Bioactive bile salt-capped silver nanoparticles activity against destructive plant pathogenic fungi through in vitro system

Abstract

Colletotrichum gloeosporioides is the most destructive endophytic plant-pathogenic fungi causing anthracnose disease in a wide number of economically important plants throughout the world. Presently there are no existing methods for effective disease management over the outbreak of anthracnose disease and it thus leads to huge economic losses for farmers and plant tissue culture laboratories. In order to find a new and effective control over these endophytes, we synthesized and characterized bioactive bile salt sodium deoxycholate (NaDC) capped silver nanoparticles. These nanoparticles were employed to control the endophytic fungus through in vitro direct and indirect model systems with time dependant/light mediated incubation manner. In our findings we achieved fivefold synergistic effect of NaDC-capped AgNPs with their bioactive capping agent against Colletotrichum gloeosporioides upon analyzing different parameters. Moreover, it was evident that NaDC-capped AgNPs did not cause any phytotoxicity to treated plants as revealed by molecular marker studies.

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Introduction

In vitro plant tissue culture offers mass cultivation and conservation of endangered and endemic flora, including medicinal and ornamental plants, principally with the production of disease-free planting material in a short time interval. In fact, a successful tissue culture mostly depends on the removal of exogenous and endogenous contaminating microorganisms,^6,8 which cause diseases in the host plant. 3–15% losses due to contamination by bacteria, fungi and viruses at every subculture in the majority of commercial and scientific plant tissue culture laboratories have been reported.^18,28,29 The explants initially undergo surface sterilization for removal of contaminants by chemicals (mercuric chloride, sodium hypochlorite, hydrogen peroxide, fungicides, bactericides and antibiotics) and surfactants, which are fundamentally phytotoxic in nature as well as retarding or inhibiting plant growth,^23,53 although using surfactants has only been effective in eliminating exogenous contaminants of explants but not endogenous contaminants.⁴³ Endophytic microorganisms are microbes that colonize within plant tissues and may lead to symptoms in their hosts. The number of endophytic species potentially associated with a plant species is often estimated as several hundred.⁴⁷ The most frequently isolated endophytes are fungi and these biotropic contaminants are particularly dangerous when they are identified as plant pathogens.⁵⁸ Among these, the fungal phytopathogen Colletotrichum gloeosporioides causes anthracnose disease,¹⁷ which has posed serious problems worldwide in the cultivation of economically important plants such as coffee,⁵⁷ olives,³² mangoes,³³ apples,⁹ citrus fruits,² strawberries, beans,³⁷ rice¹³ and other crops. With the aim of developing a model control methodology, we initially set out to find an alternative to chemically manufactured surfactants which are efficient against the endophytic fungus. We decided to turn to nanotechnology, specifically silver nanoparticles, given their numerous physiochemical properties and outstanding antimicrobial activity even on multi-drug resistant microbes. In various forms, silver compounds have been exploited as antimicrobial agents since ancient times and are now becoming more commonly used in the medical field.^38,45,48 In fact, both silver ions (Ag⁺) and silver nanoparticles (AgNPs) display multiple modes of inhibitory action against several pathogenic microorganisms,⁷ but the mechanism of action of AgNPs (Ag⁰) differs from Ag⁺ (AgNO₃ as a source). The degree of activity depends on concentration,⁴⁹ sensitivity of the microbial species to silver,¹⁴ external factors such as irradiation,¹⁶ and most significantly, surface physiochemical properties of AgNPs, which are modified by capping agents that are used to synthesize the nanoparticles.^19,55 We decided to use a naturally-occurring mammalian bile salt, sodium deoxycholate (NaDC), as a reducing agent for synthesizing AgNPs. The bile salt has a proven record as a potential bioactive agent against salmonellae,⁴⁰ enteric bacteria and sexually transmitted diseases (STDs).^{5,39,41,46,50} Generally, most AgNP-aided antimicrobial activities are concentration dependant studies, which at high levels may be toxic to the organism and environment. Alternatively, here we demonstrate time-dependant light-irradiation mediated indirect (tissue culture) and direct (agar plate assay) antifungal activity studies of NaDC-capped AgNPs against Colletotrichum gloeosporioides. In support of these experiments, we also studied uptake and movement of nanoparticles in plants, with RAPD molecular markers, so as to also investigate possible cytotoxicity and genotoxicity to treated plants.^1,35 From this study, we aim to eliminate Colletotrichum gloeosporioides contamination thorough in vitro administration using NaDC-capped AgNPs as a new strategy of plant disease management for sustainable agriculture productivity with disease free planting materials.

