Cyclic siloxane biosurfactant-producing Bacillus cereus BS14 biocontrols charcoal rot pathogen Macrophomina phaseolina and induces growth promotion in Vigna mungo L.

Rhizobacteria are vital component of soil–plant interfaces which helps in plant growth responses and disease management. Precisely, the role of biosurfactant production by rhizobacteria in biocontrol mechanisms is underscored. The current study explores the destructive effect of a biosurfactant-producing bacterium Bacillus cereus BS14 on fungal growth under in vitro experiments and showed in vivo reduction of disease severity in pulse crop Vigna mungo. In this study, B. cereus BS14 was observed as plant growth-promoting rhizobacterium (PGPR) based on abilities of production of phytohormone and HCN, phosphate solubilization and biocontrol of Macrophomina phaseolina. The purified biosurfactant from BS14 inhibited the fungal growth by arresting radially growing mycelia. Scanning electron microscope (SEM) study revealed deformities at cellular level in the mycelia of M. phaseolina. The biosurfactant of Bacillus BS14 was identified as cyclic siloxane in GC–MS spectroscopy and FT-IR spectroscopy analyses. In the pot trial studies, B. cereus BS14 proved its efficiency for the growth promotion of Vigna mungo and significantly reduced disease severity index. The present study concludes that biosurfactant of rhizobacterial origin and rhizobacteria can serve for biological control, improvement in crop production and agricultural sustainability. In future, it can be developed as biological control and biofertilizer formulations for legume crops, and commercialized for routine farming practices.


