Brassica nigra essential oil: In-vitro and in-silico antibacterial efficacy against plant pathogenic and nitrifying bacteria

The present study was aimed to examine the antibacterial potential of Brassica nigra essential oil (BNEO) against Ralstonia solanacearum, causal agent of bacterial wilt and Nitrosomonas sp., the nitrifying bacteria. In poisoned food assay, BNEO showed 100% growth inhibition of R. solancearum at ≥ 125 µg mL⁻¹. Revalidation of findings by volatile assay employing inverted Petri plate technique exhibited 100% bacterial growth inhibition caused by vapors of BNEO, even at 50 µg mL⁻¹ concentration. In the broth microdilution assay, the BNEO exhibited significant antibacterial activity only at higher concentrations (>500 µg mL⁻¹). At 500 µg mL⁻¹, BNEO showed 80% bacterial growth inhibition over control, which was at par with that of streptomycin (5 µg mL⁻¹). In resazurin microtitre-plate assay, the maximum concentration of BNEO, at which color change occurred was 512 µg mL⁻¹ (T9), and thus 512 µg mL⁻¹ was concluded as the minimum inhibitory concentration (MIC). BNEO effectively inhibited the activity of Nitrosomonas spp. with 30-65% nitrification inhibition at the dose of 400 mkg⁻¹ of Urea-N. Homology modeled protein targets assisted computational tool-based novel analysis helped to understand that the antibacterial potency of BNEO is due to preferable binding efficiency of allyl isothiocyanate (AITC), the major active ingredient of BNEO.

Keywords: Bacterial wilt; nitrifying bacteria; phytochemicals; bioprospecting; allyl isothiocyanate; Molecular modeling

Phyto-pathogenic bacteria cause significant reduction in agricultural production. Bacterial wilt caused by Ralstonia solanacearum is one of the major diseases of several cultivated crops causing huge damage worldwide.[[1]]R. solancearum is a soil-borne gram negative plant pathogenic bacteria responsible for causing up to 90% economic loss globally, besides deteriorating the quality of fruits and vegetables.[[1], [3]] Bacterial wilt disease caused by R. solanacearum is a serious constraint in cultivation of tomato (Solanum lycopersicum L; family Solanaceae), the second most important vegetable crop in the world. In India, tomato ranks second in the area (7, 89, 000 ha) with production (1,97,59,000 metric tonnes) with average productivity of 25.0 MT/ha.[[4]] The bacteria cause substantial yield loss ranging from 10-90% in tomato growing areas of the country.[[5]]

Tomato being a high N-fertilizer consuming crop (approximately 137 kg ha−1 N-uptake),[[6]] also suffers from low nitrogen use efficiency (NUE). Nitrosomonas and Nitrobacter species are the nitrifying soil borne bacteria, responsible for the low NUE of nitrogenous fertilizers and subsequent huge economic losses globally.[[7]] The available candidate molecules[[8]] for managing these microbes suffer from limitations like high toxicity, low stability, high cost and pesticide resistance, necessitating the search for alternative broad spectrum bactericidal leads.

Plants serve as natural reservoir of effective chemotherapeutants.[[9]] Extracts and exudates of number of plants are often active against a limited number of specific target species, biodegradable to nontoxic products, and thus bear potential as green pesticidal leads.[[10], [12], [14]] Essential oils (EOs) are lipophilic volatile secondary metabolites, comprising of natural mono and sesquiterpenes.[[15]] They are commonly found in various plant parts and play a crucial role in plant protection.[[16]] Diverse biological functions of predominant components of EOs have already been proven.[[17], [19]] Further, the anti-bacterial potency of EOs have been demonstrated extensively against different pathogenic bacteria.[[21]] The mechanism of their action has rarely been elucidated. Sporadic literature reports suggest major component of the oils possibly interfere with the phospholipid bilayer of the cell membrane resulting impairment of enzymatic functions through inhibition.[[23]] BNEO, the mustard isolated from Brassica campestris contains allyl isothiocyanate as the main chemical component responsible for its broad-spectrum bioactivity range.[[24]] Despite a few preliminary reports on antibacterial efficacy of BNEO, comprehensive studies on potential bactericidal action and understanding of interactions of AITC toward specific nematode proteins have been lacking. Furthermore, effectiveness of BNEO on nitrifying bacteria in order to improve nitrification efficiency has not been reported in literature.