Materials and methods

Chemicals

Silver nitrate (99.99%) and sodium deoxycholate (∼98%) were obtained from Sigma-Aldrich. Sodium hypochlorite (NaOCl), dextrose, agarose, ethidium bromide, MS medium components, hormone kinetin and lactophenol cotton blue were purchased from Himedia laboratories, India. TAE (tris-acetic acid-EDTA) and Dellaporta buffer were purchased from Sisco research laboratories and Loba chemicals. respectively, and PCR reaction mixture was obtained from Amplicon QIII. All glassware was thoroughly cleaned with aqua regia (HCl : HNO₃ = 3 : 1), rinsed with double distilled water, and dried in a hot air oven prior to use.

Synthesis of AgNPs

Stock solutions of 4 mM AgNO₃ and 0.1 M sodium deoxycholate (NaDC) were prepared using deionized double distilled water, and subsequent diluted solutions were prepared from this stock solution. To study the effect of pH on AgNPs formation, 0.15 mL of AgNO₃ stock solution was mixed with 5 mL of 0.05 M NaDC at different pH, and the reaction was carried out under UV light irradiation at 365 nm.²⁴ To synthesize AgNPs for antifungal activity, about 1.5 mL of AgNO₃ stock solution was mixed with 50 mL of 0.05 M NaDC at neutral pH, and the reaction was carried out under UV light irradiation at 365 nm with constant stirring for 7.5 h. The formation of AgNPs was observed by the gradual change in color of the solution from colorless to yellow.³ The production yield of nanoparticles was quantitatively calculated by UV/visible extinction spectra of NaDC synthesized silver nanoparticles according to ref. 34. Fixed volumes of freshly synthesized AgNPs colloidal solution were subjected to high speed centrifugation (BW-HRC-16M, No. 1 angle rotor, Lark Innovative Technologies, India) at 10 976 × g for 10 min in 15 °C to complete the sedimentation of nanoparticles. The pellet of nanoparticles was harvested and dissolved in ultra-purified water for further characterization and applications. For investigating the stability of nanoparticles, the synthesized colloidal solutions of aliquots were kept in 15–40 °C both in light and dark.

Characterization of AgNPs

UV-visible absorption spectra were measured using a Shimadzu UV-1601 spectrophotometer over the 250–700 nm range. High-resolution transmission electron microscopy images were obtained using a HRTEM instrument (FEI TECHNAI, G2 MODEL T-30 S-TWIN) at an acceleration voltage of 250 kV. The samples were drop-casted onto a carbon-coated copper grid and allowed to dry at room temperature before analysis. The hydrodynamic diameters of the synthesized AgNPs were measured using dynamic light scattering analysis (DLS-Nanotrac Ultra NPA 253 from Microtrac, USA).