Introduction
The bacterial metabolites, such as enzymes, antibiotics, vitamins, and other bioorganic acids have different applications in environment, industries, medicine, and agriculture (Lynch and Audus 1976;Singh et al. 2017). In agriculture, these are utilized to fulfil an aim of increasing crop productivity and soil-fertility management in an eco-safe way (Maheshwari et al. 2013;Maheshwari 2015). Such metabolites like biosurfactants or bio-emulsifiers are precious compounds as they are involved in soil-conditioning phenomenon to remediate soil, restrict the growth of phytopathogens and aid disease control in various field crops (Sachdev and Cameotra 2013). The rhizobacteria, the inhabitant of rhizosphere can enhance the plant health and growth via phytohormone production, secretion of iron-chelating siderophores, solubilization of insoluble phosphatic salts in the soil, induction of plant-immunity via systemic acquired resistance (SAR), and induced systemic resistance (ISR) (Sharma et al. 2018), water stress management by 1-aminocycloprapone-1-carboxylate (ACC)-deaminase activity, and biotic (densitydependent) stress regulation by biological control. Soilborne fungal pathogens attacking on below-ground parts of plants are still need to be controlled by rhizobacteria and their novel metabolites like biosurfactant (Bee et al. 2019) irrespective to cell-wall degrading enzymes.
Recent science of biocontrol has emerged to describe the role of certain biosurfactant in destructions of fungal pathogens, enhancement of rhizobacterial colonization and other means of biological control (Sarwar et al. 2018a), bacilli are often considered for elite production of a comprehensive array of biologically active molecules, such as lipopeptides (LPs), chitinase, and glucanase which protect plant from fungal diseases (Agarwal et al. 2017). Earlier, Romano et al. (2013) have purified cyclic lipopeptides from Bacillus amyloliquefaciens strain BO5A and checked its antifungal activity against pathogenic fungi Fusarium oxysporum, Aspergillus niger, Botrytis cinerea, and Penicillium italicum. Moreover, production of a wide range of antimicrobial substances by aerobic endospore forming bacteria (AEFB), such as biosurfactants has also been reported (Adu and Hunter 2021). Biosurfactants demonstrate antibacterial and antifungal activities against a wide array of pathogenic bacteria and fungi (Kumar and Johri 2012). A biosurfactant-producing Bacillus sonorensis MBCU2 was isolated from vermin compost-amended soil which showed potential antagonistic activity against M. phaseolina (Pandya and Saraf 2015). After qualitative analysis, biosurfactant from Bacillus was studied for its antifungal properties (Sarwar et al. 2018b). Ample reports have been published to establish Bacillus as a biosurfactant-producing bacteria (Sarwar et al. 2018a, b;Hafeez et al. 2019). On the other hand, Al-Ali et al. (2018) have demonstrated the production of exopolysaccharide (EPS) and biosurfactant for biofilm formation and rhizosphere colonization. Naturally, exopolysaccharide (EPS) produces silicones, when reacting with siloxanes to form organic siloxane, which is also produced by Bacillus mucilaginosus var. siliceous (Avakyan et al. 1986). In the present study, biosurfactant produced by Bacillus BS14 is structurally similar to the cyclic siloxane. Cyclic siloxanes are particularly useful to produce silicone surfactants and foam polyurethane for foam applications (Hil 2002). Recently, a US patent (US20190059385A1) has appeared to increase agronomic yield using the functional approach of siloxanes biosurfactants in agriculture (Hänsel et al. 2019).
Vigna mungo (L.) Hepper is a vital annual pulse crop and valued for its high digestibility and liberty from any harmful effect. Vigna mungo (experimental plant), commonly known as black gram, urad bean, black bean is an important crop from agricultural point of view, grown in South Asia and precisely in Indian sub-continent. It is cultivated from the distant past and praised as highly important pulse crop in India and Asian countries. The plant has the maximum height ranging from 35 to 110 cm and seedpods from 4 to 6 cm. V. mungo is more nutritious as it holds high value of protein, carbohydrates, potassium, iron, thiamine, riboflavin, niacin, folate, and other essential amino acids.
Macrophomina phaseolina (Tassi) Goid. is a soil-borne fungal pathogen causing charcoal rot in more than 500 plant species and reduce crop yield including Vigna mungo, which is a fast-growing herbaceous legume being cultivated throughout the world especially in South Asia (Arora et al. 2001;Gupta et al. 2002;Shahid and Khan 2019;Pandey et al. 2020). Several metabolites of bacterial origin have already been reported effective to control M. phaseolina (Illakkiam et al. 2013;Castaldi et al. 2021). Also, the role of biosurfactant has been evaluated to control fungal pathogens in certain plant species (Rodrigues et al. 2021). This research was aimed to evaluate the effect of biosurfactantproducing and plant growth-promoting Bacillus strains on fungal cellular integrity, in vitro control and plant growth promotion along with in vivo reduction of disease severity index. Bacillus strains have been investigated due to their versatility of spore-formation, survival in harsh conditions, and production of toxic metabolites inhibitory to phytopathogens (Bais et al. 2004). The present study was carried out to identify the beneficial roles of biosurfactant-producing Bacillus from the rhizosphere soil of V. mungo and shown their application as plant growth-promoting rhizobacteria (PGPR) for biocontrol of M. phaseolina.

Isolation of putative microorganisms
Healthy plants of V. mungo were collected from different farmer's fields of district Saharanpur, Uttar Pradesh, India (29.919° N 77.304° E), in sterile polythene bags and carried to the laboratory. Bacterial isolates recovered were identified as spore-forming after isolation via heat-treatment and serial dilution techniques as described by Agarwal et al. (2017). Micro-colonies were purified by streaking and designated with laboratory code prior to identification by cultural and biochemical tests, such as Gram-staining, spore staining, motility test, oxidase test, indole test, catalase test, citrate test, coagulase test, methyl red, and Voges-Proskauer test.

Screening of biosurfactant production
All the bacterial isolates were initially screened for biosurfactant production via various screening procedures, such as mineral salt cetyl-trimethyl-ammonium-bromide (CTAB)-methylene blue agar plate assay (Siegmund and Wagner 1991), hemolytic activity (Youssef et al. 2004), bacterial adherence to hydrocarbons (BATH) assay (Rosenberg et al. 1980), drop collapse assay (Jain et al. 1991), oil spreading assay (Rodrigues et al. 2006), emulsification stability (E24) test (Das et al. 2008), and measurement of surface tension of cell-free culture broth according to Du Nouy's ring method (Lunkenheimer and Wantke 1981), with slight modification as previously carried out in our study (Kumar et al. 2016).

