Bioinspired palladium-doped manganese oxide nanocorns: a remarkable antimicrobial agent targeting phyto/animal pathogens

Microbial pathogens are known for causing great environmental stress, owing to which emerging challenges like lack of eco-friendly remediation measures, development of drug-resistant and mutational microbial strains, etc., warrants novel and green routes as a stepping stone to serve such concerns sustainably. In the present study, palladium (Pd) doped manganese (II, III) oxide (Mn3O4) nanoparticles (NPs) were synthesized using an aqueous Syzygium aromaticum bud (ASAB) extract. Preliminary phytochemical analysis of ASAB extract indicates the presence of polyphenolics such as phenols, alkaloids, and flavonoids that can act as potential capping agents in NPs synthesis, which was later confirmed in FTIR analysis of pure and Pd-doped Mn3O4 NPs. XRD, Raman, and XPS analyses confirmed the Pd doping in Mn3O4 NPs. FESEM and HRTEM study reveals the mixed morphologies dominated by nanocorns appearance. Zeta potential investigation reveals high stability of the synthesized NPs in colloidal solutions. The developed Pd-doped Mn3O4 NPs were tested against two fungal phytopathogens, i.e., Sclerotinia sclerotiorum and Colletotrichum gloeosporioides, known for causing great economic losses in yield and quality of different plant species. The antifungal activity of synthesized Pd‐doped Mn3O4 NPs displayed a dose‐dependent response with a maximum of ~92%, and ~72% inhibition was recorded against S. sclerotiorum and C. gloeosporioides, respectively, at 1000 ppm concentration. However, C. gloeosporioides demonstrated higher sensitivity to Pd‐doped Mn3O4 NPs upto 500 ppm) treatment than S. sclerotiorum. The prepared NPs also showed significant antibacterial activity against Enterococcus faecalis. The Pd-doped Mn3O4 NPs were effective even at low treatment doses, i.e., 50–100 ppm, with the highest Zone of inhibition obtained at 1000 ppm concentration. Our findings provide a novel, eco-benign, and cost-effective approach for formulating a nanomaterial composition offering multifaceted utilities as an effective antimicrobial agent.

Metal oxides such as ZnO, CuO, TiO 2 and MnO etc. have also a great potential to have excellent antimicrobial activity 19 .Chamaecostus cuspidatus extract is used to green synthesis CeO 2 and ZnO nanoparticles (NPs) and effective antibacterial activities.The anticancer effects of CeO 2 and ZnO nanoparticles were investigated in human breast cancer cell lines 21 .Similarly, Green synthesis is used to prepare Cerium oxide nanoparticles (CeO 2 NPs) from Artabotrys hexapetalus leaf extracts.The prepared NPs exhibit excellent antibacterial activity against a variety of bacterial species.The anticancer potential of the compound was studied against the (MCF-7) human breast cancer cell line 22 .In addition to that, Zinc oxide nanoparticle (ZnO NPs) was prepared utilizing starch in a single step green synthesis and had highly porous, novel hollow multi-sphere in morphology.Because of their morphology and porosity, the synthesized ZnO NPs can be employed in a variety of drug delivery applications 19 .Mn has been reported as the transition element with the third highest abundance on earth followed by iron and titanium 30 .Among various 3d transition metal-oxides, Mn-oxides (MnO, MnO 2 , Mn 2 O 3 , Mn 3 O 4 , and Mn 5 O 8 ) have obtained key attention owing to their compositional and structural diversity 24,31 .Mn-oxides NPs also possess structural adaptability with varying physicochemical qualities 32 .Mn-oxide NPs have excessive potential for sustainable-nanotechnology research and innovation 24,30 .Mn-oxides can have applications in optoelectronics, magnetic storage devices, imaging contrast agents, magnetic materials, drug delivery, catalysis, wastewater treatment, solar cells, etc. 24 .
Sclerotinia sclerotiorum is a necrotrophic phytopathogen that harbors a broad host range and causes stem rot disease in different crops including soybean, oilseed rape, sunflower, tomato, etc., resulting huge losses of agricultural produce 33 .On the other hand, C. gloeosporioides follows the hemibiotrophic infection mode and is known for causing anthracnose in fruits like papaya, mango, avocado, apple, guava, banana, papaya, cashews, grapes, pitaya, etc., resulting in serious postharvest losses 34,35 .Although chemical fungicides have been utilized for their control, their indiscriminate utilization has serious environmental consequences that necessitate the search for novel and eco-friendly alternatives 36 .E. faecalis is known to colonize the human intestine, and its occurrence in aquatic bodies implies fecal contamination 37 .These bacteria have been reported as multidrugresistance microbial pathogens associated with hospital-acquired infections 37,38 .

