Nanohexaconazole: synthesis, characterisation and efficacy of a novel fungicidal nanodispersion

Abstract

Here, the authors describe a simple method to formulate the nanodispersion of hexaconazole (hexa); henceforth, referred to as nanohexaconazole (N‐hexa) that is water soluble and effective against several species of Aspergillus. Size and shape of the prepared nanocomposite was determined with high‐resolution transmission electron microscopy and field‐emission scanning electron microscopy. Nanohexaconazole structure was further confirmed by Fourier‐transform infrared spectroscopy and gas chromatography–mass spectrometry. The antifungal efficacy of nanohexaconazole (N‐hexa) was studied in vitro, compared with micronised hexaconazole (M‐hexa) at different doses (5 ppm, 10 ppm and control) against two food pathogenic fungi: Aspergillus niger (MTCC 282, MTCC 2196 and BDS 113) and Aspergillus fumigatus through poisoned food technique. A dose‐dependent significant growth inhibition was observed in nanohexaconazole (N‐hexa) treated fungal sample compared with that of micronised hexaconazole (M‐hexa). Micrographic studies for the morphological analysis of control and nanohexaconazole (N‐hexa) treated fungal samples were done, exhibited an alternation in fungal morphology. Results showed that nanohexaconazole (N‐hexa) is more efficacious than commercially available micronised hexaconazole (M‐hexa). In future nanohexaconazole (N‐hexa) could be a possible candidate for modern medical science and also reduce damage to the environment from injudicious use of pesticides.

1 Introduction

Intrinsic to methods of increasing food production are the use of agrochemicals and one class of agrochemicals that ranks high, both in terms of concerns regarding usage, and as environmental pollutants, is that of crop protectants? Increase in the use of agrochemicals is being viewed as a contentious issue; their production, storage and use are now strongly regulated. Since the 1980s, triazoles have been widely used as antifungal agents both against human and plant pathogens. Most researches have been directed at pathogens of human interest [1]. Triazoles have also been effective as crop protectants and used on many different types of plants including field crops, fruit trees, small fruit, vegetables and turf [2]. Hexaconazole (hexa) is a triazole fungicide commonly used against agro‐medically important fungal pathogens. It targets and blocks the enzyme, 14 alpha lanosterol demethylase in fungi, crucial for the lipid biosynthetic machinery. It is most effective against fungi of Ascomycetes and Basidiomycetes. Hexaconazole (hexa) such as other triazoles functions as a demethylation inhibitor, specifically against cytochrome P450‐mediated oxidative demethylation reactions, which are necessary for the synthesis of ergosterol. Ergosterol is a key component of fungal cell walls and hence a target for antifungal agents [3]. It is systemic in nature and used in agriculture to protect crop losses caused by several pathogens of apples, vines, coffee, peanuts, bananas, cucurbits, peppers, rice, groundnut, mango etc. In India, it is used for controlling pests such as blister blight Exobasidium vexans disease of tea [4]. However, overuse of this biocide has led to the contamination of ground waters, soil, sediments, plants and animals, besides damaging innumerable non‐target organisms necessitates their replacement by novel and more effective formulations. Moreover, many fungicides are poorly soluble in water and large amounts of organic solvents are required to solubilise them. Many of these organic solvents are not eco‐friendly and add to the cost of fungicide use [5]. Nanotechnology has gained scientific interest due to its applications in diverse fields [6, 7]. This growing interest has also been intensified in agriculture sector. A novel approach that has received increased research attention in recent years is the nano‐formulations of bulk materials. Nano‐formulation of conventional pesticide with polymers or metal nanoparticles is highly demanding area of pesticide industry. Nanoencapsulation of pesticide is advantageous in controlled and slow release of active ingredient by manipulation in outer shell of nanocapsule, which releases low dose over a prolonged time period and reduces excess run‐off of unwanted pesticide [8, 9]. Nano‐formulations in the form of nanopesticide have been modifying conventional farming practises into precision farming. Many reports have been demonstrated the positive impact of nano‐pesticides on control of plant pest and disease [10, 11, 12, 13]. Certainly, nanodispersions of fungicides offer a promising way out of the double bind of food protection and environmental depredation which makes their use problematic. Nanodispersions are formulations that use the principles of nanotechnology, whereby a nano‐conjugate is prepared. This nano‐conjugate has properties that are very different from its bulk form. It can be designed to retain all the advantages of the bulk form and in addition possess properties that would help to reduce the effective concentration of pesticides use [14, 15, 16, 17]. Gopal et al. have reported two times better fungicidal activity of nanohexaconazole (N‐hexa) than that of commercial formulations against Rhizoctonia solani at low dose, leads to low toxicity and thereby reveals the nanopesticide as eco‐friendly pesticide [18]. Therefore, in this paper, we report the synthesis, characterisation and application of nanohexaconazole (N‐hexa) for the bioefficacy of commonly used crop fungicide; hexaconazole (hexa) against food borne pathogenic fungus Aspergillus.