Indirect antifungal activity of NaDC capped AgNPs through tissue culture

Phyllanthus amarus plants were selected and used for in vitro tissue culture study. There are two valuable reasons behind the selection of this well-known medicinal plant²⁷ as a test plant: it is among the hosts for C. gloeosporioides,⁵¹ and endophytic contamination produces toxic metabolites colletotrichin and ferricrocin^21,22 that may affect the quality of the medicinal herb used as a raw formulation for drugs worldwide. The explants of Phyllanthus amarus were collected from the Herbal Garden belonging to Department of Botany, University of Madras, Chennai, India. The explants were kept under running tap water for 30 min, adding two drops of detergent (Tween 20), followed by a thorough rinse with double distilled water two times. The nodal explants were 1.5–2 cm in length incised aseptically with sterile blades, and they were surface sterilized with 1% sodium hypochlorite (NaOCl) for 4–5 min, after which the nodes were thoroughly washed with sterile distilled water 3–4 times. The plant tissue culture-specific MS medium was prepared by the standard procedure outlined by Murashige and Skoog.³⁰ The medium was also fortified with kinetin hormone (0.5 mg L⁻¹) for shoot proliferation. Borosil glass tubes (25 × 150 mm) each containing 15 mL of the culture medium and capped with plugs of non-absorbent cotton were autoclaved at 120 °C for 15 min. Initially, a few explants (which show no morphological symptoms) were screened for endophytic contamination by inoculating on the MS medium following the existing standard methodology.⁵² After positive confirmation of contamination, emerged fungi were isolated (fungal-contaminated one-week culture) by inoculating them on potato dextrose agar (PDA: peeled potato 250 g, dextrose 20 g, agar 15 g, mixed in 1 L distilled water) plates followed by incubation at 37 °C for seven days under a 12 h photo-period. Pure isolates were obtained from repeated sub-culturing of the isolates. They were then transferred to a clean glass slide and stained with a drop of lactophenol cotton blue. The stained slide was visualized under a light microscope for the identification of fungal isolates. Fungal isolates were identified using cultural characteristics and morphology and by comparing with standard taxonomic protocols indexed by Barnett and Hunter.⁴ After that, the efficacy of NaDC capped AgNPs to eradicate disease-causing organisms on plants was examined (on the basis of contamination generated) in in vitro tissue culture system. The C. gloeosporioides infected explants of P. amarus were taken into separate 20 mL test tubes and treated (completely submerged) separately with the following three solutions: NaDC capped AgNPs (1 mL), 0.1 M of NaDC (1 mL), and 1 mM AgNO₃ (1 mL), respectively, in the presence/absence of light irradiation at various time intervals (30, 60, 90, 120 and 150 min). Both AgNO₃ and NaDC-treated explants were considered as control (C) and reference (R), respectively, for AgNPs-treated explants. For this treatment, molar concentrations of the control (NaDC) and reference (AgNO₃) solutions were the same as used in AgNPs synthesis. In this experiment, the culture chamber white light (3000 lux) was used as the source of light irradiation with a distance of 0.5 m. At the completion of every incubation time both at light/dark, the treated explants were washed with dd-H₂O at two times and left to dry for a few minutes. Then all the treated explants were taken and vertically implanted in each culture tube containing MS medium. All the treatments were carried out with appropriate care inside the laminar air-flow chamber to avoid external microbial contamination. Furthermore, cultured tubes were kept inside the tissue culture chamber at a temperature of 25 ± 2 °C for 16/8 hours light/dark cycles per day. At least seven replications with six explants were maintained and evaluated for each treatment. The results were evaluated from the 4^th and 5^th day of inoculation and the percentage of contamination was calculated by measuring the width and height of the appearance of fungal growth around the explants. In relevance to this antifungal activity, fungal morphology was investigated before and after treatment of AgNPs through simple light microscopy. To monitor the possible uptake and presence of AgNPs into the treated plant, samples were analyzed by UV-vis spectra and HRTEM with EDX (energy-dispersive X-ray spectroscopy). For the UV-vis spectra analysis, AgNPs colloidal solution alone and AgNPs with incubated explants were used. For HRTEM analysis, ultramicrotome obtained semithin plant sections (1 μm) were placed on a copper grid and fixed with 4% paraformaldehyde and 2% glutaraldehyde then postfixed in 1% osmium tetraoxide, all in cacodylate buffer (0.05 M, pH 7.0). Further the samples were processed with 2% uranyl acetate, dehydrated in acetone and fixed in epoxy medium. Finally the sections were used to obtain HRTEM images and EDX was performed to determine elemental identification, specifically silver.