Molecular docking and simulation approaches are increasingly being reported as effective tools to find out the target binding sites in multiple receptors.[[25], [27]] Nematicidal action of various EOs has been explained with the help of molecular docking through target protein ligand interactions.[[28]] Given the lack of information on BNEO, this study was undertaken to assess its antibacterial potential against plant pathogenic bacteria, R. solancearum, and nitrifying bacteria, Nitrosomonas sps., vis-à-vis correlating its efficacy on bacterial target specific proteins using molecular docking and simulation studies. The present study envisions a holistic solution to the low NUE and bacterial wilt menace in tomato.

Microbial resistance to antibiotics is a common problem often encountered in the successful management of plant pathogens. This necessitates the development of new antimicrobial products to manage the microorganisms responsible for limiting the agricultural production. In the present study, the BNEO was tested for its antibacterial activity against a highly virulent plant pathogenic strain, R. solanacearum BI001. BNEO demonstrated complete growth inhibition at 125 µg mL−1 and above concentrations (Figure 1) against R. solanacearum. The lowest test concentration of 62.5 µg mL−1 was less effective in inhibiting the growth of bacteria and the level of bacterial growth in the treatment was similar to that of the negative control. However, the positive control treatment, streptomycin exhibited complete inhibition of bacterial growth as low as 5 µg mL−1 concentration.

PHOTO (COLOR): Figure 1. Evaluation of different concentrations (ppm) of essential oil E1 against R. solancearum RS-09-100 growth by poisoned food assay. A- E1 oil [i- 62.5 ppm, ii- 125 ppm, iii- 250 ppm, iv- 500 ppm and v- 1000 ppm]. B- Control (i- diluent, ii- streptomycin 5 ppm respectively).

Nevertheless, validation of the results by volatile assay employing inverted Petri plate technique (Figure 2) demonstrated that the vapors of BNEO showed complete bacterial growth inhibition even at 50 µg mL−1 concentration. Complete bacterial growth inhibition was noticed for the streptomycin treatment plates, attributed to the non-volatility of the molecule. The results indicated the significance of an appropriate technique for evaluation of antibacterial activity of a volatile component. In the poisoned food assay, BNEO being in direct contact with the bacterium, significantly inhibited the bacterial growth in comparison to the negative control, though, the effect was relatively less with respect to the conventional antibiotic, streptomycin. However, the volatile assay being more advanced, exhibited that the antibacterial effect of BNEO was highly significant over the negative control, while the antibiotic didn't show any inhibition of the bacterium. The results, therefore, suggested the potential bactericidal effect of the volatile constituents of the BNEO.

PHOTO (COLOR): Figure 2. Evaluation of different concentrations (ppm) of essential oil E1 against R. solancearum RS-09-100 growth by dual application assay. A- E1 oil [i-50 ppm, ii- 100 ppm, iii- 200 ppm, iv- 400 ppm and v- 800 ppm]. B- Control (i- diluent, ii- streptomycin 5 ppm respectively.

Since plants are prone to phytopathogen attack, it is speculated as reason for the relatively less effectiveness of the plant origin compounds against phytopathogens in comparison to that against human and animal pathogens. However, in a detailed analysis of the pitfalls in methods used for determining the antimicrobial activity of plant extracts,[[29]] indicated the use of wrong methods or the wrong plants as reasons for less effectiveness. In order to test the antimicrobial activity of phyto-extracts, agar diffusion technique is identified to have limited value mainly due to the relatively less polar nature of the antimicrobial compounds of the plant extracts, which interferes with their diffusion in the agar matrix used in the assay. The relatively less effectiveness of the BNEO in comparison to streptomycin (positive control) in the poisoned food assay performed on the agar matrix is therefore, speculated to be an interference in the diffusion of the antimicrobial compounds. In this context, the disk diffusion method is reported as the best alternative in determining the antimicrobial activity of plant extracts.[[30]]

The MICs of the antimicrobial compounds or plants extracts are usually determined by the dilution methods, which serve as reference methods in the antimicrobial susceptibility testing. Besides resistance surveillance, MICs are used to determine the extent of susceptibility of the pathogenic microbes. In the broth microdilution assay, the BNEO exhibited significant antibacterial activity only at higher concentrations (≥500 µg mL−1). At 500 µg mL−1 concentration, BNEO showed 80.4% bacterial growth inhibition over control (Table 1), which was on par with that of streptomycin (5 µg mL−1).

Table 1. In vitro evaluation of BNEO against growth of R. solanacearum RS-09-100 by broth microdilution assay.

