Effect of silver nanochitosan on control of seed-borne pathogens and maintaining seed quality of wheat

Abstract

Seeds, considered as the foundation of agriculture, are invaded by a broad spectrum of seed-borne pathogens. The current study aimed to control seed-borne fungal pathogens of wheat, Aspergillus flavus and A. niger, by using Ag⁺ nanochitosan (Ag-NC) for nano-priming of seeds and enhancing seed quality. Nanochitosan (NC) and Ag-NC were synthesized using the gelation method and characterized by UV–vis spectrophotometry, FESEM, EDXS, and HRTEM. NC and Ag-NC showed irregular surface topography with an average particle size of 275 and 325 nm, respectively. Antifungal activity of both the nanoparticles at 0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL revealed that Ag-NC at 0.5 mg/mL has completely terminated the mycelial growth of both pathogens. Malonaldehyde content increased to 77.77% in A. flavus and 82.66% in A. niger when exposed to 0.5 mg/mL Ag-NC. High-intensity fluorescence due to oxidative stress was observed in Ag-NC-treated pathogens. Ultra-structural changes in Ag-NC treated pathogenic spores under SEM displayed pronounced membrane damages. Wheat seeds were nano-primed with NC and Ag-NC at 0.5 mg/mL, and fungal load was examined to evaluate the mitigation of pathogenic stress and its effect on seedling growth promotion activity. Ag-NC priming reduced the fungal load and allowed successful seed germination. Ag-NC priming increased the albumin, gliadin, gluten, and glutenin content along with total phenol, reducing sugar and starch levels. Ag-NC priming increased the overall protein levels traced through SDS-PAGE. Seed priming with Ag-NC promotes seed germination, mean germination time, stress tolerance index, vigour, etc. NC and Ag-NC at 0.5 mg/mL showed no cytotoxic effect on the Human Embryonic Kidney (HEK293) cell line that ensures the nanoparticles are non-toxic. Thus, the synthesized nanoparticles exhibit a dual role in antifungal activity and plant growth promotion.

Background

Wheat (Triticum aestivum L.), one of the most important cereal crops, is served globally as an essential staple food. It rates the highest grain production in comparison to other crops (Khan et al. 2023). In addition to mankind, wheat is also an important animal feed. However, the decrease in the global production rate is of greatest concern to agro-researchers and farmers (Majumder et al. 2013; Islam et al. 2015). The most vital input of agriculture is the seed, which has also been considered a direct mean of conveying seed-borne pathogens over the years. Disease caused by seed-borne pathogens acutely affects plant health and are liable to deteriorate the seed quality during storage (Majumder et al. 2013). Wheat seeds are known to be distressed by a large number of seed-borne pathogens, including Aspergillus flavus, A. niger, Alternaria alternata, Fusarium graminearum, Curvularia lunata, Bipolaris sorokinaina, Helminthosporium sativum, and Penicillium chrysogenum (Rehman et al. 2011; Majumder et al. 2013; Hussain et al. 2013; Raza et al. 2014; Mehboob et al. 2015). Sheltering these seed-borne pathogens inside wheat deteriorates plant morphological development and yield when sown in the field. Seeds are often attacked by bacteria, nematodes, and rodents due to unhygienic and unscientific storage conditions (Sarwar 2015; Ali et al. 2019). Amid of all the seed-borne pathogens, diseases caused by fungal pathogens suffer 60% economic loss worldwide (Majumder et al. 2013). Seed quality and its longevity are awfully hampered by the invasion of fungal pathogens during both pre-harvest and post-harvest periods (Martin et al. 2022). Seed-borne pathogens are reported to lower the amount of reducing sugar, protein, phenolics, and amino acids in seeds (Rao et al. 2014; Tahmasebi et al. 2023). Thus, the management of seed-borne pathogens is crucial for diminishing the loss of seed quality.

Various methodologies for administrating seed-borne pathogens with techniques, such as seed priming, seed treatment or seed dressing, use of fungicides and botanicals, have been studied in recent years (Islam et al. 2015). Despite using these strategies, the success rate for combating seed-borne pathogens has not been satisfactory recently (Martin et al. 2022; Khan et al. 2023). Seed treatment with synthetic fungicides like captan (heterocyclic nitrogen), farmerzeb (zinc salt), mancozeb (ethylene bis-dithiocarbamic acid), baytan (tridimenol), and thiram (dithiocarbamate) are often observed to increase the viability of seeds (Kadegea and Lyimo 2015). But tremendous use of chemical-derived fungicides leads to the lowering of seed health, followed by disturbing the soil mycoflora, arresting seedlings' nutritional qualities, and disrupting nitrification and denitrification processes (Choudhary 2018).

In that regard, the appraisal of an eco-friendly, perishable biomolecule like chitosan and its nanometal-derivatives are highly recommended by the scientific sphere. Several scientific groups have already confirmed metal-derived nanoparticles' antifungal efficacy, viz. Ag, Fe, Zn, Cu, Ni, etc. (Eskikaya et al. 2023). Applying metallic nanoparticles on pathogenic microbes is known to produce reactive oxygen species (ROS), inducing cellular damages and hampering their protein and nucleic acid (Dananjaya et al. 2017; Nguyen et al. 2023). In order to synthesize a beneficial and cost-effective metallic nanoparticle, the choice of metal is a stiff task. One needs to consider increased bioactivity, which has a low cost value and a threshold limit of metal exposure to the seeds before synthesizing any metallic nanoparticle.