Isolation of plant growth-promoting bacteria
The determination of IAA production by bacterial isolates was done by growing the cultures on LB broth and incubating at 28 °C for 24 h at 120 rpm. Exponentially grown culture (10 8 cfu mL −1 ) was centrifuged at 10,000 rpm for 20 min at 4 °C to collect the supernatant. 2 μL of orthophosphoric acid was added to 2 mL of supernatant with the subsequent addition of Salkowski's reagent. Appearance of pink colour confirms the IAA production. Further, HCN (cyanogen) production was determined following the modified method of Bakker and Schippers (1987). Siderophore production was evaluated on Chrome-azure S (CAS) medium by spot inoculating bacterial culture and incubated at 28 ± 1 °C for 48-72 h (Schwyn and Neilands 1987). The formation of orange to yellow halo around the bacterial colonies confirmed siderophore production. Phosphate solubilization ability of all isolates was detected by spotting them separately on Pikovskaya's agar plates (De Freitas et al. 1997). These plates were then incubated at 28 ± 1 °C for 3 days and observed for the appearance of the clearing zone around the colonies. The qualitative assay for chitinase production was performed following the method of Dunne et al. (1997). Isolates were separately inoculated by spotting on the plates containing chitin minimal medium (CMM) as the sole source of carbon and incubated at 30 ± 2 °C for 7 days. These plates were examined for the development of clear zones around the bacterial colonies. For biofilm assay, sterile Muller Hinton broth (MHb) (5 mL) was poured in the pre-sterile test tubes inoculated separately with the test organisms along with proper control and incubated at 37 °C for 24 h. The broth was discarded, washed with 0.5 M phosphate buffer saline (PBS) and the internal surface of the tube was stained with 1% crystal violet solution to confirm biofilm formation (O'Toole 2011).

Biochemical and physiological characterization
The biochemical characterization of isolates was carried out followed by Bergey's Manual of Determinative Bacteriology (Holt et al. 1994). Different phenotypic characters of these isolates were compared with the standard strains, such as Bacillus sp. (MTCC 297) and Bacillus subtilis (MTCC 441) which were procured from the Institute of Microbial Technology (IMTECH), Chandigarh (India). These eight isolates showed similarity with the species of Bacillus.

Molecular characterization
For molecular characterization of the isolate BS14, genomic DNA was extracted following Green and Sambrook (2012). The 16S rRNA gene of the isolate BS14 was amplified using polymerase chain reaction (PCR). A universal primer set consisting of 27F 5′AGA GTT TGATCMTGG CTC AG3′ and 1492R 3′CGG TTA CCT TGT TAC GAC TT5′ was used to amplify 16S rRNA gene using MJ Research PTC-100 Peltier Thermal Cycler (PCR). DNA amplicons were purified and directly sequenced from Institute of Microbial Technology (IMTECH), Chandigarh, India. All the sequences were compared with 16S rRNA gene sequences (type) available in the GenBank databases of NCBI using BLAST search. The 16S rDNA sequence was submitted to the GenBank Database to get an accession number. Phylogenetic analysis was performed using MEGA 6.0 version.

Production and purification of biosurfactant
The biosurfactant-producing potential isolate BS14 was transferred to 5 mL nutrient-rich broth (NRb) containing 1% yeast extract, 1.5% nutrient broth, and 1% ammonium sulfate and incubated at 37 °C for 12 h and 120 rpm as seed culture to get the optical density of 0.5 at 600 nm (equivalent to McFarland solution). Further, 5 mL suspension of BS14 was transferred to a 1000-mL Erlenmeyer flask containing 500 mL of LB medium and incubated on a rotary shaker incubator (150 rpm) at 37 °C. The bacterial cells were removed by centrifugation at 10,000 rpm at 4 °C for 20 min and the supernatant was acidified with 6 N hydrochloric acid to get the pH 2.0. The precipitate containing biosurfactant was allowed to settle at 4 °C for overnight and collected by centrifugation at 15,000 rpm for 20 min. The precipitate was dissolved in distilled water, pH 7.0 was maintained using 1 N NaOH and re-centrifuged at 10,000 rpm for 10 min to get the final precipitate of biosurfactant (Sanchez et al. 2007).