1 Average of three replications

In the testing of any EO, the turbidity of oil-water emulsions is known to interfere with the determination of end points.[[31]] Hence, the broth microdilution method often employs an indicator to determine cell viability. The resazurin microtitre-plate assay is a simple, rapid, robust, yet a sensitive and effective method to give response for the presence of any viable cell by changing the blue color of the oxidation-reduction indicator resazurin to pink due to the action of oxidoreductase enzymes present in the viable cells.[[32]] This assay was therefore, employed to get the minimum inhibitory concentration (MIC) of BNEO at which no bacterial cell remained viable. Figure 3 suggested a complete conversion of color from blue to pink in negative controls (C2 and C3), unlike that of the sterile control (C1). In presence of BNEO, the maximum concentration at which color change occurred was found to be 512 µg mL−1 (T9) (Figure 3), and thus the MIC was determined as 512 µg mL−1. In the positive control (C4) with streptomycin, no change of color was observed for all the tested concentrations. Thus, this study clearly established the relevance of MIC as essential tool required for confirmation of the antimicrobial activity. Though the potential broad-spectrum antifungal activity of BNEO has been reported in literature in management of soil-borne pathogens[[33]] still no reports is available on the MIC of the BNEO. Thus, the study qualifies as the first report on potential activity of BNEO against highly virulent strain of R. solanacearum BI001.

PHOTO (COLOR): Figure 3. Minimal Inhibitory Concentration (MIC, ppm) of Essential oil E1 against R. solancearum RS-09-100 (0.25X 108 CFU/mL) by resazurin (Himedia) assay (Sarker et al. 2007). The color development observed after 24 h of incubation at 28 ± 2 ̊ C [Pink color indicates microbial growth and blue indicates no growth], each well contain liquid Kelman media + resazurin 0.7%.

The BNEO treated soil demonstrated higher ammonium-N content (Figure 4a) as compared to urea alone during the entire period of incubation. The respective ammonium-N content for all the BNEO treated samples were 46.66 to 71.32 and 26.44 to 36.67 mg kg−1 as compared to urea control, 44.86 and 16.23 mg kg−1 on the 7th and 14th day. Higher ammonium-N content in BNEO treated samples clearly indicated its antibacterial potency against Nitrosomonas species. The antibacterial activity of BNEO increased with the increase in its dose of application from 25 to 400 mg kg−1 Urea-N. The highest ammonium-N content, 71.32 mg kg−1 were observed in T5 (400 mg kg−1 Urea-N) which gradually decreased to the lowest 46.66 mg kg−1 for T1 (25 mg kg−1 Urea-N). The highest amount of ammonium was observed on initial days and decreased with incubation period; the lowest amount of ammonium was reported by urea control soil sample on the 14th days of incubation.

PHOTO (COLOR): Figure 4. Effect of BNEO on (a) ammonium-N production on 7th and 14th day, (b) nitrite-N production on 7th and 14th day (c) nitrate-N production on 7th and 14th day.

The nitrite-N content was low and ranged from 2.48 to 9.23 mg kg−1 for the treatments (Figure 4b). The nitrite-N contents were 2.37-3.79 for the urea alone. Significantly lower contents of nitrite-N suggested that the MEO did not have anti-bacterial activity against the Nitrobacter species. The antibacterial activity against Nitrobacter spp. could have led to the accumulation of nitrite-N. It also suggested the selective inhibition of the activity of Nitrosomonas spp. in soil by the test chemical.

Effect of different doses of BNEO to inhibit nitrification was evident from the maintenance of a lower amount of nitrate-N as compared to sample treated with urea alone throughout the incubation studies. The amounts of nitrate-N among the soil treated sample or soil without treated with BNEO are shown in Figure 4c. Concentration of nitrate-N in urea control (without BNEOs) increased during the incubation period to a maximum value of 159.19 mg kg−1 on 14th day of incubation. The rate of production of nitrate-N in BNEO treated samples were much lower as compared to urea control.

BNEO was thus able to effectively inhibit the activity of Nitrosomonas spp. and consequently the nitrification process (Figure 5). Percent nitrification inhibition for BNEO applied at different doses was 12-65% on 7th day of the incubation and 13-30% on 14th day of incubation and it was in a dose-dependent manner. The highest percent nitrification inhibition (30-65%) was observed with the BNEO applied at 400 mg kg−1 of Urea-N.

PHOTO (COLOR): Figure 5. Effect of BNEO on nitrogen dynamics in soil.