Preparation of S. aromaticum bud extract.
The procured flower buds were thoroughly washed with distilled water to remove dirt particles and dried at 40 °C for 48 h.The dried flower buds were homogenized into a fine powdered form and stored in an air-tight container until used.To prepare the extract, powdered flower buds were macerated in DDW (1:10, w/v) at 60 °C for 2 h.After cooling at room temperature, the aqueous solution of phytoextract was filtered using Whatman filter paper number 1 at stored at 4 °C temperature until use.
Green synthesis of pure and Pd-doped Mn 3 O 4 nanoparticles.The NPs were synthesized using a sol-gel method 39 , assisted by the addition of phytoextract as a potential source of capping agent (Fig. 1).Briefly, 2% (w/v) PdCl 2 was added to the aqueous solution of MnCl 2 .4H 2 O (49 mM) followed by drop-wise addition of 10 ml aqueous Syzygium aromaticum bud extract under continuous stirring at 650 rpm at 90 °C for one hour.After getting the light brown color of the solution, 25 mM aqueous solution of NaOH was added drop-wise to Antimicrobial activity of nanoparticles.The antifungal activity of NPs was tested against S. sclerotiorum and C. gloeosporioides using the poisoned food technique.The fungal cultures were procured from Indian Type Culture Collection (ITCC), Division of Plant Pathology, Indian Agriculture Research Institute, New Delhi, India.The potato dextrose agar (PDA) media was prepared for fungal growth with following composition: dextrose (2% w/v), potato starch (0.4% w/v), and agar (1.5% w/v).The pH of 5.6 ± 0.2 was adjusted using 0.1N KOH/0.1NHCl.The synthesized NPs were dispersed in PDA media to get the desired concentrations (up to 1000 ppm).The media containing NPs was poured into a Petri plate.At the next step, a ~ 8 mm piece of actively growing mycelia from 5 to 8 days old pure cultures of S. sclerotiorum and C. gloeosporioides were placed in the middle of each plate and incubated for five days at ~ 25 ± 2 °C and ~ 28 ± 2 °C temperature, respectively.The media plates without NPs treatment served as the negative control, and plates with 2 mg/ml of carbendazim + mancozeb were designated as the positive control.The % of growth inhibition was calculated using the below formula: The antibacterial activity of NPs was determined using agar disc diffusion assay (ADDA).The pure culture of E. faecalis was inoculated to freshly prepared nutrient broth media (NBM) and maintained at 37 °C for ~ 18 h.The composition of NBM was Yeast extract (0.2% w/v), Beef extract (0.1% w/v), Peptone (0.5% w/v), NaCl (0.5% w/v), and pH ~ 7.4 ± 0.2.2% (w/v) agar was added to the nutrient broth for preparing solid media plates (SPM).The pure culture of E. faecalis at the active log phase was uniformly spread on SPM.The sterile filter paper discs of about 6 mm diameter, each dipped in various concentrations of NPs (0 to 1000 ppm), were placed on SPM.The DDW-dipped discs were served as a negative control.The Petri plates were placed in an incubator overnight, and the ZOI was measured in millimetre.All the experiments on antimicrobial activity were performed in triplicates under aseptic conditions in a laminar airflow chamber.The nutrient media, glassware, and other utilities were autoclaved at 121 °C for 15 min at 15 psi pressure before use to maintain aseptic conditions throughout the assay.