Direct antifungal activity of AgNPs by in vitro Petri-plate assay

Fungal spores were picked up from 2 week-old growing C. gloeosporioides cultures with a sterile inoculation needle and suspended directly into the required number of Eppendorf tubes each containing 100 μL of AgNPs colloidal solution. Further, the tubes were incubated at different time intervals (30, 60, 90, 120 and 150 min) in both dark and light using the same conditions as described in the previous section. After incubation, 100 μL of conidia was taken from each tube and spread on Petri dishes containing PDA agar. All the Petri dishes were incubated at 26 ± 1 °C in a light chamber. After the 4^th day of incubation, direct antifungal activity of AgNPs was qualitatively assessed (visually) by the number of colonies that appeared in the Petri dishes. Subsequently, fungal morphological changes were also investigated with field emission scanning electron microscopy (FESEM-Hitachi SU600) using the same silver source at all respective incubation timings. For FESEM analysis, specimens were prepared by the standard protocol.⁵¹

Phytotoxicity analysis

The study of possible cytotoxicity generation by the silver nano-solution to treated plants was assessed by external morphological changes such as growth inhibition and necrotic spots in leaves. The treated plant parts were also subject to a genotoxicity analysis with RAPD molecular markers. A predominant technique, RAPD, has been used to reveal DNA damage or molecular alterations in all the levels of plant gene alterations.^12,54,56 From the collected plant parts, genomic DNA was extracted using the standard procedure outlined by Dellaporta et al.¹¹ The RAPD decamer, OPK-7 (5′-AGC GAG CAA G-3′), specifically obtained from Operon technologies, Inc., USA, was screened and selected for further analysis based on its ability to detect distinct polymorphic amplified products between treated and non-treated mother plants. The reactions were carried out in a DNA-gradient thermocycler (Peltier thermocycler model L196GGD, Lark Innovative Technologies, India). After the reaction was completed, 20 μL of the PCR-amplified products was subjected to electrophoresis in a 2% agarose gel immersed in 1× TAE buffer at 100 V for 2.5 h. The gel product was stained with ethidium bromide and analyzed in a UV transilluminator (Lark Innovative Technologies, India).

Results and discussion

Characteristic features of NaDC capped AgNPs

In order to optimize the reaction condition, 150 μL of AgNO₃ stock solution was mixed with an aqueous solution of 0.05 M NaDC in 5 mL and the reaction was carried out under UV light irradiation at 365 nm with different irradiation times. The color of the solution gradually changed from colorless to intense yellow. The UV-visible spectrum of the prepared AgNPs shows a characteristic SPR peak at 408 nm (Fig. 1a). Furthermore, sizes of the obtained AgNPs were examined by high-resolution transmission electron microscopy (HR-TEM) and dynamic light scattering (DLS). Fig. 1b, HRTEM images showed that the formed particles were nearly monodisperse and spherical in shape with diameter ∼21 ± 2 nm. The average size of AgNPs was found to be 22 nm according to DLS analysis and their sizes were consistent with the HRTEM observations (Fig. 1c). Regarding yield of AgNPs, the AgNPs were quantified to have a concentration of 0.02 mg in 1 mL (20 ppm) of the silver colloidal solution according to Marquis’s criterion of UV/visible extinction spectra analysis.³⁴ Time-dependent UV-visible spectra were also performed to evaluate the kinetics of the formation of AgNPs (Fig. 2a). The intensity of the SPR band gradually increased with time and finally reached saturation level within 7.5 h, after which no significant changes in the absorbance intensity were observed, that confirmed the completion of reaction. The effect of pH on AgNPs formation was studied at different pH values, ranging from 7.0 to 11.0, under UV light irradiation at 365 nm as shown in Fig. 2b. At pH 7.0 maximum intensity of the SPR peak of AgNPs was noted; however, with increase in the pH from neutral to alkaline the intensity of the SPR band gradually decreased, with a small blue shift. This phenomenon indicated AgNPs were agglomerated in basic pH while stable dispersions were observed at neutral pH. No reaction was observed at pH below 7, this is because in acidic pH sodium deoxycholate was precipitated in the form of deoxycholic acid. The synthesized silver nanoparticles were very stable and remained in monodispersed condition at 15–40 °C when kept in the dark for four months (UV-spectrum data not shown).