Researchers have recently designed various multifaceted bio-derived molecules, such as nanoparticles or nanoconjugates to combat a broad spectrum of seed-borne pathogens (Banerjee et al. 2021). A number of researchers have introduced chitosan for synthesizing nanoparticles and its conjugates in view of the fact that chitosan is a biodegradable, biocompatible, less toxic, and cost-effective molecule (Hans and Lowman 2002; Sarkar and Acharya 2020). Scientists have so far confirmed the potentiality of chitosan as an optimistic antifungal agent bearing a polycationic nature, which allows the molecule to bind with negatively charged cell components of fungal pathogens (Sathiyabama and Charles 2015; Malerba and Cerana 2016; Sathiyabama and Parthasarathy 2016). Alteration of bulk chitosan into chitosan nanoparticles increases its bioactivity as an antifungal compound, permitting greater permeability into biological membranes, and increased encapsulation efficiency with larger surface area coverage and small particle size (Kong et al. 2010). Besides nanochitosan (NC), scientists have specialized focused on establishing metal conjugates of NC through ionic gelation, emulsification, precipitation, etc. (Antonoglou et al. 2018; Yanat and Schroen 2021). Various reports suggest that, with respect to chitosan and its nanoparticles, metal variants of chitosan nanoparticles are more efficient with raised biological activity, including antifungal effect. This increase in activity is due to structural and functional aspects that have changed, with more cationic groups, the involvement of active functional groups, and the increase in condensing capacity (Yanat and Schroen 2021).

The metal Ag⁺ manifests various congestions against plant pathogens and can be used with absolute safety for the governance of plant pathogens compared to commercial fungicides (Sharma et al. 2018). When applied, nanoparticles consisting of Ag⁺ ion boost the plants' physiology by enhancing the germination rate, delaying leaf senescence, increasing biomass, and strengthening the plant immune system (Mahakham et al. 2017; Sadak et al. 2019). Ag⁺ is an established wound healer and a well-known antiseptic having a highly reactive moiety that can invade pathogenic membranes (Nagaraja et al. 2023). However, exposure to Ag⁺ beyond the threshold level may lead to phytotoxic outcomes on the morphological to molecular level in plants (Tripathi et al. 2017). Henceforth, Ag⁺ chosen under the optimum dose for synthesizing a conjugative nanoparticle, including chitosan as the mother molecule, may hypothetically lead to increased bioactivity.

Ag⁺ nanoparticle is an established anti-microbial compound against an indefinite number of crop pathogens (Kumari et al. 2017; Ibrahim et al. 2020; Manssor et al. 2021). Under in vitro conditions, several workers have already explored the antifungal efficacy of Ag-NC on various seed-borne phytopathogens (Kaur et al. 2012; Kaur et al. 2015; Wang et al. 2015). However, there are very few reports on the in vivo use of Ag-nanochitosan (Ag-NC) in defeating seed-borne pathogens and alleviation of pathogenic stress responses, particularly related to wheat. The use of Ag-NC for defeating seed-borne pathogens of wheat through solid matrix priming of wheat seeds is a novel approach to our study. Our objective is to focus on reducing the fungal load on stored wheat seeds by nano-priming approach so that the seeds can germinate into healthy seedlings after they are sown. Present research involves the synthesis of Ag-NC using very low exposure of Ag⁺ and used the same for the control of seed-borne pathogens of wheat. Experiments were designed in a comparative account between NC and Ag-NC to better understand the promotional bioactivity of Ag-NC over NC. The fungicidal activity of Ag-NC against seed-borne pathogens from the vegetative to cellular level is explored. Since, chitosan is known to promote plant growth, this study also includes the assessment of germination parameters along with biochemical aspects of the nanoparticle-primed wheat seeds. Effect of nano-priming on the profile of storage proteins of seeds were analysed through SDS-PAGE. Moreover, the cytotoxicity of the synthesized nanoparticles was checked on the mammalian kidney HEK293 cell line to confirm that the applied dosimetry of Ag is under the threshold level and has no chance of exhibiting metal toxicity to plants, soil, and humans.

Results

Synthesis and characterization of NC and Ag-NC

The synthesis of NC was stipulated by the appearance of an apparent clear solution with an absorption peak in UV–vis spectrophotometer at 300 nm. While a brownish-coloured solution for Ag-NC hydrogel with an absorption peak at 405 nm for Ag-NC (Fig. 1a, b). High Resolution Transmission Electron Microscopy (HRTEM) inspection revealed an aggregative nature for NC, with irregular shape and average particle size ranging between 68 and 384 nm (measured by ImageJ Software). Whereas, Ag-NC particles are sized between 100–365 nm with a spherical layout (Fig. 1c, d). In Field Emission Scanning Electron Microscopy (FESEM) analysis, both NC and Ag-NC showed uneven, irregular surface topography with highly compact texture (Fig. 1e, f). Ag-NC showed embedded spherical-sized nanoparticles, agglomerated in masses. EDXS spectrum confirms the presence of Ag⁺ in a very low proportion of only 1.14 atomic % of the total atomic weight of Ag-NC nanoparticle. Other necessary elements down to boron were also validated in the EDXS analysis (Fig. 1g, h).

Fig. 1

a Synthesis of NC and Ag-NC through ionic gelation method; b The characterization on the basis of UV–vis spectral absorbance; c and d HRTEM analysis and particle size distribution histogram of NC and Ag-NC; e and f FE-SEM analysis of NC and Ag-NC; g and h EDXS analysis of NC and Ag-NC

Isolation of seed-borne pathogens from infected wheat seeds

The two high-frequency seed-borne pathogens were identified as A. flavus (ID 11,117.19) and A. niger (ID 11,531.21) that showed 45% and 32.5% fungal frequency from stored seeds of wheat. Aspergillus belonging to the phylum Ascomycota is a filamentous fungi having conidiophores that bear a chain of conidia attached to a club-shaped sterigmata, as observed under a microscope, A. flavus and A. niger (Additional file 1: Figure S1).