Although synthetic chemicals are able to inhibit nitrification for a longer period of time[[34]] but at a much higher concentration in comparison to natural products.[[36], [38]] Different materials derived from neem (Azadirachta indica, A. Juss), karanja (Pongamia glabra, Vent), mint (Mentha spicata, Mentha arvensis L.), and mahua (Madhuca longifolia, L.) have been reported effective against nitrifiers.[[7]] The utility of various neem products in improving the nitrogen use efficiency of prilled urea in different crops has been exhaustively studied.[[36], [38]] These experiments had led to the development of neem oil coated urea in which neem oil was used for coating urea for enhancing its nitrogen use efficiency. Products based on olive mill wastewater, hydroalcoholic extracts of Mentha piperita L. and Artemisia annua L., were also reported to be natural alternative for synthetic nitrification inhibitors.[[39]] These studies established the utility of natural products in reducing the activity of nitrifying bacteria. The present study for the first time has established potential of BNEO as nitrification inhibitory plant product and will be utilized to develop new generation fertilizer coats.

Three receptor proteins of R. solancearum and Nitrosomonas spp., each, were screened against AITC (Figure 6). The binding energies of the screened AITC-target complexes are given in Table 2. The in-silico activity in decreasing order followed the trend: AITC-glmU > AITC-brg11 > AITC -ppnP (R. solancearum); AITC-amoB1 > AITC-amoA1 > AITC-petC (Nitrosomonas spp.).

PHOTO (COLOR): Figure 6. 3-D and 2-D representation of the AITC bound target sites of proteins constituting the AMO Complex. A) amoA1 B) amoB1 C) petC and D) Best docked pose in amoB1 cavity site (Highest Inhibition).

Table 2. Molecular docking results of AITC and the target proteins of R. solanacearum and N. europeaea studied in silico.

2 Hbond is Hydrogen Bond energy - (lower the better); Hphob is the hydrophobic energy in exposing a surface to water (lower the better); VwInt is the van der Waals interaction energy (sum of gc and gh van der waals). Current version of the score uses explicit van der Waals interaction energy calculation (no grids). (lower the better); Eintl is internal conformation energy of the ligand. (lower the better); Dsolv is the desolvation of exposed h-bond donors and acceptors (lower the better); SolEl is the solvation electrostatics energy change upon binding (lower the better)

The predicted activity of AITC against the target proteins in R. solancearum clearly represents multi-modal action (Figure 7), as it is in the case of streptomycin, an antibiotic that is commonly used against R. solancearum. The activity of the streptomycin, against R. solancearum, showed stronger interactions (-24.391, −19.857, −20.723 kcal mol−1) with glmU, brg11 and ppnP, respectively (Table 3). In addition, the binding energy results clearly show the favorable binding of the molecule to the brg11-TAL receptor protein (Figure S6). This is in contrast to the selectivity shown by AITC in its binding to the N-terminal of the glmU receptor protein. This suggests that AITC may be inhibiting the conversion of N-acetylglucosamine-1-phosphate (GlcNAc-1-P) to UDP-N-acetylglucosamine (UDP-GlcNAc). Molecular interactions in the ligand-protein complexes (Tables 4 and 5) revealed that a diverse nature of bonds was responsible for the binding of the ligand in the target pocket. Three types of H-bonding, viz., conventional, carbon-hydrogen and π-donor were found to be present along with two types of hydrophobic interactions (alkyl and π-alkyl) and one type of electrostatic interaction (π-cation) (Figure 7). Very close binding energies of the ligand-protein complexes are an attestation to the fact that AITC acts on the potential target sites in a way that points toward the hypothesis that the mechanism of action of AITC is multi-modal in nature.

PHOTO (COLOR): Figure 7. 3-D and 2-D representation of the AITC bound target sites of Ralstonia spp. proteins, A) brg11-TAL effector protein, B) Bifunctional protein GlmU, C) Pyrimidine/purine nucleoside phosphorylase (ppnP) and D) Best docked pose in glmU cavity site (Highest Inhibition).

Table 3. Molecular docking results of streptomycin and the target proteins of R. solanacearum studied in silico.

Table 4. Molecular interactions detail between AITC and the R. solanacearum target proteins.

Table 5. Molecular interactions between streptomycin and the R. solanacearum target proteins.

