3.1. Characterization of synthesized nanocomposites
The structural and crystalline attributes of the Cu:ZnS-lignocellulose nanocomposites (1 h- 5 h variants) and the native Cu:ZnS nanocomposite (2 h variant), were analyzed through FTIR and XRD techniques (Fig. 2a, b). In Fig. 2a, demonstrates the dominant absorption band at 545 cm− 1 and 976 cm− 1 corresponding to the ZnS vibration and resonance interaction between Cu and Zn respectively . Additionally the prominent peak at 1218 cm− 1 for the Cu:ZnS-lignocellulose nanovariants corresponds to CH2 vibrations at C-6 or O-C- stretching of cellulose I suggesting an interaction between lignocellulose and ZnS [20, 21]. The crosslinking between the -OH group of lignocellulose and ZnS through hydrogen bonding, is evident from the shifting of the characteristic band for the inter and intramolecular -OH stretching vibration (3401 cm− 1) for the Cu:ZnS nanocomposite to 3428 cm− 1 in Fig. 1b . The band observed at 1450 cm− 1 corresponds to the aromatic skeletal vibration of the phenolic group of lignin [23, 24]. Also, the characteristic signature for the presence of capping agent SC is represented by the band at 1601 cm− 1 which corresponds to the stretching vibration of the citrate group . Figure 2b depicts the XRD diffractograms of the Cu:ZnS-lignocellulose nanovariants (1 h – 5 h) and 2 h variant of Cu:ZnS nanocomposite for all the reflux variants. The diffractograms for Cu:ZnS and Cu:ZnS-lignocellulose nanocomposites display characteristic peaks at 28°, 47°, and 58° corresponding to (111), (220), and (311) diffraction plane respectively which are in good agreement with the cubic Zinc blende phase structure of ZnS for the synthesized variants . However, an additional peak at 34.5° corresponding to the (004) diffraction planes respectively was found in Cu:ZnS-lignocellulose nanovariants indicating major characteristic diffraction peaks of typical cellulose I structure . The lower intensity of peaks in the Cu:ZnS-lignocellulose nanovariants compared to Cu:ZnS signifies the reduction in crystallinity, owing to the crosslinking with the amorphous lignocellulose matrix [28, 29].
To investigate the chemical states of the elements and to characterize the functional groups present in lignocellulose XPS analyses were performed and the XPS results are depicted in Fig. 2c. The XPS survey spectra of the 2 h variant of Cu:ZnS-lignocellulose nanocomposite show the peaks of the elements Zn, Cu, S, C, and O. The spectrum shows the Cu 2p3 binding energy values at 934.03 eV for the presence of copper. The peak at binding energy 1021.08 eV and 168.93 eV corresponds to the Zn 2p and S 2p in the sulfide phase respectively, corroborating the existence of ZnS (Fig. 2c) . The constituent elements C and O for the lignocellulose component of the nanocomposite were confirmed with the presence of photoelectron peaks at binding energy 540.5 and 293.5 eV corresponding to O 1s and C 1s respectively (Fig. 2c). The elemental specific spectra of Cu have been depicted in Fig. 2d. The photoelectron peak at binding energy 934.24 eV and 954.20 eV of Cu spectra indicates the formation of Cu2+ oxidation state. However, the additional peak around 940 eV and 960 eV results from a shakeup process owing to the open 3d9 shell of Cu2+ . The elemental spectra for Zn show peaks at 1021.43 and 1044.65 eV corresponding to Zn 2p3/2 and Zn 2p½ belonging to the Zn2+ oxidation state (Fig. 2e) . The binding energies 168.4 eV and 169.6 eV) in Fig. 2f correspond to S 2p3/2 and S 2p1/2, in accordance with the binding energies for sulfur in metal sulfides (M-S), indicating the formation of ZnS in the nanocomposite . The high-resolution C 1s spectra were resolved into four individual component peaks at 284.3 eV, 285.8 eV, 286.2 eV, and 287 eV corresponding to –C-(C, H)/C = C (i.e. C singly-bound to C/H or doubly bound to C), -C-(O, N) (C bound to C or N), -C = O (C doubly bond to O) and O-C = O (C singly and doubly bound to O), respectively (Fig. 2g) . In contrast, the O 1s XPS spectra exhibit two deconvoluted peaks at 533.1 and 531.7 eV, signifying C = O and C–OH/C–O–C groups, respectively (Fig. 2h) . The existence of these functional groups in the O 1s XPS spectra is consistent with the FTIR results, indicating the presence of O-C- stretching vibrations supporting the crosslinking reaction scheme between lignocellulose and ZnS . The elemental composition of the 2 h variant of Cu:ZnS-lignocellulose nanocomposite obtained from XPS analysis are listed in Table S1.