Effect of NC and Ag-NC on mycelium radial growth, lipid peroxidation, ROS production, and ultra-structural changes in major seed-borne pathogens A. flavus and A. niger

The assessment of the fungicidal activity of the synthesized NC and Ag-NC was evaluated by checking the mycelial growth of A. flavus and A. niger under a gradient of concentrations of the nanoparticles in vitro. The antifungal efficacy of NC and Ag-NC was estimated by measuring the colony diameter of both the seed-borne pathogens grown in PDA treated with 0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL concentrations of both nanoparticles. The radial mycelial growth of A. flavus and A. niger was observed to be decreased with the rise in the concentration of the nanoparticles (Fig. 2a). After seven days post-incubation, the control plates showed a colony diameter of 9 cm and 8.7 cm for A. flavus and A. niger, respectively. Colony diameter of both pathogens was reduced successively with the hike in the concentration of nanoparticles. At 0.5 mg/mL, NC-treated plates for A. flavus showed 7.1 cm of colony diameter, equating to only 22.3% of Percent inhibition of radial growth (PIRG), and for A. niger, claiming 59.12% inhibition, showing 3.2 cm colony diameter (Fig. 2b, c). While plates treated with 0.5 mg/mL of Ag-NC exhibited complete termination of fungal growth, leading to 100% PIRG of both pathogens (Fig. 2c). Aqueous solution of AgNO₃ was served as positive control, and it was observed that individual effect of AgNO₃ against A. flavus and A. niger was lower in comparison to Ag-NC and NC against the same pathogens (Additional file 2: Figure S2).

Fig. 2

a Effect of NC and Ag-NC on mycelium radial growth of A. flavus and A. niger with different concentrations (0.1, 0.2, 0.3, 0.4 and 0.5 mg/mL) of NC and Ag-NC on PDA plate. b Graphical representation of colony diameter of A. flavus and A. niger with different concentrations of NC and Ag-NC in comparison with control at 7 days post-incubation; c The percent inhibition of radial growth (PIRG) of A. flavus and A. niger under tested concentrations of NC and Ag-NC. Values are averages of three replicates (n = 3), error bars indicate standard deviation (SD) and different letters (a, b, c, etc.) indicate significant differences between treatments at p ≤ 0.05 by Duncan’s Multiple Range Test

A significantly higher level of Malonaldehyde (MDA) was generated with the increasing dosimetry of NC and Ag-NC. NC at 0.5 mg/mL showed 82.66% and 47.11% of MDA for A. flavus and A. niger, respectively (Fig. 3a). At the same aforesaid concentration, Ag-NC produces increased percentage of MDA in both the seed-borne pathogens in comparison to NC. A. flavus gives rise to 77.77% of MDA as a result of the treatment of 0.5 mg/mL of Ag-NC, whereas, A. niger generates 83.16% of MDA at the maximum used concentration of the same (Fig. 3a).

Fig. 3

a Effect of different concentrations of NC and Ag-NC on MDA percentage of A. flavus and A. niger. Fluorescence microscopic observations showing generation of oxidative stress in b–d A. flavus and e–g A. niger sporangium and mycelium due to the effect of b, e distilled water, c, f NC (0.5 mg/mL), and d, g Ag-NC (0.5 mg/mL); Morphological changes of A. flavus spores directly exposed to h distilled water, i NC (0.5 mg/mL), j Ag-NC (0.5 mg/mL); and A. niger spores treated with k distilled water, l NC (0.5 mg/mL) and m Ag-NC (0.5 mg/mL) observed under SEM. Data are represented as the averages of three replicates (n = 3), error bars indicate SD and a, b, c, etc.indicate significant differences between treatments at p ≤ 0.05 by Duncan’s Multiple Range Test

The visualization of fluorescence through the Dichloro-dihydro-fluorescein (DCFH) staining method further confirmed the generation of oxidative stress in A. flavus and A. niger after 72 h of nanoparticle treatment. Figure 3b–g represents fluorescence microscopic images related to the generation of oxidative stress in distilled water (control), NC (0.5 mg/mL), and Ag-NC (0.5 mg/mL) treated fungal mycelium and sporangium of both A. flavus (Fig. 3b–d) and A. niger (Fig. 3e–g). Distilled water-treated fungal mycelium showed no appearance of fluorescence. Mycelium given NC treatment shows moderate to weak intensity of fluorescence. In contrast, fungal mycelium treated with Ag-NC showed the generation of maximum intensity of fluorescence, demarcating high production of oxidative stress.

Alterations in the morphology of the nanoparticles treated spores at the ultra-structural level were traced under Scanning Electron Microscopy (SEM) for both pathogens. Spores treated with distilled water (control) revealed no morphological deformities. Negligible structural abnormalities were noticed in NC (0.5 mg/mL) treated pathogenic spores. In contrast, spores influenced by Ag-NC (0.5 mg/mL) treatment exhibit pronounced disruptions in their membrane as observed in both the couple of pathogens. Severely damaged spore walls with visible pores were also observed in Ag-NC feted A. flavus and A. niger spores. Aggregation of the fungal spores on masses was interestingly observed in Ag-NC treatment (Fig. 3h–m).

Fungal load in nano-primed wheat seeds

The effect of nano-priming with NC and Ag-NC on stored wheat seeds was checked to evaluate the mitigation of pathogenic stress generated by seed-borne pathogens on the seeds. Non-primed control seeds, when placed on PDA medium, liberated mycoflora harboured with seed-borne pathogens, which terminated the germination of the seeds (Fig. 4a). Similarly, seeds primed with NC (0.5 mg/mL) also remained ungerminated and liberated seed-borne mycoflora on the media (Fig. 4b). In contrast, seeds primed with Ag-NC (0.5 mg/mL) showed no liberation of any mycoflora in the media and successful germination of all the seeds placed on the plate (Fig. 4c). Statistically, the fungal load was found higher in non-primed seeds. NC priming in the seeds showed a reduction in fungal load by 21.42%, whereas Ag-NC priming showed a 100% reduction of fungal load in the seeds (Fig. 4d).