Talking about the predicted nitrification inhibition activity of the compounds containing C = S, which comprise speciality class of powerful N. europaea ammonia oxidation inhibitors.[[40]] CS2 (CDS), thiourea (TU), and allylthiourea (ATU) are three compounds that may be termed as the best known among these compounds. It has been argued that functional groups primarily dictate the mechanism for these inhibitors, which is why, the existence of a C = S bond in all of the above compounds is critical for their activity as ammonia monooxygenase (AMO) inhibitor. Therefore, a mechanism proposed for one compound should be generally applicable to all the compounds of this category. Keeping this assumption in place, AITC (with an active –N–C = S moiety) was subjected to molecular docking in order to understand the molecular mechanism toward the three proteins of the AMO enzyme complex and its similarity/dissimilarity with the established AMO inhibitors (Figures 8–10). The results for binding with amoA1, amoB1, and petC suggested the following trends: TU (-16.021 kcal mol-1) > AITC (-14.91 kcal mol-1) > ATU (-13.059) > CDS (-7.383 kcal mol-1); ATU (-20.226)> TU (-19.923) > AITC (-15.33) > CDS (-5.275); ATU (-14.899) > AITC (-11.66) > TU (-11.647) > CDS (-7.397), respectively (Table 6).

PHOTO (COLOR): Figure 8. 3-D and 2-D representation of the TU bound target sites of proteins constituting the AMO Complex. A) amoA1 B) amoB1 C) petC and D) Best docked pose in amoB1 cavity site (Highest Inhibition).

PHOTO (COLOR): Figure 9. 3-D and 2-D representation of the CDS bound target sites of proteins constituting the AMO Complex. A) amoA1 B) amoB1 C) petC and D) Best docked pose in petC cavity site (Highest Inhibition).

PHOTO (COLOR): Figure 10. 3-D and 2-D representation of the ATU bound target sites of proteins constituting the AMO Complex. A) amoA1 B) amoB1 C) petC and D) Best docked pose in amoB1 cavity site (Highest Inhibition).

Table 6. Molecular docking results of allyl thiourea, thiourea and carbon disulfide binding to the ammonium monooxygenase of N. europeaea.

Clearly, AITC as a new entry to the folds of already established nitrification inhibiting compounds performed quite satisfactorily (2nd best in binding to amoA1 and petC). In contrast to CDS, TU inhibits the activity of AMO completely.[[40]] Interestingly, as per the in-silico predictions of the present study, in comparison to TU's fully inhibitory effects, AITC showed similar/higher binding to the protein subunits of the AMO complex. This is in contrast to CDS's mode of action, in which AMO functioning is significantly slowed but not defuncted. Allosteric inhibition or a diversion of the catalytic reaction to a slower, less effective pathway may result in incomplete inhibition.[[41]] The molecular modeling findings suggest that the mechanisms of those two compounds (AITC and CDS) are distinct and the link between the functional group and the inhibitor's mechanism is quite complicated t. In contrast to CDS, AITC and TU inhibit the activity of AMO completely. A likely mode of action for thiourea-based compounds involves their ability to tautomerize and form reactive thiols.[[42]] Our in-silico findings show that the effects of AITC are substantially similar or superior to those seen in the known potent sets of nitrification inhibitors screened. The chemical basis of distinction between different inhibitors is described in Tables 7 and 8. Our analysis results showed that the major difference between the binding of AITC and CDS seemed to be the presence of π-sulfur bond in the case of the latter. The participation of the partial positive charge on the nitrogen of –N–C = S moiety of AITC in the formation of a strong conventional H-bond with ARG 109, TYR 341, and TYR 119 of amoA1, amoB1, and petC, respectively, comprised a unique feature of AITC binding to the AMO complex.

Table 7. Molecular interactions detail between AITC and the N. europeaea target proteins.

Table 8. Molecular interactions detail of the complexes formed between three proven nitrification inhibitors (TU, ATU and CDS) and the N. europeaea target proteins.

The BNEO exhibited significant antibacterial activity at 500 µg mL−1 with 80% bacterial growth inhibition over control against R. solancearum. BNEO was able to effectively inhibit the activity of Nitrosomonas spp. with 30-65% nitrification inhibition at the dose of 400 mg kg−1 of Urea-N. The molecular modeling studies revealed that the inhibitory action of AITC on Nitrosomonas spp. and Ralstonia solanaceraum was multi-modal in nature. The study presents novel perspective on chemistry behind the cumulative efficacy of AITC on the AMO complex. The results of in-silico and in-vitro studies against soil borne bacteria, R. solanacearum and Nitrosomonas spp., revealed a unique connection in the form of efficacy, with varied degree of effectiveness. Wet lab enzymatic assays need to be conducted to validate the findings presented here and to develop ready-to-use BNEO based multicomponent formulation(s) augmented with other bioactives for development of next generation biobactericides.