Fig. 1 (a) Photo and UV–vis absorption spectra of NaDC capped AgNPs colloidal solution, (b) HRTEM micrographic image of AgNPs (100 nm scale bar), (c) DLS histogram of NaDC capped AgNPs.

Fig. 2 Kinetics of formation of NaDC capped AgNPs: (a) time-dependant UV-visible spectra, (b) pH-dependant UV-visible spectra.

Indirect antifungal activity of NaDC-capped AgNPs

Endophytic fungus isolated from contaminated in vitro cultured plants (5^th day of explants inoculation) was grown on fungus-specific medium (PDA) and had a grayish-white appearance. The isolated fungus was further identified through microscopic observation based on its morphological structure.⁴ Light microscopic images show that the conidiogenous cells are acervular, enteroblastic, phialidic, separate, composed of hyaline to dark brown septate hyphae of Colletotrichum gloeosporioides. A similar identification of Colletotrichum gloeosporioides is also seen in two reports.^15,52 From 4^th to 5^th day of inoculation of explants, inhibition effect was measured on formation of radial hyphal growths around the treated plants within the tube and the data obtained were the calculated mean from the seven replicates. The AgNO₃ (R) solution treated plants show decreased level of contamination, light treated: 94.74–70.41%; dark: 98.54–86.81% from 30 to 150 min (Fig. 3a). In NaDC (C) with light irradiation, treated explants showed 99% fungal contamination level around the plants at both 30 and 60 min, but showed decreased contamination level of 94.9–90.7% at 90 to 150 min. However, dark-treated NaDC (C) exhibited 99% contamination growth in all the tubes (Fig. 3b). Interestingly, NaDC-capped AgNPs treated in the presence of light alone showed a gradual decrease in the contamination level (90.21–50.52%) from 30–90 min; amazingly no fungal colonies were observed for 120 and 150 min treated plants (Fig. 3c). We also obtain clear evidence for fungal growth inhibition through light microscopic analysis, Fig. 3d and e shows fungal morphology before and after AgNPs treatment (150 min incubation) respectively. For dark treated samples with AgNPs, contamination appeared in all the tubes and the rate of contamination merely decreased (98.5–92.2%) from 30 to 150 min, which is clearly represented in the bar graph of Fig. 3c. From the cumulative results, neither well-established antimicrobial agent NaDC^5,39 or AgNO₃²⁰ alone were very effective in eliminating the endophytic fungal contaminants residing inside the plants. However, NaDC-capped AgNPs under long time light exposure was highly effective in fungal inhibition activity, and clearly light irradiation played a crucial role in the activation of AgNPs to inhibit Colletotrichum gloeosporioides growth. This phenomenon correlates very well with a previous report:¹⁶ irradiation may induce the photo-ejection of electrons, causing oxidation or ionization at the NP surface and segregation of metal ions.

Fig. 3 Histogram along with photographs showing the percentage of contamination for in vitro tissue culture treated explants in the presence/absence of light at various time exposures with: (a) AgNO₃, (b) NaDC, (c) NaDC capped AgNPs. Each value shown was the mean of seven determinations and error bars represent standard deviation (±sd). (d) and (e): Images of fungal structures of Colletotrichum gloeosporioides before and after treatment with AgNPs (150 min light exposure), respectively.