The morphological structure of the surface of the 2 h variant of the synthesized nanocomposite was obtained with SEM-EDX analysis. The SEM micrograph for the 2 h variant of Cu:ZnS-lignocellulose nanocomposite (Fig. 3a) shows a quasi-spherical morphology. The existence of C, O, S, Cu, and Zn as major elemental constituents in the nanocomposite was confirmed from SEM-EDX studies (Fig. 3b) and the weight % and atomic % of the elemental compositions are listed in Fig. 3b inset. Pseudo-color elemental mapping for the 2 h variant was carried out wherein C, O, S, Cu, and Zn demonstrate a uniform distribution of the elements within the area under investigation (Fig. S1 a-e).
3.2. Effect of solvent pH on the hydrodynamic diameter and electrostatic stability of the synthesized nanocomposites
The particle size distributions and electrostatic stability of Cu:ZnS-lignocellulose nanocomposites under different solvent pH, were estimated using Photon Correlation Spectroscopy (PCS) to analyze the particle behaviour (Fig. 4a, b). From Fig. 4a. it is evident that there is no significant change in the hydrodynamic diameter of the different variants, across the pH range indicating their stability across pH 2–12. It has been reported that colloidal dispersions with a ζ potential of ≥ ± 20 meV are generally regarded stable . The ζ potential of the synthesized nanocomposites were found to be in the − 22 meV to -34 meV range (Fig. 4b), across the solvent pH 2–12. Here, the 2 h reflux variant was found to be the most stable with the highest ζ potential. Further, the sizes of the variants were well within the desirable range and is indicative of their well dispersed state in the solvent. The high electrostatic stability and stable hydrodynamic diameter of the dispersed nanocomposite could be attributed to the crosslinking of Cu:ZnS core to the lignocellulose matrix. This is likely to address the concerns of low water solubility, oxidation, and agglomeration associated with the conventional copper-based fungicidal formulations.
3.3. Antifungal assay
The as-synthesized Cu:ZnS-lignocellulose nanocomposites were initially evaluated for their antifungal efficacy by performing the disk diffusion assay. It is evident from the disk diffusion assays, that the nanocomposites when used at concentrations of 0.5 mg/ml, 0.8 mg/ml and 1 mg/ml, result in a significant concentration dependent inhibition of E. vexans hyphal growth (Fig. 5a). Among the nanocomposites, the 2 h variant of Cu:ZnS-lignocellulose shows the (Fig. 5b) maximal antifungal efficacy with the zone of inhibition measuring ~ 1.76 cm2. In comparative studies with the common commercial fungicides for blister blight control, viz. copper oxychloride and fluconazole, Cu:ZnS-lignocellulose nanocomposites also show better antifungal efficacy . The nanocomposites demonstrate the zone of inhibition for concentrations as low as 0.5 mg/ml, while copper oxychloride is effective only at a concentration of 4 mg/ml (Fig. 5b). On the other hand, fluconazole was unable to inhibit E. vexans growth even at the concentration of 4 mg/ml. Further, the MIC50 and MFC (48 h) values derived for the nanocomposite variants are depicted in Fig. 5c. The lowest MIC50 of Cu:ZnS-lignocellulose nanocomposites, which inhibited E. vexans growth by 50%, was achieved at concentration 0.05 mg/ml for the 2 h variant. Likewise, the lowest MFC value was at the concentration 0.25 mg/ml for the 2 h variant of Cu:ZnS-lignocellulose. In addition to this an activity index of the nanocomposite variants was calculated at a concentration of 4 mg/ml, based on the antifungal activity of copper oxychloride (Fig. 5d). A > 10-fold increase in the activity was observed for the 1 h − 4 h variants of the nanocomposites (Fig. 5d). Here the 2 h variant of Cu:ZnS-lignocellulose, was most potent as it exhibited a > 12 fold increase in activity against E. vexans.