In vitro antagonistic activity
The fungal pathogen M. phaseolina was procured from the culture repository of Department of Botany and Microbiology, Gurukula Kangri (Deemed to be University), Haridwar (India) (Singh et al. 2010). Antagonistic properties of bacterial isolates were tested against M. phaseolina on potato dextrose agar (PDA) plates following the dual culture technique of Skidmore and Dickinson (1976). 5-day-old mycelial discs of 5 mm diameter were placed in the center of solidified medium in plates containing modified PDA by adding 2% sucrose. Culture of the isolate BS14 (7 × 10 6 cfu mL −1 ) was spotted 2 cm apart from the fungal disc and incubated at 28 ± 1 °C for 5 days. Growth inhibition was calculated by measuring the distance between the bacterial and fungal colonies as compared to the control. The fungal growth inhibition (%) was calculated using the formula: 100 × C − T/C, where C = radial growth of fungus in control and T = radial growth of fungus in dual culture. Purified biosurfactant of isolate BS14 was used to test the fungal growth inhibition by disc-diffusion method (Kirby et al. 1957;Jorgensen and Turnidge 2015).

Post-interaction events
Fungal mycelia growing towards the zone of interaction were processed for scanning electron microscopic (SEM) studies. Agar discs of 1 mm thickness were cut and isolated from the zone of interaction and placed on a glass slide. These were treated with 2% glutaraldehyde solution at 20 °C for 24 h. The samples transferred to copper stubs over double adhesive tape were coated with gold in POLARON, AU/PD sputter coater, and scanned by microscope at 30 kV. The electron microscopic study was carried out at Wadia Institute of Himalayan Geology, Dehradun (India).

Fourier transform-infrared spectroscopy (FT-IR) spectra of the dried biosurfactant
FT-IR spectra of the dried biosurfactants were recorded on an 8400S, FT-IR spectrometer (Shimadzu) (available at Patanjali Research Institute, Haridwar), and equipped with a mercury-cadmium-telluride (MCT) detector and cooled with liquid nitrogen. About 2 mg of dried biomaterial was milled with 200 mg of KBr to get a fine powder. The powder was compressed into a thin pellet to be analyzed by FT-IR spectra measurement in wavelength of 400-4000 cm −1 . The analysis of FT-IR spectra was carried out using OPUS 3.1 (Bruker Optics) software.

Gas chromatography-mass spectroscopy (GC-MS) analysis
GC-MS analysis of biosurfactant was done using a Varian 4000 Mass Spectrometer employing DB5 type capillary column and helium as a carrier gas at a flow rate of 0.5 mL/ min. The sample volume was 1 µL and the temperature was gradually increased from 40 °C to 280 °C to identify the compound. The total run time was 45 min. The MS transfer line was maintained at a temperature of 280 °C. GC-MS analysis was done using electron impact ionization at 70 eV and data were evaluated using total ion count (TIC) for identification and quantification of the compound. A comparative study was done between the identified compound spectra and that of known compounds of the GC-MS library NIST.