Statistical analysis.
All of the experiments were completed in triplicates and recorded data presented as mean ± standard deviation using Microsoft Excel®.

Results and discussion
Green synthesis of nanoparticles and FTIR analysis.Pure and Pd-doped Mn 3 O 4 NPs were synthesized using an aqueous Syzygium aromaticum bud (ASAB) extract.Preliminary phytochemical analysis indicated the presence of phenols (ferric chloride test) 42 , flavonoids (lead acetate test) 43 , alkaloids (Wagner test), carbohydrates (fehling's test), and tannins (ferric chloride test) 44 .The results were in concordance with the findings of Jimoh et al. 43 , which established the suitability of the tested plant as a potential substrate for developing Phyto inspired nanoparticles [45][46][47] .Through FTIR analysis, Rajesh et al. 45 predicted the role of metabolites present in S. aromaticum bud extract, such as flavonoids, tannins, alkaloids, and carotenoids, in the green synthesis of CuNPs.We have compared the FTIR spectra of ASAB extract, pure and Pd-doped Mn 3 O 4 NPs to validate the capping and stabilizing potential of bioactive compounds present in phytoextract (Fig. 2).
(1) FTIR spectrum of ASAB extract revealed a broad band at ~ 3431.62 cm −1 , which corresponds to the OH group 45 , alkyl CH stretching (sp 3 ), and C-O ester group was observed at ~ 2931.95 and ~ 1711.92cm −1 , respectively 48 .A sharp peak at ~ 1611.99 cm −1 belongs to -C = C aromatic stretching vibrations and C = O stretching vibrations of proteins denoting amide linkages 45 .The aromatic groups were indicated at ~ 1511.36 cm −148 , while two separate peaks at ~ 1366.60 cm −145 and ~ 1051.25 cm −148 denoted the C-O group.These characteristic FTIR spectral peaks suggest for eugenol presence in ASAB extract 48 .The peak at ~ 640 cm -1 are attributed to the Mn-O in synthesized nanoparticles 49

Structural analysis.
The XRD confirms the crystalline structure of the prepared manganese oxide NPs with two distinct phases, as shown in Fig. 3 220), (312), (321), (224), and (400) crystal planes of Mn 3 O 4 phase respectively 50 .The above (hkl) planes correspond to the Hausmannite phase of the Mn 3 O 4 crystal structure (JCPDS 24-0734) 51 .The intensity of XRD peaks was decreased in the case of Pd doping, indicating peak shifting towards lower diffraction angles and crystalline lattice expansion 52 , suggesting the successful incorporation of Pd in Mn 3 O 4 .There is no shifting of XRD peaks in the case of Mn 3 O 4 NPs, indicating that as-synthesized NPs are comprised of the tetragonal Hausmannite phase 53 .The sharp peaks confirmed the highly crystalline nature of NPs 54 .There were no other peaks in the XRD pattern that demonstrated the phase purity of the produced NPs 55 .
The crystallite size of synthesized NPs were calculated by Scherrer's formula at a extremely intense peak (Eq.5), and the values were ~ 32 and ~ 28 nm for pure and Pd-doped Mn 3 O 4 NPs, respectively 56,57 .The Pd-doped  www.nature.com/scientificreports/Mn 3 O 4 NPs were smaller than the pure Mn 3 O 4 NPs, which could be owing to the fact that ionic radii of Pd (0.137 nm) are much larger than that of Mn (0.082 nm) 58 .
where D is crystallite size, λ is the wavelength, θ is Bragg's angle, and β is FWHM.where α, hν, E g , and B are the absorption coefficient, photon energy, the band gap energy, and constant, respectively.The value of index 'n' calculated from Tauc's Plot was 2 (Fig. 5).The estimated band gap was ~ 3.79 and ~ 3.75 eV for pure and Pd-doped Mn 3 O 4 NPs, respectively, which is in agreement with the previous reports for Mn 3 O 4 NPs 61 .It was noticed that the band gap decreased in Pd-doped Mn 3 O 4 NPs, compared to pure Mn 3 O 4 NPs.A red shift was observed in the band gap of Pd-doped Mn 3 O 4 NPs.This may be due to intermediate levels forming between the CB and VB of the host Mn 3 O 4 matrix 62,63 .Pd atoms act as an accepter to decrease the band gap of Mn 3 O 4 NPs 64,65 .Therefore, variation in the energy band gap of Mn 3 O 4 NPs by Pd doping may have applications in photocatalytic activity 66 .