Fig. 4

Determination of fungal load in stored wheat seeds on 3rd day a non-primed, b NC (0.5 mg/mL), c Ag-NC (0.5 mg/mL); d Assessment of fungal load in treated wheat seeds. Values in the line graph are the average of three replicate plates (n = 3), error bars indicate SD and a, b, c, etc.indicate significant differences between treatments at p ≤ 0.05 by Duncan’s Multiple Range Test

Effect of seed nano-priming on seed quality, germination, and disease incidence of wheat seeds

The effect of solid matrix priming with NC and Ag-NC on wheat seeds was evaluated by the estimation of essential biochemical parameters that affect seed quality. The albumin content in the non-primed seeds was traced to be 1.6 mg/g FWT, which increased by 2.75 folds in seeds primed with 0.5 mg/mL NC. Whereas the albumin content was significantly raised by 4.25 folds in seeds primed with 0.5 mg/mL Ag-NC. The globulin level in the NC primed seeds rose by more than 3 folds when compared to non-primed seeds. This level of globulin is again increased by 6.5 folds in seeds primed with Ag-NC. The gliadin content increased by 52.90% and 315% in NC and Ag-NC primed seeds, respectively, in comparison to non-primed seeds. Similarly, the gluten content rose by 4.64 folds in NC primed seeds and 5.78 folds in Ag-NC primed seeds when compared with the non-primed seeds (Fig. 5a). The phenol content in the non-primed seeds was estimated to be 1.23 µg/mg FWT which is found to be increased by 19% in NC primed seeds and by 40% in Ag-NC primed seeds (Fig. 5b). On the other hand, the reducing sugar content raised by 1.40 folds and 1.87 folds, respectively, in NC and Ag-NC primed seeds, with respect to non-primed seeds (Fig. 5c). The starch content in the NC primed seeds increased by 46.15% in comparison to non-primed seeds. This starch content increased by 126% in seeds primed with Ag-NC (Fig. 5d).

Fig. 5

Effect of solid matrix priming of wheat seeds on a total protein content, b total phenol content, c reducing sugar, and d starch content. Values are represented as the mean of three replicates (n = 3), error bars indicate SD and a, b, c, etc.indicate significant differences between treatments at p ≤ 0.05 by Duncan’s Multiple Range Test

Analysis of the SDS banding pattern of NC and Ag-NC primed wheat seeds resulted in 14 major bands (Fig. 6a). The intensity of the band is significantly higher in Ag-NC primed seeds, which demarcates that the expression of a particular protein is quantitatively higher in seeds primed with Ag-NC (Fig. 6a). The integrated density of band at 63 kDa is increased by 20% in NC-primed seeds and by 44.39% in Ag-NC primed seeds, with respect to non-primed seeds (analyzed through ImageJ software) (Fig. 6b). Also, the integrated density of the band at 35 kDa is increased by 36.87% and 40.30% in NC and Ag-NC primed seeds, respectively, in comparison to non-primed seeds (Fig. 6b).

Fig. 6

Analysis of storage protein profiling through SDS-PAGE after nano-priming of stored wheat seeds. Non-primed seeds, NC (0.5 mg/mL) primed seeds, and Ag-NC (0.5 mg/mL) primed seeds

The effect of solid matrix priming with the synthesized nanoparticles on wheat seed physiology was examined through the evaluation of germination-related parameters (Table 1). Non-primed seeds given treatment with distilled water showed only 32% of seed germination (SG), whereas NC and Ag-NC primed seeds revealed 62% and 90% germination, respectively (Table 1). The MGT in NC-primed seeds decreased by 6.25% from the MGT in non-primed seeds. The MGT in Ag-NC primed seeds is further decreased by 9.27% in comparison to non-primed seeds (Table 1). Similarly, the GSTI of the seedlings whose seeds were primed with Ag-NC significantly increased to 200% with respect to non-primed (18%), where seeds were manifested with pathogens and without any priming (Table 1). PI was also found to be elevated by 4.88 folds in Ag-NC primed seeds and by 3.44 folds in NC primed seeds when contrasted with non-primed seeds (Table 1). Results of VI proclaimed that non-primed seeds germinated with retarded root-shoot length (500.23 unit), whereas NC priming resulted in increased VI by 50% and by 170% for seeds primed with Ag-NC as compared with non-primed seedlings (Table 1).

Table 1 Effect of solid matrix priming of wheat seeds on seed germination (SG) %, mean germination time (MGT), germination stress tolerance index (GSTI), promptness index (PI), vigour index (VI), and disease incidence (DI) % of variably treated wheat seedlings at 7th day of germination

On the 7th day of germination, the NC and Ag-NC primed seeds revealed a significant difference in the reduction of disease incidence and promotion of germination parameters of the seedlings. The non-primed seeds showed significant establishment of disease in the seedlings, with 88.66% disease incidence, whereas NC primed seeds displayed 46.66% disease incidence. A remarkable reduction in the disease incidence was traced in the seedlings whose seeds were primed with Ag-NC, which showed only 10% disease incidence (Table 1).

Cytotoxicity of NC and Ag-NC

The cytotoxic effect of the synthesized nanoparticles was examined on the mammalian HEK293 cell line through an (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) MTT assay. Mammalian cells were exposed to an increasing range of dosimetry, from 0.1–2 mg/mL of NC and Ag-NC. Results revealed that both the nanoparticles possess no cytotoxic effect on the seeded cells up to 1.4 mg/mL concentration. Cell proliferated successfully in the medium treated with 0.1–1.4 mg/mL of both NC and Ag-NC. But beyond 1.4 mg/mL of NC and Ag-NC, a cytotoxic effect was observed in the seeded cells leading to the loss of reproductive ability and further proliferation. Treatment of the cells above 1.4 mg/mL of NC showed up to 10% cytotoxic effect, whereas Ag-NC treatment showed 30% cytotoxicity up to 2 mg/mL dosage, with respect to the cells treated with distilled water imposing no toxicity (Additional file 3: Figure S3).

Discussion

Chitosan is counted as one of the most powerful chelating agents that are able to form complexes in the company of heavy metals and transition metals with ease (Jiang et al. 2011; Pivarciova et al. 2014; Pincus et al. 2021). Reports have stated that chitosan-involved metal complexes with transition metals (Ag, Cu, Fe, Ni, etc.) revealed enhanced antimicrobial properties in comparison to bulk chitosan because of the modifications in the physical structure of chitosan bearing -NH₂ and -OH group as predominant reactive sites (Wang et al. 2005; Ardean et al. 2021). Chitosan, with its nano-metal derivatives, exhibits a broad spectrum of antifungal properties against Aspergillus niger, A. flavus, Alternaria solani, Fusarium oxysporum, Penicillium spp., Candida spp., Cordyceps militaris, etc. (Qiang et al. 2011; Ilina et al. 2017; Liu et al. 2018). Therefore, chitosan has emerged as a key biodegradable molecule for the management of seed borne pathogens in food crops.