Pot trial experiments
Pot trial experiments were carried out in triplicate and twenty seeds of V. mungo var. U-31 (procured from Plant Pathology Department, IARI, Delhi, India) were sown per pot randomly in the rabi season from November to April. A total of five treatments were used during pot trial. Treatment 1 was seed bacterization with antibiotic-resistant strain of Bacillus cereus BS14 Cam+Ery+ . The antibiotic-resistant mutant of B. cereus BS14 was developed using Chloramphenicol and Erythromycin by following the method of Dheeman et al. (2020). For seed bacterization, healthy pre-sterilized seeds soaked in sterile lukewarm water for overnight were used . Treatment 2 was designed by infesting the seeds of V. mungo with sclerotia of M. phaseolina by mixing 0.5 mg sclerotia in 1% CMC or bio-priming. Treatment 3 was a mixture of treatment 1 and treatment 2 (include bacterization and infestation as a multistep protocol). In treatment 4, 0.5 mg/mL of purified biosurfactant in 1% CMC (in the ratio of 1:1 v/v) (for seed coating; at the rate of 10 mL semi-solid suspension per 100 g seed) was used. For treatment 5, treatment 2 was mixed with treatment 4 (include bacterization and biosurfactant treatment). Non-bacterized seed acted as a control. After all the treatments, seeds were sown in sterile pots in triplicates. The rhizosphere soil, sampled from treatments 1 and 2 were used to monitor root colonization and estimation of B. cereus BS14 Cam+Ery+ at 30, 60, and 90 days after sowing (DAS). B. cereus BS14 Cam+Ery+ strain colonizing V. mungo roots was screened on chloramphenicol and erythromycin amended medium. Colony numbers of indigenous bacteria were monitored on nutrient agar plates. Seed germination and plant growth parameters, such as root/shoot (length) weight (dry/fresh) were measured. As a sample ten fungus-infested plants were taken out after 60 days after plantation and symptoms of defoliation and wilting due to charcoal rot in plant roots were monitored. Disease index was calculated based on a scale 0 to 2, where 0 = no wilting/root rot, 1 = chlorosis and yellowing of leaves/wilting or root rot, 2 = dead plants. Disease severity was calculated as follows: disease severity = Σ (number of diseased plants at each index value × disease index value)/(total number of plants investigated × 2) × 100% (Sotoyama et al. 2015;Agarwal et al. 2017).

Statistical analysis
The data were analysed statistically for the mean differences in values of control and treated plants using Microsoft excel and Graph pad prism 5.0 software. The data were subjected to two-way analysis of variance (ANOVA) to determine the effect of treatment conditions, period and its interaction on various parameters. The data were analysed employing the Duncan's Multiple Range Test (DMRT) by taking p ≤ 0.01 as a significant level.

Isolation of putative Bacillus
Based on morphological, physiological, and biochemical characteristics the isolates were found Gram-positive, rodshaped endospore former, and producers of white, dry and folds, opaque and irregular edged colonies on NAM plates. Eight isolates showed positive reaction for catalase and oxidase test, utilized glucose, glucosamine, and sorbose. The isolates were negative for H 2 S production and methyl red test (Supplementary Table 1).

Screening of biosurfactant
Eight isolates were screened for biosurfactant production exhibited positive β-haemolytic activity, BATH assay, drop collapse test, oil spreading assay, emulsification assay, and surface tension assay. All the isolates except BS21 and BS24 displayed positive CTAB-methylene blue agar plate assay. Among all the isolates BS14 was found to show the best biosurfactant-producing properties (Table 1).
A dark blue halo zone with a sharply defined edge around the culture well was observed after 24 h in CTAB-methylene blue agar assay. In BATH assay, the bacterial cells indicated their affinity towards the hydrophobic substrate. BS14 showed β-haemolysis displaying the maximum haemolytic zone of ~ 2.94 cm. Cell adherence of BS14 to crude oil was 80.23%. Emulsification assay is an indirect method used to screen biosurfactant production. The cell-free culture broth of BS14, BS24, BS27, and BS41 showed more emulsification activity with petrol oil than the other isolates. BS14 showed significant emulsification activity with the emulsification index (E 24 ) of 70.58. In contrast, BS27 and BS41 displayed a better drop collapse test than the other isolates. Furthermore, the isolates BS12 and BS14 showed the maximum reduction in surface tension by 67.14 D/CM than the other isolates. BS14, BS27, and BS41 showed the maximum oil spreading activity forming the clearing zone (Table 1).

Plant growth-promoting (PGP) activities of isolates
Eight Bacillus isolates produced IAA and solubilized phosphate, whereas BS12, BS14, BS24, and BS27 produced HCN. The development of pink colour with and without tryptophan in cell-free supernatant indicated IAA production. Change in colour of filter paper from yellow to moderate and reddish-brown by adding FeCl 3 indicated HCN production by the isolates. Siderophore was produced only by BS12, BS14, BS28, BS40, and BS41. The formation of orange halos around the spots on CAS agar medium indicates siderophore production. However, all the isolates were solubilized phosphate. Formation of clear halos around bacterial spots in Pikovskaya's medium after 48 h displayed phosphate solubilization. Moreover, none of the isolates produced chitinase except BS12 and BS14 (Table 2). All the isolates exhibited biofilm formation except BS12, BS40, and BS41. Bacterial cells adhered to the surface of test tubes which showed biofilm production.