2 Experiment section

2.1 Materials

Hexaconazole (hexa) was obtained in technically purified from Division of Agrochemicals, Indian Agricultural Research Institute, India and used as the source material. Ethyl acetate, PEG‐400 GR grade were procured from Merck, Mumbai, India. Potato dextrose agar was obtained from Hi‐Media Laboratories, Pvt. Ltd. India. Deionised water was sourced from 18 MΩ, arium (61316 reverse osmosis system), Sartorius Stedium Biotech, Aubagne, France.

2.2 Synthesis of nanohexaconazole (N‐hexa)

Nanodispersion of N‐hexa was prepared by modifying protocol from earlier work [19]. In brief, 5 g hexaconazole (hexa) was dissolved in 100 ml ethyl acetate (solution A). In another beaker, PEG and water were mixed in 9:1 ratio to get a 200 ml solution (solution B). Solution A was added dropwise to solution B under continuous stirring at 40°C for 240 min. Finally, the nanodispersion was subjected to rotary evaporation to remove ethyl acetate. The end product was stored at 4°C which was referred as N‐hexa.

2.3 Physicochemical characterisation of nanohexaconazole (N‐hexa)

The synthesised N‐hexa was characterised by high‐resolution transmission electron microscopy (HR‐TEM) (FEI, Technai, USA) to record size dimensions. Field‐emission scanning electron microscopy (FE‐SEM) (FEI Quanta‐200 MK‐2, OR, USA) was used to observe changes in the morphology of fungal structure and to record dimensions of N‐hexa. Fourier‐transform infrared (FTIR) (Shimadzu) spectroscopy was used to characterise and identify the encapsulation of N‐hexa within PEG. The identity of hexaconazole (hexa) was confirmed by gas chromatography–mass spectrometry (GC–MS) study.

2.4 Fungal strain

Aspergillus niger strains, namely MTCC 282 and MTCC 2196 were purchased from Microbial Type Culture Collection, IMTECH, CSIR (Chandigarh, India). The wild strain (BDS 113) was collected from a batch of affected potato bought from a vegetable vendor in Kolkata, India. It was purified to single spore level and identity was established at Microbial Type Culture Collection, IMTECH, CSIR Chandigarh, India by phenotypic features, ITS/5.8 rRNA (HQ293217) and partial gene sequences for β ‐tubulin (HQ293218). A. fumigatus strain CBS542.75 was provided by Dr. S. Mandal, A.E.R.U. Indian Statistical Institute, Kolkata. This was purified to single spore level and identity was established at Microbial Type Culture Collection, IMTECH, CSIR Chandigarh, India by β ‐tubulin gene sequence.

2.5 Fungal cultivation and antifungal assay

Modified poisoned food technique was used to carry out efficacy studies on select species of Aspergillus [20]. About 3.9% potato dextrose agar was used as the growth substrate. In this experiment, low doses of N‐hexa were used to evaluate its bioefficacy compared with M‐hexa. Five sets were prepared with 3.9 g potato dextrose agar and 100 ml water; first set was the control and in rest of four set doses of 5 and 10 ppm N‐hexa and M‐hexa was added after autoclave with the help of micropipette. Pure hexaconazole (M‐hexa) dissolved in ethyl acetate served as the positive control. Three replicates of each dose were made. There were three plates of control for each round of experiment. Aliquots of a spore suspension at the concentration of ∼104 spores/ml were used as inoculums [21]. About 5 µl from the suspension was used every time to inoculate the plates. Each plate was inoculated in three places. The plates were incubated for a total of 72 h and observations of radial growth were taken with the help of zone diameter scale after 48 and 72 h.

2.6 Fungal sample preparation for FE‐SEM study

For FE‐SEM studies, after 72 h of treatment, fungal samples (A. niger) were isolated and washed several times with deionised water and fixed with 2% glutaraldehyde solution at 4 °C for 2 h, followed by post‐fixing the specimen for 2 h with 1% osmium tetroxide solution, and then the samples were dehydrated with graded ethanol [22].