Essential oil extracted from black mustard [Brassica nigra (L.) Koch] seeds (BNEO) was provided by Moksha Lifestyle ProductsTM (New Delhi, India) and used in the study without further purification. Allyl isothiocyanate (AITC) was the principal constituent (87.3%) of BNEO.

A highly virulent Ralstonia solanacearum strain BI001 responsible for causing bacterial wilt in tomato was obtained from Indian Type Culture Collection of Division of Plant Pathology, ICAR-Indian Agricultural Research Institute (ICAR-IARI), New Delhi, India and used as the test pathogen. A loopful of the bacteria was streaked onto Triphenyl Tetrazolium Chloride Agar (TZC, 0.1% casein, 1% peptone, 0.5% glucose and 1.7% agar agar amended with 0.005% v/v 2,3,5-triphenyl tetrazolium chloride) in Petri dishes and the dishes were incubated at room temperature (28-30 °C). Virulent bacterial colonies developed after 48-72 h of incubation as irregularly round, fluidal, creamy white clusters with light pink centers were sub cultured to obtain single colonies. The single colonies were subsequently cultured in Kelman's broth (Kelman, 1954) for 16 h (O.D = 1.2) in an orbital shaker (180 rpm) at room temperature (28-30 °C). The bacterial cells were then washed free of medium (5000 rpm 20 min−1) and re-suspended in same volume of sterile distilled water. The pathogenicity of the purified bacterium was then established as described [[3]] with modifications by dipping injured roots of apparently healthy seedlings of 30-days old tomato plants (cv. Pusa Rohini) grown under controlled conditions in the bacterial suspension (1 × 108 cfu mL−1) for 2 h. The treated plants were then transplanted in individual 20 cm diameter and 20 cm depth earthen pots containing 6 kg of steam sterilized (121 °C, 30 min for two consecutive days) soil and were maintained in a polyhouse for a week. Plants inoculated with sterilized water served as a control.

Antibacterial activity of the BNEO was initially assessed by poisoned food technique on solid Kelman medium. Calculated quantities of BNEO was aliquoted from a stock of 10,000 µg mL−1 and added separately in Kelman-TTC media to get 62.5, 125, 250, 500 and 1000 µg mL−1 concentrations to and was poured into sterile Petri dishes of 45 mm diameter. A drop (10 uL) of RS-09-100 culture (1.5 × 108 CFU mL−1) was placed at the center of each plate and the inoculated plates were incubated at 28 ± 2 °C for 48 h. The experiment was carried out in completely randomized design and the treatments and control were replicated thrice.

The effect of BNEO as a volatile entity was investigated using inverted Petri plate method.[[43]] Kelman media was poured in Petri plates and the bacterial suspension was spread on it. Different doses of BNEO (1, 2, 4, 8 and 16 uL per Petri plate which is equivalent to 50, 100, 200, 400 and 800 µg mL_SP_−1_sp_, respectively) were soaked in pre-sterilized filter paper disks and were placed into the inner surface of Petri plate lids. The inoculated plates were incubated at 28 ± 2 °C and the bacterial growth was observed at 48-72 h. The experiment was carried out in completely randomized design and the treatments and control were replicated thrice.

The antibacterial efficacy of BNEO was also confirmed by broth microdilution assay. Liquid Kelman media (90 uL) was pipetted in each well of a sterile 96 well plate. BNEO stock emulsion was poured into the plates by two-fold serial dilution technique in descending order (125, 250, 500, 1000 and 2000 µg mL_SP_−1_sp_) in such a way that each well received 50 μL of the test emulsion. Finally, 10 μL of bacterial suspension (1 × 108 cfu/mL) was added to each well to achieve a concentration of approximately 6.6 × 105 cfu/mL and a total volume of 150 μL. The plates were prepared in triplicates and incubated at 28 ± 2 °C for 24 h. After incubation, the absorbance of each well was taken at 600 nm using ELISA plate reader (BIOTEK EL X 800 – MS). The bacterial suspension in plain Kelman media was used as control.