Molecular identification
The 16S rRNA gene sequence of the BS14 incorporated 1425 bp (NCBI GenBank Accession No. KU991962). It showed 99.44% sequence similarity with Bacillus cereus ATCC 14579. Therefore, the isolate BS14 has further been referred to as B. cereus BS14.

In vitro antagonistic activity of Bacillus cereus BS14 and pure biosurfactant
The pure culture of B. cereus BS14 and its biosurfactant inhibited the radial growth of M. phaseolina by 70.10% and 53.6%, respectively, after 7 days of incubation at 28 ± 1 °C (Fig. 1A, B). However, fungal inhibition was more pronounced in dual culture as compared to that of pure biosurfactant. Further, fungal growth inhibition corresponded with the incubation period.

Post-interaction events in mycelia of M. phaseolina
B. cereus BS14 in the zone of interaction resulted in halo cell formation and caused mycelial deformities and hyphal degradation of M. phaseolina. Formation and development of M. phaseolina sclerotia were arrested towards the zone of interaction; consequently, such mycelia and sclerotia lost their vigour. The SEM study shows the dissolution of fungal septa, hyphal fragmentation, and perforation in the cell wall of M. phaseolina ( Fig. 2A-C).

Chemical analysis of biosurfactant
The chemical structure of the purified biosurfactant from B. cereus BS14 was preliminarily investigated using FT-IR spectroscopy. The peak at 1568.02 cm −1 indicates the chemical structure identical to that of a cyclic compound consisting of a hexane ring. The peak at 2329.85 cm −1 indicates the Si-H group. The peak between 3394.48 and 3558.42 cm −1 shows the relatedness of the Si-NH 2 group. The FT-IR analysis shows the similarity with the cyclic compound produced by B. cereus BS14 (Fig. 3). FT-IR responsiveness within different absorption regions, absorbance peak heights corresponding to the Si-O bond and Si-CH 3 bond were evidenced and indicated a strong correlation with the expected concentration in the siloxane. For external verification of the calibration, FT-IR results were compared to those produced by GC-MS. The GC-MS analysis of the concentrated methanol extract resulted in many compounds. The peaks in the chromatogram were integrated and were compared with the database of the spectrum of known components stored in the GC-MS library of NIST to confirm the FT-IR structure analysis. Based on GC-MS analysis, the compound showed the relatedness with cyclic compound 'cyclic siloxane' (Fig. 4).

Pot trial experiments
Seed germination was enhanced maximally with the treatment of B. cereus BS14, while the germination of the fungal-infested seeds significantly decreased followed by mixed treatment of M. phaseolina and B. cereus BS14. Following the pattern of robustness, treatment BS14 evidenced approximate 25% enhancement in shoot length over control but significantly lowered in the fungal treatment after 90 days of sowing. It is also evidenced that the disease severity index reached at a peak in the fungal infestation. The development of roots can never be ignored as the attained maximum length in the bacterial treatment is similar to the treatment    improve the growth parameters of V. Mungo, however, reduced the disease severity index as compared to bacterial inoculation (Table 3). B. cereus BS14 Cam+Ery+ displayed an effective root colonization of V. mungo as evidenced by the recovery of significant bacteria population from V. mungo rhizosphere at 90 DAS (Table 4).