2.7 Statistical analysis

Data was carried out with Statistical Package for the Social Sciences (Version A) by Dr. Debashis Roy at Computer and Statistical Service Centre, Indian Statistical Institute, Kolkata, India.

3 Result and discussion

3.1 Physicochemical characterisation of nanohexaconazole

HR‐TEM micrographs revealed that the synthesised product N‐hexa was indeed in the nano‐dimension (Figs. 1 a and b). FE‐SEM micrographs show that the agglomerated particles are spherical in structures and sticky in nature, which is expected from PEG encapsulation of hexaconazole (Fig. 1 c) [23, 24]. FTIR studies revealed that there was no change in the chemical structure of hexaconazole (hexa) as evident from the presence of peaks due to same functional group which was more intense around 3420 cm⁻¹ due to the presence of polyethylene glycol in nanohexaconazole (N‐hexa + PEG) (Fig. 1 d). This is the characteristic peak of hexaconazole (hexa) and its presence in the encapsulated sample indicates that hexaconazole (hexa) has been incorporated in the PEG micelles. The fact that N‐hexa is not degraded was also confirmed by GC–MS (Fig. 2). The fragmentation pattern of the prepared N‐hexa tallied with the pattern available from national institute of standards and technology (NIST) library [25, 26].

Fig. 1.

Synthesis and characterisation of N‐hexa

(a) HR‐TEM image of N‐hexa, (b) Close view HR‐TEM image of N‐hexa, (c) Typical of FE‐SEM image of the PEG encapsulated N‐hexa, (d) FTIR analysis of N‐hexa

Fig. 2.

Further characterisation was done by GC–MS of N‐hexa

3.2 Modified poison food technique

The radial growth (vegetative growth) of fungal samples was measured. The parallel antifungal activity of M‐hexa was also studied. Two different concentrations (5 and 10 ppm) of N‐ and M‐sized hexaconazole were used throughout the experiment. A dose‐dependent reduction in radial growth was observed in all strains of fungi for both N‐ and M‐hexa treatments. However, N‐hexa was more potent than M‐hexa (Figs. 3 a and b). After 72 h of incubation, radial growth was significantly inhibited by 44.56% in BDS113, 32.45% in MTCC 282, 36.84% in MTCC 2196 and 50% in A. fumigatus at 5 ppm N‐hexa treatment compared with M‐hexa‐treated fungal samples, whereas 62.71% in BDS113, 74.41% in MTCC 282, 69.11% in MTCC 2196 and 80.645 in A. fumigatus growth reduction were observed at 10 ppm N‐hexa‐treated samples compared with that of M‐hexa. However, 98–99% of growth inhibition was observed at 10 ppm N‐hexa‐treated all fungal strains compared with control. Therefore, the result showed that nano‐form of hexaconazole is more effective in killing fungus rather than its bulk form.

Fig. 3.

Comparative antifungal effect of N‐hexa and M‐hexa against the different fungal sample

(a) A. fumigates, (b) A. niger (MTCC 282) after 72 h of incubation, (c) Reduction in radial growth of N‐hexa and M‐hexa‐treated fungi A. niger [BDS 113, MTCC 282, MTCC 2196] and A. fumigatus after 48, (d) 72 h incubation (mean of zone diameter ± standard error)

One‐way analysis of variance analysis revealed that changes in the zone diameter among nano‐ and micron‐sized hexaconazole, and the control was statistically significant (Figs. 3 c and d): for BDS113 (after 48 h F  = 1488.706, P  < 0.001 and after 72 h F  = 5703.108, <0.001); for MTCC 282 (after 48 h F  = 22.639, P  < 0.001); for MTCC 2196 (after 48 h F  = 863.500, P  < 0.001 and after 72 h F  = 4389.684, P  < 0.001); for A. fumigatus (after 48 h F  = 807.500, P  < 0.001 and after 72 h F  = 3337.820, P  < 0.001). Corresponding statistical analysis was shown in Table 1.

Table 1.