Ag⁺ imparts deleterious reverberations on multiple pathogenic microorganisms along with the physiological upliftment of plants towards vigor. Ag⁺ induces the resistance of plants to various diseases and abiotic stresses (Kale et al. 2021). An ion of this compassion, when impregnated with chitosan nanoparticles, hiked the antifungal activity along with plant developmental metabolism and stress resistance capacity (Anusuya and Banu, 2016; Santiago et al. 2019; Alghuthaymi et al. 2020). Our results demonstrated that Ag-NC could stand as a promising antifungal agent and can successfully combat seed-borne pathogens of wheat. In this context, it is important to better understand the nanoparticle's structural configuration. The UV–vis spectral peak obtained for NC and Ag-NC suggested the formation of desired nanoparticles, as it corresponds to the previous work done by Kalaivani et al. (2018); Gohary et al. (2021), and Mirda et al. (2021). It is believed that the smaller is the particle size, the easier its penetration into the biological membrane (Labhasetwar et al. 1997; Bhattarai et al. 2006). The range of particle size distribution of NC and Ag-NC acquired through HRTEM analysis strongly tally with the results published in the reports of Hoang et al. (2022) and Ali et al. (2011). Various research supports the idea that particle size within 500 nm is efficient for ingression into the fungal cell membrane with ease (Desai, 2016). Irregular to spherical aggregative topography of both the nanoparticles obtained through FESEM analysis strongly coincides with the results obtained in the previous reports (Murugan et al. 2017; Sen et al. 2020). Furthermore, EDXS analysis also confirmed the presence of Ag⁺ in Ag-NC, which could be regarded as a metallic nano-composite (Mirda et al. 2021).

In the present study, A. flavus and A. niger showed high frequency in the screened seeds, and treatment of seeds with NC and Ag-NC resulted in their remarkable reduction. Various workers have experimentally proved the high occurrence of Aspergillus spp. as a dominant seed-borne pathogen in stored wheat (Baka 2014; Mohmed et al. 2019; Kumar et al. 2023). Examination on percent inhibition of radial growth (PIRG) discloses complete termination of radial mycelial growth of A. flavus and A. niger on the application of 0.5 mg/mL of Ag-NC on PDA plate. Inversely, at the highest used dosage (0.5 mg/mL) of NC, both A. flavus and A. niger showed only 22.3% and 59.12% of PIRG, which infers NC to be barely efficient as an antifungal agent in comparison to Ag-NC. Thus, Ag-NC exhibited potentially more antifungal properties in contrast to NC. Various other groups of researchers also agreed that Ag-NC is a more potent antifungal agent than NC due to the add-on competence of Ag⁺ ion (Dananjaya et al. 2017). The promotive impact of metal-dopped nanoparticles or chitosan-metal conjugates on various seed-borne pathogens has already been established by several researchers (Sangeetha and Sudha, 2019; Encinas et al. 2020). With the gradual rise in the dosimetry of Ag-NC on the PDA plate, the diameter of the radial colony of A. flavus and A. niger was observed to be decreased. This gradual decrease in the colony diameter confirms the dose-dependent functioning of metal-derived nanoparticles (Tareq et al. 2018). Several groups of workers reported the fungicidal activity of Ag⁺-chitosan nanoparticles at a maximum of 1 mg/mL concentration against Aspergillus spp. and other related seed-borne pathogens (Kaur et al. 2012; Kalaivani et al. 2018; Shehabeldine et al. 2022). Whereas, our study demonstrated that Ag⁺ complexed with chitosan nanoparticles delivers complete depletion of both A. flavus and A. niger only at 0.5 mg/mL concentration. An in-depth review on the implementation of Ag⁺ ion on chitosan meshwork expresses a promotive inhibitory effect not only against a large number of seed-borne pathogens but also against several phytopathogens. It is believed that the amino group of the polycationic chitosan coalesce with the negatively charged fungal cell components, thereby suppressing the fungal ramification by chelation and inhibiting fungal enzymes (Shehabeldine et al. 2022).

The consequence of different concentrations of NC and Ag-NC on lipid peroxidation of A. flavus and A. niger was estimated by determining the MDA content generated due to oxidative stress on the application of the aforesaid nanoparticles. It is claimed that the polyunsaturated fatty acids (PUFA) residing in the fungal membrane containing methylene (-CH₂-) group is critically affected by ROS produced as a result of lipid peroxidation of the pathogens (Kalagatur et al. 2018). Our investigation reveals the generation of high MDA content in Ag-NC-treated fungal systems in comparison to NC treatment. The obtained results are in agreement with the previously performed works suggesting the successful generation of MDA by Ag-NC and some other metallic nano chitosan in variedly tested pathogenic organisms (Farooq et al. 2022).

The level of intensity of fluorescence generated in the tested seed-borne pathogens is directly equivalent to the degree of ROS produced due to oxidative stress for the exposure nanoparticles (LeBel et al. 1992; Kumar et al. 2016). Our experiment clearly concludes the fact that metal-derived nanoparticles can generate preferably more ROS than their bulk analogues (Li et al. 2012). In accordance with this, our current investigation also displayed maximum fluorescence intensity in Ag-NC-treated mycelium as well as in the sporangium of A. flavus and A. niger. The treatment of NC at the same dosage on A. flavus and A. niger does not satisfactorily generates oxidative stress. It is known that -conjugated nanoparticles can successfully uplift the generation of high intensity of oxidative stress, disrupting fungal membrane integrity. An illustration revealed that uplifted ROS content leads to an imbalanced level of antioxidants in the fungal cell surplus, which results in the release of cytochrome C, followed by cell apoptosis (Kalagatur et al. 2018). The input of Ag⁺ ion into the chitosan network successfully proved the emission of high levels of fluorescence in some of the previously tested pathogens that can tally with our study (Dananjaya et al. 2017).