To identify the minimum inhibitory concentration (MIC) of BNEO, a modified resazurin microtiter-plate assay method was adapted with slight variations.[[31]] Briefly, 80 μL of liquid Kelman media and 10 μL of resazurin indicator solution (0.7%) was pipetted in each well of a sterile 96 well plate. BNEO stock emulsion was poured into the plates by two-fold serial dilution technique in descending order in such a way that each well received 50 μL of the test emulsion. Finally, 10 μL of bacterial suspension (1 × 108 cfu/mL) was added to each well to achieve a concentration of approximately 6.6 × 105 cfu/mL and a total volume of 150 μL. A set of controls was also maintained in a plate, viz. C1: sterile control without addition of test emulsion and bacterial suspension, complemented with 60 μL of liquid Kelman media instead; C2: a negative control without addition of test emulsion, complemented with 50 μL of liquid Kelman media instead; C3: a negative control with a surfactant solution (Atlas G5002, 2% w/v) instead of test emulsion; and C4: positive control with the addition of streptomycin suspension (5 µg mL−1) instead of test emulsion. The plates were prepared in triplicates and incubated at 28 ± 2 °C for 24 h. Visual assessment of any color change from purple to pink (or colorless) was recorded as positive. The lowest concentration at which color change occurred was noted as the MIC value. MIC value has been interpreted as the lowest concentration of the test emulsion that did not show any fungal growth.

Test chemical, BNEO were evaluated for its effect on nitrifying bacteria in a soil incubation study under laboratory conditions. The soil was collected from the 0-15 centimeter surface layer of institute's experimental farm, New Delhi, India. The experiments were conducted following a completely randomized design (CRD) with three replicates.[[34]] Fertilizer-N was applied @ 200 mg kg−1 urea-N in each sample. For each set of treatments, a control (treated with only 200 mg kg−1 urea-N without the test chemical) was taken. Finely grounded sieved soil sample (100 g) was taken in 250 mL of polyethylene plastic beakers. The ccalculated amount of urea-N solution was added to each beaker to get soil containing 200 mg urea –N kg−1 followed by application of different doses 25 (T1), 50 (T2), 100 (T3), 200 (T4) and 400 (T5) mg kg−1 urea –N) of BNEO.

Distilled water was added to each beaker to bring the moisture to one-half of the water holding capacity of the soil and mixed thoroughly. All the beakers were accurately weighed, labeled, and incubated in a biological oxygen demand (BOD) incubator at 27 ± 1 °C, and 98% RH. For maintaining the moisture of incubated soil samples throughout the study, the required volume of distilled water was added in each alternate day and mixed well. Samples were drawn on 7th, and 14th day of incubation. Five grams of soil was withdrawn from the beaker and extracted with aqueous sodium sulfate (50 mL, 1 M) by shaking on a reciprocal shaker for one hr. The extracted soil samples were filtered and estimated for ammonium, nitrite and nitrate-N by indophenol blue, sulfanilic acid, and phenol-disulfonic acid methods respectively using a spectrophotometer (LABMAN, model- LMSP-UV1000B, serial no. L1_8256). The contents of ammonium, nitrite, and nitrate-N were obtained from the standard curves and expressed in milligrams per kilogram. The nitrification rate (NR) and percent nitrification inhibition (NI) were calculated using Sahrawat's formulas for assessing the effectiveness of test chemicals on nitrifying bacteria, Nitrosomonas and Nitrobacter spp. After sampling, incubation was continued in the same beaker.

To understand the mechanism of BNEO's anti-bacterial potential, its major volatile constituent, AITC, was subjected to in silico molecular docking for three putative target proteins of R. solanaceae and Nitrosomonas spp.

Three target proteins viz., amoA1, amoB1 and petC, which together constitute the Ammonia Monooxygenase (AMO) complex, were chosen as targets for the in-silico evaluation of AITC as a nitrification inhibitor against Nitrosomonas spp. The functions of the selected targets are described in Table 9. Similarly, three targets, brg11-TAL effector protein, bifunctional protein GlmU, and pyrimidine/purine nucleoside phosphorylase were shortlisted as potential targets (responsible for plant pathogenicity) for screening AITC for anti-bacterial activity against R. solancearum, whose functions are described in Table 9.

Table 9. Functional targets for the selected bacteria.

