powdery mildew disease [39]. A B. velezensis strain NJAU-Z9, isolated from pepper rhizosphere, showed growth promotion effects in pepper [40].
In the present study, the isolated strain Lle-9 showed considerable antagonistic effects against fungal phytopathogens like Fusarium oxysporum, Botryosphaeria dothidea, Botrytis cinerea, and Fusarium fujikuroi. These pathogens have previously been reported to cause serious diseases in several crop plants [41]. These phytopathogens may cause diseases in Lilium species as revealed by a test confirming pathogenicity potential against cultivated species/varieties like Lilium davidii and Tresor. One of these phytopathogens, Fusarium fujikuroi, was isolated and identified from in vitro bulbs of Lilium wardii, a Lilium species. This could be a diseasecausing agent in Lilum wardii and other Lilum species. Interestingly, the isolated endophytic strain Lle-9 exhibited higher antifungal activities against all tested fungal pathogens and was found very effective against growth and proliferation of Fusarium fujikuroi.
The ability of the strain Lle-9 to suppress the growth of phytopathogens could be due to the presence of compounds and metabolites with antimicrobial properties. To provide evidence of this, the ethyl acetate fraction was assessed for potential secondary metabolites with bio-control properties. A number of secondary metabolites were identified and showed close homologies with the already known compounds with bio-control properties. Some of the prominent compounds and group of compounds, putatively identified from the isolated strain were diketopiperazines, cyclo-peptides, linear peptides, latrunculin A, 5alpha-hydroxy-6-ketocholesterol, (R)-Slactoylglutathione, triamterene, rubiadin, moxifloxacin, 9-hydroxy-5Z,7E,11Z,14Z-eicosatetraenoic acid, Derythro-C18-sphingosine, citrinin, and 2-arachidonoyllysophosphatidylcholine. Isolation of these secondary metabolites and compounds is evidence that the Lle-9 has a high potential of restricting the growth and proliferation of disease-causing fungal pathogens. Our results are supported by previous identification of these bio-control compounds and metabolites from other Bacillus species. Diketopiperazines and linear di-and tripeptides were previously recovered from cultures of endophytic bacterial isolates. Syed-Ab-Rahman et al. [42] found diketopiperazines in the cultures of Bacillus amyloliquefaciens, Bacillus velezensis, and Acinetobacter sp. They further reported that the presence of these bioactive compounds resulted in resistance to phytophthora infection and plant growth promotion. A number of other studies also reported isolation of diketopiperazines (cyclo di-peptides) that resulted into broad-spectrum antimicrobial activities [43,44]. Cyclo (Leu-Leu) found in Lactobacillus plantarum AF1 isolated from kimchi was found with antifungal activity against Aspergillus flavus [45]. Cyclo (Pro-Phe) in Bacillus amyloliquefaciens Q-426 was found to have a significant antifungal activity [46]. In addition, dipeptides and tripeptides have also been proved effective in conferring fungal resistance [47]. In the present study, latrunculin A was detected in the Lle-9 extract. This compound was previously shown as having fungistatic, fungicidal and fungilytic effects on the budding yeast Saccharomyces cerevisiae [48]. Triamterene, found in the ethyl acetate fraction of Lle-9 was previously found in ayurvedic medicine, Salmali niryasa, from a medicinal plant, Bombax ceiba [49]. The salmali resin had shown strong antimicrobial and antioxidant activities. Rubiadin, a bioactive compound detected in the present study was isolated from the roots of Morinda elliptica L. (Rubiaceae) [50]. This compound demonstrated anti-HIV, cytotoxic and antimicrobial activities [51]. Recently, rubiadin (AQ1) and rubiadin 1-methyl ether (AQ2), two photosensitizing anthraquinones (AQs) isolated from Heterophyllaea pustulata, showed reduction in biofilms formation of Candida tropicalis, a common cause of fungal infections [52]. Moxifloxacin was another important bioactive compound detected in the present study. This compound was previously shown as having antimicrobial effects [53]. The presence of these bioactive compounds might be responsible for the significant antifungal activities of Lle-9 against the tested fungal pathogens.
Previous studies have revealed that the plant growth-promoting traits in isolated strains of Bacillus species were correlated with several mechanisms such as production of organic acids, siderophores, lowering plant ethylene levels by ACC deaminase production, synthesis of plant growth-regulating hormones like indole-acetic acid and cytokinins, nitrogen fixation and phosphate solubilization [54]. In the present study, we confirmed through various tests that the isolated strain Lle-9 possessed plant growth-promoting traits. The isolated strain Lle-9 was capable of producing organic acids as revealed by a qualitative test. Organic acids play an important role in plant growth promotion and defense against phytopathogens [55].
Moreover, production of 1-aminocyclopropane-1-carboxylate (ACC) deaminase is one of the important characteristics of plant growth-promoting microbes and endophytes. ACC deaminase cleaves ACC, the immediate precursor of the plant hormone ethylene, to produce α-ketobutyrate and ammonia [56]. Ethylene serves as an important signaling molecule in plants under biotic and abiotic stresses and results in plant growth inhibition [57]. Previous studies have reported that inoculation of plants with ACC deaminase-producing microbes decreased ethylene levels that resulted in decreased inhibition of plant growth under biotic and abiotic stresses [3,58]. Previous studies showed that improvement of several crops, inoculated with Bacillus species, might partly be due to the production of ACC deaminase. The B. velezensis strain BACO 3 was reported to produce ACC deaminase, indole acetic acid, and ammonia, resulting in improved plant growth in terms of high biomass of leaves and roots in radish [59]. Bacillus species isolated from seeds of commercial tomato varieties exhibited multiple plant growth-promoting traits such as production of ACC deaminase, IAA, siderophores, and potential of nitrogen fixation and phosphate solubilization [60]. One of the isolates, HYT-12-1, identified as Bacillus subtilis, showed the highest ACC deaminase activity, which resulted in growth enhancement of tomato seedlings under greenhouse conditions. Consistent with the mentioned reports, the isolated strain Lle-9 in the present study exhibited ACC deaminase activity that might partly contributed to the improvement of growth of lily varieties upon inoculation.