The application of polycationic chitosan, in addition to super positive Ag⁺ ions, results in the binding of the same to the negatively charged cellular components of the pathogens. Our outcome was compatible with the reports suggested by Alghuthaymi et al. (2020) where Ag⁺-chitosan nanocomposite produced defined damages on the spore membrane of another seed-borne pathogen, Penicillium expansum but at a slightly higher concentration of 0.9 mg/mL. It was endorsed in some of the published works that nanoparticles comprised of metal lead to the docking of metal ions on the surface of pathogenic organisms, leading to the loss of linearity of the spores and the formation of spore aggregates (Alghuthaymi et al. 2020). From the above experiments, it can be concluded that the chitosan-Ag⁺ duo is predominantly successful in imparting fungicidal activity by controlling the growth of seed-borne pathogens.

Moreover, our study also aimed to investigate the plant growth-promoting criteria of chitosan-derived nanoparticles. Experiments conducted in vivo revealed that 0.5 mg/mL of Ag-NC can provide maximum protection against infection of seed-borne pathogens on wheat seeds through solid matrix priming. Tarakanov et al. (2023) discussed the role of metal-derived chitosan nanoparticles as a resistance elicitor and in plant growth promotion. Our results on the check of fungal load in stored wheat seeds revealed that Ag-NC priming can suppress the growth of imminent seed-borne pathogens and promote successful germination of the seeds. Enormous reports have been filed against seed-borne pathogens for executing stress on wheat seeds that lead to failure of its germination or seed abortion (Rehman et al. 2011; Hussain et al. 2013). Under this scenario, it is obligatory to establish a potential antifungal compound like Ag-NC to mitigate the detrimental effect of seed-borne pathogens from seeds.

Essential storage proteins like albumin and globulin are affected by the interference of seed-borne pathogens, resulting in the depletion of the seed quality (Kumar et al. 2023). In our study, it is experimentally proved that Ag-NC priming of stored wheat seeds enhances the levels of albumin, gliadin, glutenin, and gluten, thereby promoting seed quality. Our findings tally with the previous report that concludes the successful application of chitosan-Ag⁺ nanoparticles in the elevation of total protein in monocot seeds (Anusuya and Banu, 2016). With the application of Ag-NC in our experiment, the total phenol content in the seeds raised manifold, irrespective of NC and non-primed seeds. The use of Ag-NC as a priming agent also uplifted the reduced sugar content, and the inclusion of Ag⁺ ion into the plant system enhances the reduced sugar level has already proved (Siddiqi and Husen, 2022). Wheat endosperm is comprised of 70% of starch, which is one of the important by-products of gluten production (Kim and Kim 2021). Infection caused by seed-borne pathogens reduces the starch content up to 70% (Gebeyaw 2020). On exposing wheat seeds to Ag-NC priming, starch levels increased despite pathogenic interference. In the contrary, seed priming with NC does not satisfactorily promote the overall protein, reducing sugar and starch levels in wheat seeds.

Previous studies have demonstrated the fruitful application of Ag⁺ chitosan nanoparticles in plant growth promotion activity. These nanocomposites promote germination, vigour and biomass of the model plant studied (Pereira et al. 2021; Gowda and Sriram 2023). Similar results were also attributed to our study, showing a high seed germination percentage in the set of seeds primed with Ag-NC followed by NC. The MGT taken by the seeds primed with Ag-NC is comparatively less than the MGT required by non-primed seeds to germinate. GSTI of the germinating wheat seedlings affirmed that Ag-NC incites maximum pathogenic stress tolerance capacity by accelerating seed germination percentage, promoting plant height, and root length with healthy vigor. Stress results due to the infection of seed-borne pathogens triggering seed germination and affecting the proliferation of vigor, showing retarded growth. Seeds without any priming could not withstand pathogenic stress, resulting in failure of seed germination and spotting of fungal colonies on the seed surface. On the other hand, seeds primed with NC showed moderate stress tolerance capacity with respect to seeds primed with Ag-NC. Results obtained from our investigation denote the promotive effect of the Ag⁺ ion, implemented in the chitosan network. The input of Ag⁺ into the chitosan body has been proven to be significant as it induces a growth-promoting effect and has antifungal properties compared to raw chitosan (Dananjaya et al. 2017). The elevated growth promotion activity traced in our study concludes that seed priming with Ag-NC can efficiently combat seed-borne pathogens and pass the seed to a successful germination regime.

Data for the cytotoxicity assay does not portray any toxic effect, with the increased dosimetry of both NC and Ag-NC at up to 1.4 mg/mL. The cytotoxicity of both NC and Ag-NC was noted from 1.5 mg/mL and beyond. The toxicity of Ag-NC on the seeded mammalian HEK293 cells was obtained due to the inclusion of metal Ag in the nanoparticle. There are also reports that nanoparticles involving Ag⁺ exhibit dose-dependent phytotoxicity on plant physiology and accumulate in the soil, hampering the soil microbiota (Yan and Chen, 2019). Ag⁺ nanoparticles are reported to show a phytotoxic effect on wheat seed germination, primary seminal root, coleoptile and biomass production at 10 to 40 mg/mL concentration (Lahuta et al. 2022). The threshold level of Ag⁺ exposed to living organisms should not exceed 20 µg/100 g of dry matter. The U.S. National Institute for Occupational Safety and Health has set the upper extent of all Ag⁺ forms to be 0.01 mg/m³ (Antsiferova et al. 2023). In our experiment, the bioactive dose of Ag-NC i.e., 0.5 mg/mL contains 1.14% of Ag⁺ (analysed through EDXS), which is an approximate of 0.0057 mg/mL Ag⁺ used for seed priming. The applied dosimetry of Ag⁺ is extremely low and has no chance of imparting a phytotoxic effect. However, Ag⁺ is considered one of the expensive metals and its large-scale application on agricultural practices could be strenuous for farmers and governments in developing countries like India. Thus, it is important to consider the cost-effectiveness of Ag⁺-derived nanoparticles. Since, we have used a very low percentage of Ag⁺ for configuring the nanoparticle, Ag-NC can also be regarded as a cost-effective fungicidal agent.