Electronic states and elemental composition analysis.
The oxidation states of Pd-doped Mn 3 O 4 NPs were determined by XPS analysis 67 .The survey spectrum (Fig. 6a) revealed the presence of Pd, Mn, and O, confirming their existence in the product, i.e., Pd-doped Mn 3 O 4 NPs.Further analysis of the Pd 3d spectrum showed a doublet feature 68 , providing evidence of Pd species' presence in the material.The peaks observed at 332.17 eV and 335.56 eV in the Pd 3d 5/2 region, along with 340.57eV and 344.58 eV in the Pd 3d 3/2 region, corresponded to Pd (II) and Pd (IV) states 68 .Moving on to the Mn 2p core-level spectrum, two distinct peaks were observed at binding energies of 654.94 eV, 653.56 eV and 643.53 eV, 642.13 eV for pure and Pd-doped Mn 3 O 4 nanocorns, respectively.These peaks were associated with Mn 2p 1/2 and Mn 2p 3/2 in Mn 3 O 4 NPs and indicating a spin-orbital splitting of 11.4 eV (Fig. 6b) 69,70 .Additionally, the O1s spectrum peaked at 529.68 eV for pure Mn 3 O 4 NPs and 532.89 eV for Pd-doped Mn 3 O 4 NPs (Fig. 6d) 70 .This peak confirmed the presence of oxygen in both materials.The XPS analysis provided conclusive evidence that the prepared manganese oxide material was indeed Pd-doped Mn 3 O 4 NPs, with the oxidation states of Pd (II) and Pd (IV) and specific Mn 2p states characteristic of Mn 3 O 4 .
Transmission electron microscopy analysis.The TEM micrograph of the green synthesized Mn 3 O 4 NPs in Fig. 7a shows that the Mn 3 O 4 NPs were composed of nearly uniform types of particles.The SAED patterns of Mn 3 O 4 NPs in Fig. 7b displayed bright rings with some bright spots, suggesting the high crystallinity of the materials 71 .Figure 7c represents the high-resolution TEM images of the Pd-doped Mn 3 O 4 NPs, and the magnified calibrated lattice fringes of Mn 3 O 4 NPs for the crystal plane of ( 103) and ( 211) revealed an interplanar spacing (d-spacing) of 2.7 and 2.4 Å in Fig. 7d.These planes were also observed in the XRD analysis, and the reduction of the d-spacing of these planes is in good agreement with the shifting of the XRD peaks.Therefore, these findings indicate the successful formation of Pd-doped Mn 3 O 4 NPs, which was also consistent with the XRD patterns 72,73 .
Raman Spectroscopy analysis.Figure 8 presents the Raman spectra of pure and Pd-doped Mn 3 O 4 NPs.
Two characteristic peaks at ~ 629 and ~ 630 cm −1 were observed, corresponding to the skeletal vibrations for pure and Pd-doped samples, respectively.The strongest peaks at ~ 629 and ~ 630 cm −1 are consistent with the reported data 74 for both materials.These sharp peaks can be assigned to the A1g mode, representing the Mn-O breathing vibration of divalent manganese ions in tetrahedral coordination.This mode is a characteristic feature of Hausmannite 75,76 .The comparison of the Raman spectra between pure and Pd-doped Mn 3 O 4 NPs reveals similarities in the characteristic peaks, indicating that the introduction of Pd did not significantly alter the skeletal vibrations and Mn-O breathing vibrations in the tetrahedral coordination.The Raman spectra analysis of pure and Pd-doped Mn 3 O 4 NPs confirmed the presence of specific vibrational modes and provides key insights into the structural properties of these materials.The similarities in the Raman spectra between pure and Pd-doped samples suggested that the Pd-doping did not cause significant changes in the observed vibrational features.