Discussion
Rhizobacteria as soil ecological population is a heterogeneous group of bacteria and beneficial for the improvement of legume crop production (Baliyan et al. 2018). Biosurfactantproducing Bacillus with plant growth-promoting traits as a rationale was studied to prove a better alternative for biocontrol agents of phytopathogens, bioinoculants for plant growth promotion and overall improvement in legume crop yield via eco-balanced ways. In this study, B. cereus BS14 was found the most feasible biosurfactant-producing plant growthpromoting rhizobacteria among the other eight isolates. B. cereus BS14 established a new functional niche in the  rhizosphere or soil habitat, as able to produce biosurfactants and involve in soil-conditioning of indigenous farms. We have observed that, B. cereus BS14 has plant growth-promoting characters, such as IAA, HCN and siderophore production. Earlier, Bacillus is reported to bear a PGPR's characteristics as evidenced in our study . In this trend, plant hormones like indole-3-acetic acid (IAA), gibberellins, cytokinin, and certain volatiles have also been reported in PGPRs (Mehta et al. 2010). Bacillus has significant effects on the biocontrol of phytopathogenic fungi (Dheeman et al. 2020). Similarly, B. cereus BS14 was found effective to restrict the growth of phytopathogenic fungi, and caused cytopathic effects in the fungi. In vitro inhibition of fungal pathogen by the purified biosurfactant of B. cereus BS14 is interesting to note the biocontrol behaviour of biosurfactants. The biocontrol of phytopathogens by such metabolites has been evidenced from the study conducted on antifungal activity and characterization of Bacillus against M. phaseolina (Hussain and Khan 2020). Under our investigation, we have observed phosphate solubilization efficiency of biosurfactant-producing bacteria. Previously, impact of biosurfactant production has been correlated to the phosphorus solubilization activity in certain rhizobacteria (Mishra et al. 2020). Phosphorus (P) is a very crucial plant growth-limiting nutrient and available in insoluble form in a substantial amount in the soils for plant growth nutrient. The abilities of B. cereus BS14 in phosphate solubilization was observed as an important trait of PGPR, which most probably improved the available P in the soil and directly affected the plant growth. However, in the biosurfactant as responsible mechanisms of P-solubilization has not been understood well and still in the nutshell. Still, the role of biosurfactants in phosphate and other mineral solubilization as the bioremediation approach is underscored. Besides, Prakash and Arora (2019) evidenced the abilities of Bacillus strains for solubilization of phosphate and other mineral nutrients. Further, this study opens a scope to utilize bacterial origin surfactant for soil remediation and enhancement of soil nutrition improvement. Furthermore, iron is a crucial nutrient for all forms of life and all microorganisms require iron for their growth and metabolism too (Neilands 1995). B. cereus BS14 was found a prominent siderophore-producing bacterium, in vitro. Many rhizobacteria have been reported to produce antifungal metabolites like HCN (Bhattacharyya and Jha 2012). In the present study, B. cereus BS14 has been shown to produce HCN; similar ability of Bacillus has been reported by Dheeman et al. (2020). B. cereus BS14 produced a clear zone in the chitin-containing growth medium and showed chitinase producing abilities, as the desired mechanisms of the biocontrol agent . Chitinase production by rhizosphere bacilli from rice (Chen et al. 2010) and Phaseolus vulgaris ) has been reported earlier.
B. cereus BS14 possesses all necessary characters of PGPR along with biosurfactant producing abilities. Beneficial rhizobacteria can promote plant growth not only by facilitating mineral nutrient uptake and phytohormone production but also more indirectly by protecting against the infection of fungal pathogens. They can antagonize pathogens by producing low molecular-weight toxin or extracellular lytic enzymes (Haas and Keel 2003) and more indirectly by triggering the defensive capacities in the host plant (Cameotra and Makkar 2004). We found that B. cereus BS14 inhibited the growth of M. phaseolina in vitro and resulted in several types of abnormalities in mycelia. It may be due to the secretion of many inhibitory compounds leading to multifarious abnormalities in fungal hyphae as observed by scanning electron microscopy (SEM). Similar work on biocontrol of M. phaseolina has been carried out by . Furthermore, fungal inhibition was more pronounced in dual culture as compared to pure biosurfactant. Earlier, Bacillus species have been reported for the biological control of fungi including Fusarium species and M. phaseolina . The broad-spectrum antagonistic activity of bacilli has been executed by the secretion of several metabolites including antibiotics (Haas and Keel 2003), volatile HCN (Chen et al. 2010) and siderophores (Gupta et al. 2002).