Conclusion

An overview of the role of nano-priming in restraining the growth of seed-borne pathogens of wheat seeds has been depicted in Fig. 7 and it has been observed that nano-priming with Ag-NC modulates the physiological and biochemical properties of the seed. Concomitantly, Ag-NC also alters the fungal spore structure, thereby arresting its further growth and preventing seedlings from contracting seed-borne diseases. On the other hand, nano priming alleviates different proteins inside the seed by mitigating the deleterious effect of seed-borne pathogens and rendering the seedlings with greater vigor and health by lowering the disease incidence.

Fig. 7

Prospective role of nano-priming in modulation of wheat seed physiological that affects the growth of seed-borne pathogens and lowering disease incidence

Methods

Synthesis of NC and Ag-NC

Nanoparticles of chitosan were synthesized using an ionic gelation method using sodium tri-polyphosphate (STPP) as a cross-linker (Asgari-Targhi et al. 2018). Synthesis was carried out by using 80% N-deacetylated chitosan of low molecular grade (50–190 kDa; Sigma Aldrich). In brief, 0.1% chitosan (w/v) solubilized in 1% (v/v) aqueous acetic acid was subjected to 1 h of constant stirring followed by the dropwise addition of Sodium tri-poly phosphate (STPP) solution of 1 mg/mL. The resulting opalescent solution was centrifuged at 10,000 rpm for 15 min, and the gel-like sediments harbouring the nanoparticles were diffused in distilled water and kept at 4 ºC for future use.

Ag-loaded chitosan nanoparticles were developed by the chemical reduction method of Dananjaya et al. (2017) with soft modifications. 20 mM aquo-silver nitrate solution (AgNO₃; Sigma-Aldrich) was mixed with 0.1% chitosan in 0.05:3 ratio. The solution was agitated in a magnetic stirrer for 30 min followed by the addition of 50 µL sodium borohydrite of 0.2 M imparting immediate brown colouration. The solution was allowed to undergo complete reduction through the continuous stirring of 1 h. To enable cross-linking of the chitosan molecules, STPP salt was added to the aforesaid method. Centrifugation was carried out at 10,000 rpm, collecting the pellet as particles of Ag-NC.

Characterization of NC and Ag-NC

For obtaining the spectral absorbance, NC and Ag-NC were scanned between 200–800 nm on UV–vis spectrophotometer (Aligent Technologies, Carry 100 UV–Vis). High-resolution transmission electron microscopy (HRTEM) analysis was employed to detect the morphology and structure of the nanoparticles on JEOL JEM 2100F using 200 kV of accelerating potential and 50 × –1.5 M × power of magnification. The exterior topography of the nanoparticles was studied through Field emission scanning electron microscopy (FESEM); Schottky JSM-7900F, JOEL, 0.1–30 kV. Detection of elements following boron was confirmed through Energy dispersive X-ray spectrometry (EDXS), especially for Ag-NC.

Isolation of seed-borne pathogens from infected wheat seeds

Infected wheat seeds of Sonalika cultivar that had typical symptoms of seed-borne diseases were collected from wheat-growing fields and seed preservatories of North Bengal, India, for the isolation of seed-borne pathogens. Permissions were granted from the wheat growers to collect infected seeds from the fields. Seed-borne fungal pathogens were isolated through the agar plate method. Infected wheat seeds were surface sterilized and seeded on sterile Petri dishes containing autoclaved potato dextrose agar (PDA) media. The isolates were further sub-cultured after 7 days of incubation to obtain pure cultures. Pure cultures of all the seed-borne fungal pathogens were maintained and sent for identification in IARI (Indian Agricultural Research Institute, New Delhi, India).

Effect of NC and Ag-NC on mycelium radial growth, lipid peroxidation, ROS production, and ultra-structural changes in major seed-borne pathogens A. flavus and A. niger

Poisoned food bioassay was carried out against A. flavus and A. niger using different concentrations (0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL) of NC and Ag-NC (Dananjaya et al. 2017). Precisely, an aqueous solution of each concentration of both nanoparticles was blended with 20 mL of autoclaved PDA media in such a manner that the desired concentration remained unchanged in the resultant plates. A 5 mm mycelium disc of both pathogens, excised by a sterile cork-borer, was taken as inoculum. After solidification of the media, the mycelium disc was seeded at the center of a 90 mm petri plate. Also, a plate was served to both the fungus without the treatment of any nanoparticle. Plates were made in triplicates and incubated at 28 ºC for 7 days. The percentage inhibition of radial growth was calculated as follows.

$$\text{PIRG }\left(\text{\%}\right)=\frac{\text{Radial}\, \text{growth} \,\text{of} \,\text{colony} \,\text{in} \,\text{control} \,\text{plate}-\text{Radial} \,\text{growth} \,\text{of} \,\text{colony} \,\text{in} \,\text{treated} \,\text{plate}}{\text{Radial} \,\text{growth} \,\text{of} \,\text{colony} \,\text{in} \,\text{control} \,\text{plate}}\times 100$$

The quantitative estimation of lipid peroxidation of both the seed-borne pathogens on application of NC and Ag-NC was determined by the synthesis of malonaldehyde (MDA), an indicator of lipid peroxidation in the pathogens (Subban et al. 2019). Mycelial mat cultured in potato dextrose broth (PDB), treated with the aforesaid range of dosimetry of both the nanoparticles, were harvested after 7 days of incubation at 28 ºC. The harvested fungal mat was washed and dried with blotting paper. The mat was homogenized with chilled sodium phosphate buffer (0.1 M, pH 7) in a ratio of 1:5. Resultant homogenate was cold centrifuged at 10,000 rpm for 12 min. 100 µL of the supernatant was added to 3 mL of thiobarbituric acid (0.335%) prepared in 10% trichloroacetic acid. The developed reaction mixture was subjected to a boiling water bath until a pink colour is observed. After cooling the mixture, spectrophotometric readings were taken at 530 nm and MDA levels were calculated using 1.56 × 10⁵ as molar absorption co-efficient.