Verification of silver dissolution by light and its consequent uptake into plants was studied through UV-vis spectral analysis. Measurements were carried out with the AgNPs solution alone and AgNPs with explants, incubated separately at light exposure times (0, 30, 60, 90, 120 and 150 min) the same as previously examined. Obtained spectral data (Fig. 4a and b) reveals the dissolution of ions and the capping agent. The degree of dissolution is found to increase with the time of light incubation. and AgNPs alone did not dissolve in contrast to AgNPs incubated with explants. The “dissolution” solution can be regarded as a “nanomixture”. Since this was preliminary data, we intensively followed the TEM and EDX analysis to determine the fate of uptaken AgNPs into treated plants. For TEM analysis of prepared AgNPs treated plant specimens, we had a track of silver nanoparticles and their dissolution in the vascular region (Fig. 4c and d) where silver ions appear to be generated from the AgNPs at vascular tissues.

Fig. 4 Assessment of NaDC capped AgNPs uptaken into treated plants: (a) UV-vis spectral data of AgNPs incubated with light for various times (0, 30, 60, 90, 120 and 150 min) and (b) UV-vis spectral data of AgNPs incubated with explants in presence of light for various times (0, 30, 60, 90, 120 and 150 min). (c) TEM image of plant specimen depicts vascular region contains silver metal trace (1 μm bar scale), (d) TEM image of plant specimen depicts AgNPs dissolution state (20 nm bar scale). (e) EDX spectrum represents silver element signal from vascular region of plant specimen.

Fig. 4e shows the EDX spectrum of a particular region of AgNPs dissolution indicating a strong signal of silver element. It was confirmed that both light exposure and explant proximity accelerated the dissolution of AgNPs to metal ions along with the capping agent. With extension of time for adsorption via the stomata of nutrient medium into tissue culture the concentration will increase. Once taken up, transport within the plant occurs by mass flow via the stomata.¹⁰ Hence, there is a rapid uptake of the nanomixture solution into plant cells leading to internalization and localization of the endophytic environment which might disrupt the metabolic activity by accumulation and interactions with macromolecules found in the fungal body. Long-time exposure of the nanomixture solution to microorganisms, would increase the surface contact to microorganisms, which will alter significant biochemical pathways so as to kill the organisms.^18,31

Direct antifungal activity of NaDC capped AgNPs

In order to reveal the effect of AgNPs directly against endophytes on the 4^th day post-inoculation, inhibition levels on colony formation of C. gloeosporioides decreased on PDA plates when the light incubation time was increased (Fig. 5a–f). Apparent colony formation is seen at 90 min incubation time and none thereafter. Dark incubation showed the highest number of colonies on all the PDA plates (figure not shown). The result demonstrates an excellent AgNP activity only in the presence of light as shown in the graph (Fig. 5g). These assay results bear close similarity with previous results which were obtained from the explant-treated assay. The FESEM investigation on the same blends at the same incubation times is pictorially shown in Fig. 6a–f. The microscopic images clearly show the cell depletion (shrinking and cracking) of fungal structures at the respective incubation times in a progressive manner. AgNPs irradiated by light form a nanomixture solution which is understood to be the mechanism behind this activity. The nanomixture is subsequently excited by prolonged light irradiation and is directly involved in the catalytic activity on the surface contacting endophytes. Approach on the surface of C. gloeosporioides disrupts the cell structures through the generation of reactive oxygen species that react with molecular oxygen present on the surface of endophytes.²⁵

Fig. 5 Direct antifungal activity of NaDC capped AgNPs on Colletotrichum gloeosporioides on PDA plates at; (a) 0, (b) 30, (c) 60, (d) 90, (e) 120 and (f) 150 min light irradiation and (g) data (mean ± sd) expressed over all activity of AgNPs against fungus.

Fig. 6 Direct antifungal activity: FESEM images of Colletotrichum gloeosporioides incubated with NaDC capped AgNPs at various times showing degree of cell damage: (a) 0, (b) 30, (c) 60, (d) 90, (e) 120 and (f) 150 min light irradiation.