Statistical representation of the reduction in the radial growth of A. niger (BDS113, MTCC 292 and MTCC 2196) and A. fumigatus after treatment with M‐hexa and N‐hexa at different concentrations after 48 and 72 h

Fungal Strains	Concentration, ppm	Control after 48 h	Control after 72 h	M‐hexa after 48 h	M‐hexa after 72 h	N‐hexa after 48 h	N‐hexa after 72 h	Significance
BDS113	0	27.20 ± 0.37	37.60 ± 0.24					***
	5			8.60 ± 0.24	9.20 ± 0.20	4.60 ± 0.24	5.10 ± 0.10
	10			5.80 ± 0.20	5.90 ± 0.24	2.20 ± 0.20	2.20 ± 0.12
MTCC282	0	26.40 ± 0.40	38.40 ± 0.40					***
	5			10 ± 0.31	11.40 ± 0.24	5.60 ± 0.24	7.70 ± 0.20
	10			5.20 ± 0.20	8.60 ± 0.24	1.50 ± 0.22	2.20 ± 0.20
MTCC2196	0	24 ± 0.31	34.80 ± 0.20					***
	5			9.80 ± 0.20	11.40 ± 0.24	5.80 ± 0.37	7.20 ± 0.20
	10			4.60 ± 0.24	6.80 ± 0.20	2 ± 0.31	2.100 ± 0.100
A. fumigatus	0	20.40 ± 0.24	36.50 ± 0.31					***
	5			7.40 ± 0.24	9 ± 0.15	3 ± 0.31	4.50 ± 0.31
	10			5 ± 0.3162	6.200 ± 0.200	1.2 ± 0.2	1.20 ± 0.20

Mean of zone diameter ± standard error (nm) and significance at (***) p  ≤  0.01.

3.3 Morphological study of fungal cells by SEM

Fungal samples from control colonies and 10 ppm (M‐ and N‐hexa)‐dosed colonies were subjected to FE‐SEM. Both the M‐ and N‐hexa‐treated fungi showed significant fungicidal action in comparison with the control. However, the situation is more abrasive in case of N‐hexa‐treated fungal sample. A healthy, uniform, regular, vasiform, uninterrupted conidiophores were seen for the control hyphae under FE‐SEM, whereas the treated samples had shrivelled, inchoate morphology. However, additional dramatic changes were observed in the hyphal cell surface of N‐hexa‐treated fungi (Fig. 4). The mechanistic interpretation of the abrasive action of N‐hexa might be due to the size specific penetrating capacity of nano‐formulation to cross the intact cell membrane via pores and caused severe damage to the cell wall [27].

Fig. 4.

FE‐SEM image of the treated fungal sample

(a) Control conidia, (b) M‐hexa‐treated fungal sample showing malformed conidia, (c) N‐hexa showing additional damage to the membrane of the treated fungal sample. Arrows demonstrated the membrane damage

3.4 Benefits of nanohexaconazole (N‐hexa) over the oldest known antimicrobial agent hexaconazole

Hexaconazole (hexa) is a commonly used plant antifungal drug. The bulk formulation has a distinct disadvantage. It is poorly water soluble, adding these many fungal species are becoming resistant to azole, necessitating the use of organic solvents that adds to the environmental burden [28]. PEG is well known as a bio‐friendly polymer [29]. The nanodispersion with PEG is water soluble, and therefore has increased applicability. Moreover, it has better solubility in water than the M‐hexa and preliminary studies indicate that it is more photo‐stable than its bulk counterpart. The performed antifungal studies proved that N‐hexa was more efficacious than the M‐hexa in controlling the growth of several species of Aspergillus. The cell wall penetrating capacity of nano‐formulation made it more efficacious. It is also more eco‐friendly and cost‐effective too. It is attempted to increase the scope of use of a common fungicide such that the novel nanodispersion facilitates: (a) increased solubility, (b) used against species that are recalcitrant to currently used bulk formulations and (c) decreased overuse of hexaconazole. Hexaconazole (hexa) is not the antifungal of choice in clinical applications, but this study reveals the possibility of developing, non‐traditional azole nanodispersions as possible candidates for drug development against human opportunistic fungal pathogens.

4 Conclusion

To the best of our knowledge, this is the first report of fungicidal activity of N‐hexa against Aspergillus sp. Fungicidal activity against A. fumigatus an opportunistic human pathogen is significantly notable. N‐hexa was found to be more abrasive than their micron form in all the fungal strains. The bioactivity of N‐hexa was many folds higher as compared with conventional formulations against all the strains of Aspergillus. This report presents the preliminary studies on the toxic impact of N‐hexa (synthesised by a simple step at neutral pH) on food borne pathogen Aspergillus at different concentrations which are significantly effective in low doses, thereby reveals N‐hexa as an eco‐friendly pesticide. Furthermore studies would be needed to validate this preliminary study.