The generation of reactive oxygen species (ROS) in both the seed-borne pathogens upon exposure of NC and Ag-NC, was spotted by using a non-fluorescent probe-2’,7’-dichlorodihydrofluorescein diacetate (H₂DCFH-DA); purchased from Sigma Aldrich, India (Chen et al. 2020). Both the pathogens were cultured in PDB treated with 0.5 mg/mL of NC and Ag-NC for 72 h. Post incubation, the hyphae were collected from the broth, washed rigorously with phosphate buffer saline (PBS), and left suspended in the same. 40 µL of H₂DCFH-DA was added to 500 µL of the hyphal suspension under dark conditions and laid for 2 h at 28 ºC with sudden shaking at regular intervals. The production of ROS was spotted visually under a fluorescence microscope (Nikon Eclipse E200, Nikon, Tokyo, Japan) using an excitation filter at 485 nm and an emission filter at 525 nm.

The morphological changes in the spore membrane of both the seed-borne pathogens at the ultra-structural level were diagnosed under a scanning electron microscope (SEM) (JSM-IT 100; JEOL). The spores cultured in PDB for 72 h, treated with 0.5 mg/mL of NC and Ag-NC were pre-treated with chemical fixatives before observation under SEM, as prescribed by Babu et al. (2018).

Effect of seed nano-priming on fungal load

Stored wheat seeds of Sonalika cultivar, collected from the regional sub-station of National Seed Corporation of India Ltd., were surface sterilized and primed with 0.5 mg/mL of NC and Ag-NC as defense elicitors, using Celite (O₂Si; Himedia) as an inert solid matrix agent. Seeds and celite were taken in 1:1 proportion, followed by maintaining 10% matrix moisture with the priming agent used. Seeds were primed for 24 h in air-tight zipper bags and held undisturbed in a seed germinator (REMI) at 22 ± 2 ºC. After 24 h, the celite was sieved off, and seeds were collected (Sen et al. 2020). Four primed seeds from both treatments were placed in each petri plate containing 20 mL sterilized PDA media. A plate containing non-primed seeds was used as a control. Every treatment was made upon three replicates. Plates were incubated at 22 ºC for three days. Colony Forming Units (CFU) were recorded, and the fungal load was calculated by the following equation (Andualem et al. 2019).

$$\text{N}=\frac{5a*10000}{bt}$$

where, N = fungal CFU/m³; a = number of colonies per Petri dish; b = surface area of dish (cm²); t = exposure time (min).

Effect of seed nano-priming on seed quality, germination and disease incidence of wheat seeds

Estimation of total reducing sugar in the nano-primed seeds were carried out through the DNSA method suggested by Miller (1959) using maltose as standard. Total glutenin, gliadin, globulin, and albumin present in the nano-primed seeds were estimated by method Zhao et al. (2021) with minor modifications. Profiling of total seed protein were conducted through SDS-PAGE (Zhao et al. 2021). For starch estimation, 50 mg of nano-primed seeds were homogenized in 80% ethanol and subjected to 80 ºC for 1 h. The starch is extracted with 52% perchloric acid in a hot, acidic medium. Colorimetric observations were recorded at 620 nm after the addition of Anthrone reagent (Clegg 1956). Changes in the total phenol content after nano-priming in the wheat seeds were estimated using the Folin-Ciocalteu method using the gallic acid standard as prescribed by Malick and Singh (1980).

Stored wheat seeds were primed with 0.5 mg/mL of NC and Ag-NC and germinated by following standard blotter paper method in sterile petri plates for seven days at 22 ± 2 ºC (Khan et al. 2023). Each plate was seeded with 30 seeds. Petri plates were fixed with three layers of soaked blotter papers. Parameters such as seed germination (SG), mean germination time (MGT), germination stress tolerance index (GSTI), promptness index (PI), and vigour index (VI) were studied (Abdul- Baki & Anderson, 1972; Sen et al. 2020) in NC and Ag-NC primed seeds.

A number of 30 stored wheat seeds were nano-primed with the aforesaid dosimetry of NC and Ag-NC and allowed to germinate for seven days in sterile petri plates at 22 ± 2 ºC by following the standard blotter method, as mentioned above. Percentage of disease incidence was evaluated as follows (Guha and Sindhu 2011):

$$\text{Disease}\, \text{incidence }\left(\text{DI}\right){\% }=\frac{\text{Total} \,\text{no}.\, \text{of} \,\text{diseased} \,\text{or} \,\text{ungerminated} \,\text{seeds}}{\text{Total} \,\text{no}. \,\text{of} \,\text{seeds} \,\text{placed}}\times 100$$

Determination of cytotoxicity of NC and Ag-NC

The mammalian kidney cell line (HEK293) was purchased from the National Centre for Cell Science (NCCS), Pune, India. The cytotoxicity of both nanoparticles was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Mosmann 1983; Denizot & Lang, 1986). Briefly, a rapidly multiplying mammalian kidney cell line was placed in 96 well microtiter plate using 6 × 10³ cells/well. The wells were maintained in 5% CO₂ using DMEM (Dulbecco’s Modified Eagle Medium) Ham F-12 cell culture medium at 37 ºC. After 24 h of proliferation, wells were treated with different concentrations (0.1–2 mg/mL) of NC and Ag-NC in triplicate. After 24 h of further incubation, the culture media was aspirated, followed by the addition of 10 µL MTT dissolved in 1 × PBS in each well and laid for 3 h. Before recording the absorbance in the ELISA reader at 620 nm, 50 µL of isopropanol was added to each well with occasional shaking for 10 min.

$$\text{Cytotoxicity } \% = \frac{\text{Mean}\, \text{optical} \,\text{density} \,\text{of} \,\text{untreated} \,\text{cells}-\text{Mean} \,\text{optical} \,\text{density} \,\text{of} \,\text{treated} \,\text{cells}}{\text{Mean} \,\text{optical} \,\text{density} \,\text{of} \,\text{untreated} \,\text{cells}}\times 100$$

Statistical analysis

The results obtained from the experiments conducted are the mean of five different observations with standard deviation (SD) (Mean ± SD). Statistical differences were investigated by using Duncan’s Multiple Range Test (DMRT) at p ≤ 0.05 (DSAASTAT ver. 1.022); treatments differing significantly are represented by letters a, b, c, etc.