Zeta potential studies.
A particle's surface potential substantially impacts its dispersion stability 77 , which may also influence its bactericidal potential 78 .Without proper surfactants or capping agents, nanoparticles tend to agglomerate, and their surface area-to-volume ratio decreases due to their increased size 79 .The zeta potential studies allow us to investigate nanoparticles' surface charge and stability in colloidal solutions 80 (Fig. 9).The surface charge of NPs can be influenced by the charged dopants 77 , which is also observed in the present study.We have obtained highly stable NPs (ZP > 30 mV) 81 , with recorded values of −33.2 ± 0.404 and −36.6 ± 1.74 mV for pure and Pd-doped Mn 3 O 4 , respectively.The NPs with greater ZP values (negative or positive) prevent agglomeration via electrostatic repulsion, hence conferring stability 80 .

Antifungal activity
The developed NPs showed mycelium growth inhibition in a dose-dependent manner.In the case of Mn 3 O 4 , NPs, we have observed maximum antifungal activity at 1000-ppm concentration with over 50% inhibitions in the growth of S. sclerotiorum and C. gloeosporioides was recorded at 500 ppm dose (Fig. 10).Overall, S. sclerotiorum exhibited higher sensitivity to the Mn 3 O 4 NPs treatment than C. gloeosporioides.The inhibition of mycelial growth in the case of Pd-doped Mn 3 O 4 NPs was higher than pure Mn 3 O 4 NPs against both fungal strains (Fig. 11).This could be due to the significant modification in structural properties of Mn

Antibacterial activity
The antibacterial activity of pure and Pd-doped Mn 3 O 4 NPs was also investigated against E. faecalis to establish their broad spectrum of antimicrobial potential, in terms of pathogen diversity, i.e., phytopathogens and animal pathogens.As stated earlier, E. faecalis is a well-known human pathogen known for hospital acquired infections.This bacterium colonizes the intestine of animals including humans, and its presence in waterbodies is an indicative of fecal contamination.The resistance of E. faecalis against various antibiotics necessitated the search of novel materials possessing significant antibacterial potential against such nosocomial pathogens.In the present work, both pure and Pd-doped Mn 3 O 4 NPs showed dose-dependent increment in ZOI (Fig. 12).The ZOI values showed effect of Pd doping on improving antibacterial activity of Mn 3 O 4 NPs.When compared to pure Mn 3 O 4 NPs, Pd doping showed ~ 14%, ~ 17%, and ~ 16% higher ZOI values at 50, 100, and 200 ppm doses of Mn 3 O 4 NPs respectively.