The secondary structures of selected bacterial protein targets were homology modeled using protein sequences downloaded from the NCBI genbank and UNIPROT databases, assisted by homologous templates available at NCBI and RSC PDB Protein Data Bank databases (Figure S1). The query sequence's molecular and biological feature was searched and annotated using the BLAST servers (http://blast.ncbi.nlm.nih.gov). The NCBI Blast tool was used with the PDB database to find templates for modeling the query sequences' secondary structures. For homology modeling of the three-dimensional structures of all the target proteins, the Modeler software (version 9.24) was used. ProSA-Web was used to calculate an overall quality score which can be used to check whether the z-score of the input structure is within the range of scores typically found for native proteins of similar size (Figures S2 and S3). In order to assess the stereochemical validity of the modeled protein, Ramachandran Plot analysis was carried out using PROCHECK-Saves (Figures S4 and S5). The Ramachandran plot showed the phi-psi torsion angles for all residues in the ensemble.

The ligand's (AITC) molecular structure was prepared as per the previously reported method.[[44]] Hydrogen molecules were added, and the incomplete side chains were substituted using the Dunbrack rotamer library.[[45]] Charges were measured using ANTECHAMBER, with regular residues receiving AMBER ff14SB charges and other residue forms receiving Gasteiger charges. The receptor molecules were prepared with the same methods but without the addition of ANTECHAMBER. The developed ligand and receptors were further saved in their respective.mol2 and.pdb before docking studies.

The Autodock Tools 1.5.7rc1 software was used to convert the protein structures to the pdbqt format[[46]] The target proteins were docked to Allyl isothiocyanate (AITC) using AutoDock Vina (version 1.1.2) software and a 30 30 30 (x, y, z) grid box that was continuously moved 10 in the x, y, and z directions to cover the three-dimensional structures of the enzymes[[47]] All parameters were left at their default values, with the exception of the exhaustiveness parameter, which was set to 32.

The authors are thankful to Director, ICAR-IARI, New Delhi for support and encouragement during the course of this investigation. A highly virulent strain of the R. solanaceraum culture provided by Dr. R. Ramesh ICAR - Central Coastal Agricultural Research Institute, Goa to verify the results obtained with that of the ITCC strain BI001 used in this study is gratefully acknowledged. This work was supported by the Indian Council of Agricultural Research-Niche Area of Excellence Programme (ICAR-NAE) (Grant number: Edn. 5(17)/2017-EP&HS), Ministry of Agriculture & Farmers Welfare, Government of India, India.

Abhishek Mandal and Anupama Singh designed the research; Abhishek Mandal, Adil Siddiqui and Amrendra Chaudhary conducted the experiments; Aditi Kundu, Anirban Dutta, Supradip Saha, Sukanta Dash and Anil Kumar analyzed the data; Abhishek Mandal, Rajesh Kumar and Veerubommu Shanmugam wrote the manuscript.

The authors declare that they have no conflict of interest.

Graph: Figure A1. Homology modelled structures of the proteins studied as Nitrosomonas target sites (Top row) and Ralstonia target sites (Bottom row): (A) amoA1, (B) amoB1, (C) petC, (D) brg11-TAL effector protein, (E) Bifunctional protein GlmU, (F) Pyrimidine/purine nucleoside phosphorylase (ppnP).

Graph: Figure A2. ProSA-web overall model quality for the recognition of errors in three-dimensional structures of proteins constituting the AMO Complex of Nitrosomonas. (A) AMOA1, (B) AMOB1, (C) petC. The negative z-scores suggests overall satisfactory modelled protein quality.

Graph: Figure A3. ProSA Z-scores for the (A) brg11-TAL effector protein, (B) Bifunctional protein GlmU, and (C) Pyrimidine/purine nucleoside phosphorylase (ppnP). The z-scores indicates overall model quality of the modelled proteins were quite satisfactory.

Graph: Figure A4. Ramachandran Plot for the homology modelled three-dimensional structures of proteins constituting the AMO Complex. (A) AMOA1, (B) AMOB1, (C) petC. The colouring/shading on the plot represents the different regions: the darkest areas (here shown in red) correspond to the "core" regions representing the most favourable combinations of phi-psi values.

Graph: Figure A5. Ramachandran Plot for the homology modelled three-dimensional structures of the modelled Ralstonia spp. proteins, (A) brg11-TAL effector protein, (B) Bifunctional protein GlmU, and (C) Pyrimidine/purine nucleoside phosphorylase (ppnP).

Graph: Figure A6. 3-D and 2-D representation of the streptomycin bound target sites of Ralstonia spp. proteins, (A) brg11-TAL effector protein, (B) Bifunctional protein GlmU, (C) Pyrimidine/purine nucleoside phosphorylase (ppnP) and (D) Best docked pose in brg11 cavity site (Highest Inhibition).