3.4. Antisporulant activity
The basidiospores, as a propagative unit, considerably aid in the rapid proliferation and pathogenicity of E. vexans on host tea. Basidiospore germination being the first significant phase in blister blight infestation, its suppression is linked to inhibition of disease at the early stages. To further appraise the fungicidal efficacy, the 2 h Cu:ZnS-lignocellulose nanocomposite variant, having the highest activity index was investigated for its antisporulant activity. Figure 6 (a-d) shows the light micrographs depicting the germination of basidiospore after incubation with the 2h Cu:ZnS-lignocellulose nanocomposite suspensions and under control conditions. As expected, the control samples (basidiospores in sterile water) showed the germination of basidiospores with extensive hyphal growth 8 h post-incubation (Fig. 6a). The presence of the Cu:ZnS-lignocellulose nanocomposite resulted in arrested germination and hyphal growth of E. vexans basidiospores in a concentration dependent manner (Fig. 6b-d). As such, the highest concentration of 1 mg/ml, completely inhibited the E. vexans basidiospore germination, while also exhibiting sporicidal effects. Figure 6e depicts the calculated rate of inhibition of basidiospore germination when incubated with and without nanocomposites. An inhibition of 0%, 65%, 80% and 100% is evident for the concentrations 0, 0.5, 0.8 and 1mg/ml, respectively. To determine the efficacy of antisporulant activity a scale was introduced, based on the microscopic observations, to grade the activity wherein -, + and + + indicated no inhibition, partial inhibition, and full inhibition respectively. The score of inhibition for the 2 h variant of Cu:ZnS-lignocellulose nanocomposite at different concentrations against basidiospore of E. vexans is shown in Fig. 6f.
The 2 h variant of Cu:ZnS-lignocellulose nanocomposite was evaluated for its optical/fluorescent properties using UV-PL analysis. The absorption peak was obtained at a wavelength of 205 nm, while the emission peak appeared at the excitation wavelength of 390 and 425 nm (Fig. S2 a, b). The photoluminescence (PL) excitation spectra of the nanocomposite corroborated the fluorescent property of the variant and provided the avenue for the fluorescent microscopic studies of the nanocomposite. To investigate the effect of the nanocomposites on the E. vexans hyphal growth, the morphological alterations were microscopically monitored (light and fluorescent) in the presence of 2 h variant of Cu:ZnS-lignocellulose nanocomposites and control conditions. As can be seen in Fig. 6g the germinated hyphae from the control samples retain a complete and homogeneous tube-like morphology. Nanocomposite treatment of E. vexans basidiospores resulted in adverse morphological alterations starting with the deposition of the nanocomposites on the germ tube leading to membrane disruption is evident in the bright field and fluorescent micrographs (Fig. 6h-i). Figure 6j represents the merged image of bright field and fluorescent micrograph wherein we can confirm the membrane impairment of the basidiospores owing to the deposition of the nanocomposites.
With respect to the antifungal effect of the Cu:ZnS-lignocellulose nanocomposites it is worth mentioning that the net negative charge of the nanocomposites facilitates its adherence to positively charged chitin of the fungal cell wall through electrostatic interaction. Following the adherence, the anti-fungal activity is likely mediated by the classical pathway of cell wall disintegration, electrolytic imbalance, inhibition of nucleic acid, and protein synthesis impairment (Fig. 7) [35, 36]. Cu-based antifungal agents are widely reported to induce oxidative stress on fungal cell walls leading to the disruption of cellular membrane and leakage of cytoplasmic components. One of the primary causes of membrane disruption is lipid peroxidation which is induced by reactive oxygen species (ROS). This formation of reactive oxygen species (ROS) is mediated by a variety of mechanisms including the generation of superoxide via inhibition of the mitochondrial electron-transport chain and the inactivation of enzymes to generate hydrogen peroxide [37, 38]. The presence of ROS in turn is known to cause DNA damage and impairment of proteins involved in DNA replication process, in addition to the disruption of the cellular membrane. The micrographs present strong evidence of the membrane adherence of the Cu:ZnS-lignocellulose nanocomposites followed by the membrane disintegration which can be inferred from the morphological deformation of the E. vexans basidiospore and germ tube (Fig. 6j).