Mechanism of antimicrobial activity of Pd-doped Mn 3 O 4 NPs
The plausible routes of inducing antimicrobial activity by Pd-doped Mn 3 O 4 NPs are illustrated in Fig. 13 36,66,82 .The NPs in fungal cells usually gain entry via diffusion and endocytosis 83 and may cause growth inhibition through multiple actions such as DNA damage, protein denaturation, breakdown of the cell wall and cell membrane, ROS-mediated lipid peroxidation, ribosome disassembly, denaturation of enzymes, perforations in the cell wall and cell membrane, mitochondrial damage, release of cytochrome-c from mitochondria to cytosol, and increase levels of metacaspase and promotes cell death 36 .Similar effects have been proposed in the case of bacterial cells where NPs can cause protein and enzyme denaturation, damage to chromosomal and plasmid DNA, ribosomal depolymerization, interference in ETC, the release of cellular contents, disruption of the cell membrane, etc. 84,85 .
In general, NPs have direct and indirect effects on microbial cells.The direct damage occurs via the electrostatic interaction of NPs with cell membrane resulting in membrane depolarization and loss of membrane integrity leading to the disruption in ETC and cell lysis 39,86 .The indirect damage to microbial cells is reported via ROS generation (Eqs.7, 8, 9, 10, 11, 12) 39,87 .The doping in pure nanomaterial leads to lattice defects (alters band gap), causing overlapping of Fermi levels, variation in cellular redox potential, promotes ROS generation (Fig. 14) and can impart enhanced antimicrobial properties 39 , which was observed in the present study as well, where Pd-doped Mn 3 O 4 NPs showed higher antifungal and antibacterial activity compared to pure NPs.In addition, doping can improve the binding capacity and cellular internalization ability of NPs.NPs generate ROS outside the cellular environment or can produce it inside the cell due to interference in ETC 39 .The oxygen molecules that are not reduced in the water get oxidized into free radicals (such as superoxide anion, singlet oxygen, or hydroxyl radicals) in mitochondria (Eqs.7, 8, 9, 10, 11, 12) 39,88

Conclusion
The present investigation demonstrates a successful green chemistry approach to synthesis pure and Pd-doped

Figure 1 .
Figure 1.Schematic of the green synthesis of pure and Pd-doped Mn 3 O 4 NPs.

Figure 4 .
Fig.4a, b appeared to be rod-like nanostructures.In contrast, Fig.4d, e showed the surface of Pd doped-Mn 3 O 4 NPs with the likely appearance of nanocorn-like structures.The morphological changes from rod (Mn 3 O 4 NPs) to nanocorn-like nanocorn (Pd-doped Mn 3 O 4 NPs) could be owing to the decoration of Mn 3 O 4 with Pd 2+ .EDAX analysis revealed Pd, Mn, and O in their suitable stoichiometric proportion, as given in Fig.4c, f59 .

3 O 4
NPs as a result of Pd doping, such as the reduction in crystallite size and nanocorn-like morphology.At 1000 ppm concentration, Pd-doped Mn 3 O 4 NPs caused ~ 92% and ~ 72% growth inhibition of S. sclerotiorum and C. gloeosporioides, respectively.Interestingly, C. gloeosporioides was more sensitive to Pd-doped Mn 3 O 4 NPs treatment, specifically at lower doses, and showed ~ 65%, ~ 23%, and ~ 10% higher inhibition compared to pure Mn 3 O 4 NPs at 500, 100, and 10 ppm concentration, respectively.The Pd-doped Mn 3 O 4 NPs at 1000 ppm showed antifungal activity comparable to those of positive control (2 mg/ml of carbendazim + mancozeb; commercial chemical grade fungicide formulation).Hence, doping Mn 3 O 4 NPs with Pd favoured their antifungal potential 66 , which is highly explicitly recommended in the case of C. gloeosporioides to decrease the effective dose.Overall, the bioinspired fabrication of nanocorn-like Pd doped Mn 3 O 4 NPs can be used as an effective antifungal nano-pesticide against different necrotrophic and hemibiotrophic phytopathogens, known for causing enormous loss to agricultural food crops globally.

Mn 3 O 4
NPs via utilizing an aqueous extract of S. aromaticum buds.Adding Pd in Mn 3 O 4 resulted in significant changes to their structural attributes, including morphology, crystallite size, and lattice defects.The Pd-doped Mn 3 O 4 NPs exhibited antimicrobial activity in a dose-dependent manner and provides higher inhibitory effects than pure Mn 3 O 4 NPs against S. sclerotiorum, C. gloeosporioides, and E. faecalis.The outcome of this study provides a novel, cost-effective method to develop Pd-doped Mn 3 O 4 based nanomaterials for highly effective antimicrobial applications against tested microbial pathogens.This breakthrough opens up new possibilities in the area of green nanotechnology to develop sustainable and multifaceted antimicrobial agents.