Biosurfactant produced by B. cereus BS14 showed antifungal activity against M. phaseolina. Earlier, Mnif et al. (2015) isolated Bacillus sp. SPB1 that produced a lipopeptide biosurfactant and exhibited antifungal activity against Rhizoctonia bataticola (perfect state M. phaseolina) and Rhizoctonia solani. Already, the concept of microbial biosurfactants and their antimicrobial activity has been propounded (Goswami and Deka 2021). We have observed that B. cereus BS14 produced cyclic siloxanes type biosurfactant, which are especially important and valuable compounds from the industrial application point of view. The cyclic siloxanes are used in the manufacture of silicones, carriers, lubricants, and solvents in a variety of commercial applications (Si 2006). The importance of the cyclic siloxane type biosurfactant has rarely been covered in the literature. This study is the first report on Bacillus capable of producing siloxane biosurfactants. The FT-IR and CG-MS analyses evidenced that biosurfactant produced by B. cereus BS14 has similarity with cyclic siloxanes. Extracellular polysaccharide plays a crucial role in silica release, especially in the case of quartz. Such polysaccharides can react with siloxanes to form organic siloxanes. It can be of bacterial provenance for example B. mucilaginosus var. Siliceous (Avakyan et al. 1986). Strains of Bacillus thuringiensis (Bt) produce crystalline proteins (δ-endotoxins) during their stationary phase of growth. Many authors used surfactants (1,2-benzisothiazolin-3-one) of the inert ingredients in Foray 48 B; the siloxane (organosilicons) Triton-X-100, Tween 20, and Latron CS-7 as surfactants for Btk formulations (Helassa et al. 2009). Biosurfactants are also known to play multifarious roles in biofilm formation. In the present study, B. cereus BS14 produced biosurfactants and formed biofilm. The best biofilm-forming activity was found in other isolates also those produce biosurfactants too. The role of bacterial biofilms and surface components in plant-bacterial associations has been evidence by Bogino et al. (2013).
Pot trial experiments illustrated the potential of B. cereus BS14 to be developed as an effective commercial biological control agent. Bacterial broth culture of B. cereus BS14 Cam+Ery+ effectively enhanced plant growth and decreased charcoal rot disease. Effect of cyclic siloxane biosurfactant was observed in disease reduction irrespective of a direct impact on plant growth promotion. This may be due to living actions and the involvement of other traits of bacteria in the rhizosphere. Thus, B. cereus BS14 played a distinguished role in declining charcoal rot disease along with plant growth promotion of V. mungo. Increase in bacterial dynamic and assemblage of B. cereus BS14 in V. mungo rhizosphere with a high number of colonies forming unit, evidenced by lower fungal infestation, which may be due to rhizospheric effect leading to higher bacterial load in the rhizosphere for natural competition in the rhizospheric niche. Also, the synergistic effect of rhizobia and Bacillus (Menéndez and Paço 2020) cannot be ruled out but the possibility of this factor canno't be ignored under this investigation. Further, the effect of fungal load in soil influences bacterial community or how bacteria adopt r-strategy for high reproduction and colonization under fungal stress is a future research. As per evidence from the results, the isolate was putative to produce novel cyclic siloxane type of biosurfactants. This study reflects beneficial gears of biosurfactant production as an indirect approach of PGPR, like enzyme production, antibiotic production responsible for biocontrol. Furthermore, the study concreted with pot trial assay in which exclusive growth and health improvement were acquainted by plants, and disease severity index was reduced significantly in the biosurfactant treatment. Therefore, we postulate the effects of siloxane biosurfactant in biocontrol of phytopathogens and growth promotion of leguminous crop.

Conclusions
It may be concluded that biosurfactant-producing bacteria like Bacillus cereus BS14 are available in the rhizosphere of legume crops and exhibit strong plant growth-promoting properties and biocontrol potential against M. phaseolina causing charcoal rot in V. mungo. Further use of biosurfactants(s) or biosurfactant-producing bacteria for biocontrol of charcoal rot disease is important alternative. Future of this study has insights of biosurfactant-producing PGPR-bioinoculants in various carrier materials for agricultural and environmental sustainability.