Indole acitic acid is one of the important auxins that directly support plant growth and productivity. The ability of PGPRs including Bacillus species is partly attributed to their potential of IAA production that directly promotes growth of associated plants. In this connection, the role of the tryptophan precursor of IAA is important as considerable amounts of IAA are produced in the presence of excess tryptophan [61]. In the present study, the exogenous tryptophan concentrations had no negative impact on IAA production in Lle-9 and a positive correlation was observed between the tryptophan concentrations and IAA production. It seems the isolated strain Lle-9 produced IAA in a tryptophan-dependent pathway. Our results are supported by previous reports where application of exogenous tryptophan enhanced the IAA production. The Bacillus amyloliquefaciens strain FZB42, now a B. velezensis strain, was reported with production of IAA and its capacity increased five-fold with application of exogenous tryptophan [62]. Bacillus thuringiensis and Bacillus cereus isolated from soil samples exhibited IAA production in the absence and presence of L-tryptophan [63]. It was reported that the IAA production in the isolates increased with increasing concentrations of exogenous tryptophan.
Iron is an essential element necessary for growth of plants and microorganisms. However, it is abundantly present in soil in the form of insoluble Fe 3+ oxy-hydroxides. Plant-associated microbes reduce Fe 3+ to Fe 2+ with the help of ferrireductases or solubilize it with extracellular Fe 3+ chelators called 'siderophores' [64]. These soluble Fe 3+ -siderophore complexes are then available to both plants and microbes. Species of the genus Bacillus were previously reported with production of siderophores. Chen et al. (2007) reported siderophore bacillibactin production in B. amyloliquefaciens FZB42, now B. velezensis, and described that the siderophore synthesis in the strain was catalyzed by nonribosomal peptide synthetases. They further reported that production of high concentrations of siderophore bacillibactin in the FZB42 strain inhibited the growth of phytopathogenic bacterial and fungal competitors by depriving them of essential iron ions. Some recent studies also reported production of siderophores in Bacillus species [65,66]. Kesaulya et al. [65] reported siderophore production in the isolated Bacillus sp. from potato rhizospheric soil. They further reported that the isolated strain inhibited the pathogen causing banana wilt disease. These reports reveal that siderophore production by the plant-associated microbes not only helps plants acquire access to limited iron supply in the soil but also confers plants with a selective advantage over the pathogenic microbes by depriving them of essential iron that leads to disease resistance. In the present study, the high siderophore production in the isolated strain Lle-9 might partly be responsible for considerable antagonistic effects against the tested pathogenic strains. Quantitative evaluation of siderophores was conducted under different iron concentrations. Maximum siderophore production was observed in the absence of Fe(III) citrate in the medium. However, increasing iron concentrations in the medium resulted in reduction in siderophore accumulation. These results are supported by previous reports where an inverse relationship was observed between siderophore production and different iron concentrations in the medium [67,68].
Nitrogen is an essential and vital element for the normal growth and developments of plants. Many of the isolated PGPRs including species of Bacillus were found with the ability to fix atmospheric nitrogen. Syed-Ab-Rahman et al. [42] reported isolation of bacterial species including B. amyloliquefaciens (UQ154) and B. velezensis (UQ156) from soils of Arabidopsis plants. The Bacillus species were found with nitrogen-fixing ability. Other Bacillus species, which were isolated from Sophora Alopecuroides root nodules and rice showed nitrogen-fixing potential [69,70]. Likewise, in the present study, the isolated endophytic B. velezensis strain Lle-9 showed nitrogen fixation ability by growth on nitrogen-free medium. In addition, the strain was able to solubilize inorganic phosphate on a solid NBRIP medium supplemented with Ca 3 (PO 4 ) 2 as the sole source of phosphate. Phosphate solubilization is one of the most important plant growth-promoting traits associated with endophytic bacteria.
The different strains belonging to the genus Bacillus have been proved very effective as biocontrol agents and biofertilizers in sustainable agriculture [38,71]. Utilization of these bacteria has been found effective in controlling pathogenic microbes, exerting beneficial effects on plant growth and facilitating nutrient accessibility and assimilation [72][73][74]. Having these beneficial effects, the isolated strain Lle-9 was assessed for growthpromoting effects on the Asiatic lilium hybrid 'Tresor' under greenhouse conditions. Inoculated plants exhibited growth improvement and significant increases were observed for several growth parameters between the inoculated and non-inoculated control plants. Inoculated plants not only showed improved plant growth and bulb production but also produced a high number of flowers per plant. The improved growth performance in the Asiatic lilium hybrids 'Tresor' upon inoculation of Lle-9 might be due to the growth-promoting effects as were evaluated through several qualitative and quantitative tests. Previous studies also reported similar growth improvement when plants were inoculated with the isolated strains of Bacillus velezensis [75,40]. The isolated B. velezensis strain Lle-9 showed antagonistic effects against the broad-spectrum fungal pathogens. These enhanced antifungal activities might be due to the presence of a number of bioactive compounds, which have previously been recognized as antimicrobial in nature. Moreover, the strain Lle-9 exhibited several plant growth-promoting traits, which were reflected in the improved vegetative and reproductive growth of lily plants upon inoculation. Owing to these beneficial antifungal and plant growth-promoting properties, the B. velezensis strain Lle-9 may be a good choice to be utilized as a source of bio-fertilizer and bio-control agent in sustainable agriculture.