Phytotoxicity impact of AgNPs

AgNO₃ (R)-treated explants left some silver metal-like trace all over the explants which later progressed to give a gray black colored stem with reduced growth (figure not shown). Hence, ionic Ag causes discoloration by itself or allows other organic materials to cause undesirable coloration while directly treated. However, in the case of NaDC (C)-treated explants for both light/dark, no morphological damage was observed, but the original contamination reduced the growth of the explants. The light-irradiated AgNPs treated explants showed no undesirable changes on plant growth and no negative morphological changes were observed. After 45 days, AgNPs treated Phyllanthus plants grew up to 11 cm with no detrimental morphological changes and they were successfully acclimatized in field. According to the result, it was affirmed that the nanomixture-containing bioactive capping agent might reduce the toxicity of dissolved Ag⁺ ions in treated plants. With the help of RAPD molecular markers, genotoxicity of treated plants was revealed intensively at Fig. 7. The gel image shows a 100 bp marker in the end lane, and the remaining four lanes show PCR-obtained RAPD products which were sourced from all the three treated (lane 2-AgNPs, lane 3-NaDC and lane 4-AgNO₃) explants and one untreated plant as control (lane 1), respectively. The sizes of the amplified fragments are obtained from the 100 bp to 1 kb range. A similarity index was calculated on all four banding patterns which were counted by the presence (indicated as ‘1’), absence (indicated as ‘0’), and the presence of faint or unclear bands (which were not considered part of the count). A total of 10 bands of different kb were obtained from the control untreated plant, and the same numbers of bands of similar molecular size were found in both NaDC-capped AgNPs and NaDC-treated plant (lanes 2 and 3). Adversely, polymorphic bands were identified only in AgNO₃-treated plants (lane 4). According to the similarity index calculation, the NaDC-capped AgNP-treated plant clearly shows no mutational changes on genomic DNA; genetic stability was retained at 100% level in the NaDC-capped AgNPs treated plant as well as in NaDC-treated plant. Therefore, only ionic AgNO₃ caused cytotoxicity and mutational changes at the gene level confirming genotoxicity to the plant, as previously reported.⁴²

Fig. 7 Genotoxicity analysis by RAPD markers; fluorogram image shows banding pattern of PCR amplified products. Lane 1: control treated, lane 2: NaDC capped AgNPs treated, lane 3: NaDC treated, lane 4: AgNO₃ treated, lane 5: 100 bp marker.

Conclusions

This experimental studies conducted here revealed that prolonged light exposure of NaDC-capped AgNPs in close proximity to plant tissues led to gradual dissolution of silver ions along with capping agent. HRTEM with EDX analysis confirmed the dissolution of AgNPs inside the treated plant and UV-vis spectra also confirmed that the explant treated solutions contain a mixture of Ag ions, AgNPs, and capping agent in different ratios. The mechanism behind the successful antifungal activity of a nanomixture was facilitated by prolonged irradiation which causes transport via stomatal opening and localization in the endophytic environment which effectively reduced the Colletotrichum gloeosporioides contamination, with no other toxicity on treated plants. Prior to this work, there have been no reports attaining 100% removal of Colletotrichum gloeosporioides fungal contamination with use of fungicides and even by silver nanoparticles.²⁶ Further, bioactive salt NaDC capped AgNPs still led to 100% genetic uniformity and stability, in contrast to AgNO₃ treated plants, as analyzed by RAPD markers. This in vitro model study could offer a potential key to use AgNPs as potential antifungal agents to maintain a high degree of sterility against endophytes in tissue culture and agricultural crops, and represent a viable alternative to toxic chemical usage. Therefore, bioactive salt capped AgNPs may be used with relative safety for control of various non-specific plant pathogens when compared to highly toxic synthetic fungicides.^36,38,44 The efficacy of the NaDC capped AgNPs provides long-term perfectly safe residue and acts quickly compared with other antimicrobial agents. This technology could effectively minimize the expenditure for plant disease management.

Abbreviations used

AgNPs	Silver nanoparticles
NaDC	Sodium deoxycholate
AgNO3	Silver nitrate
MS	Murashige and Skoog medium
RAPD	Random Amplified Polymorphic DNA

Acknowledgements

The authors thank Centre for Nanoscience and Nanotechnology, University of Madras for Instrumentation facilities.

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