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Applied Nanoscience

, Volume 8, Issue 3, pp 297–307 | Cite as

Preparation, characterization, and evaluation of azoxystrobin nanosuspension produced by wet media milling

  • Junwei Yao
  • Bo Cui
  • Xiang Zhao
  • Yan Wang
  • Zhanghua Zeng
  • Changjiao Sun
  • Dongsheng Yang
  • Guoqiang Liu
  • Jinming Gao
  • Haixin Cui
Open Access
Original Article
  • 504 Downloads

Abstract

To improve the bioavailability of the poorly water-soluble fungicide, an azoxystrobin nanosuspension was prepared by the wet media milling method. Due to their reduced mean particle size and polydispersity index, 1-Dodecanesulfonic acid sodium salt and polyvinylpyrrolidone K30 were selected from six conventional surfactants, the content only accounting for 15% of the active compound. The mean particle size, polydispersity index, and \(\zeta\) potential of the nanosuspension were determined to be 238.1 ± 1.5 nm, 0.17 ± 0.02 and − 31.8 ± 0.3 mV, respectively. The lyophilized nanosuspension mainly retained crystalline state, with only a little amorphous content as determined by powder X-ray diffraction. Compared to conventional fungicide formulations, the nanosuspension presented an increased retention volume and a reduced contact angle, indicating enhanced wettability and adhesion. In addition, the azoxystrobin nanosuspension showed the highest antifungal activity, with a medial lethal concentration of 1.4243 μg/mL against Fusarium oxysporum. In optical micrographs, hyphal deformations of thinner and intertwined hyphae were detected in the exposed group. Compared to the control group, the total soluble protein content, superoxide dismutase, and catalase activities were initially increased and then decreased with prolonged exposure time. The azoxystrobin nanosuspension reduced the defensive antioxidant capability of Fusarium oxysporum and resulted in the generation of excessive reactive oxygen species. This study provides a novel method for preparing nanosuspension formulation of poorly soluble antifungal agents to enhance the biological activity and decrease the negative environmental impact.

Keywords

Azoxystrobin Nanosuspension Antifungal activity Fusarium oxysporum Wet media milling 

Introduction

Pesticides are vital to ensure modern agricultural harvests by controlling pests, diseases, and weeds. The problems of low bioavailability and extensive pesticide residues have caused the pollution of product, water and soil as well as the development of pesticide resistant (Bai et al. 2013; Radović et al. 2015; Castro et al. 2016). Currently, exceeding 40% of commercially available drugs and 90% of novel active pharmaceutical components are extremely lipophilic/hydrophobic and exhibit poorly aqueous solubility (Krupa et al. 2016). As shown in Fig. 1, azoxystrobin, [methyl (E)-2-{2-[6-(2-cyanophenoxy)-pyrimidin-4-yloxy]phenyl}-3-methoxyacrylate], is a strobilurin fungicide. A mitochondrial respiration inhibitor, azoxystrobin, is used to protect crops, vegetables, and fruits in the worldwide (Rodrigues et al. 2013; Kumari et al. 2015; Qin et al. 2016). Compounds with aqueous solubility less than 100 μg/mL have been defined as highly insoluble (Taylor and Zhang 2016). With an aqueous solubility of 6.7 μg/mL, azoxystrobin has the characteristic of poorly soluble, typically resulting in a low bioavailability (Ghosh and Singh 2009; Kumari et al. 2015; Symonds et al. 2016). A new fungicide formulation with excellent aqueous solubility and low cost is essential for more effective utilization of azoxystrobin. Therefore, the enhancement of solubility is a challenging task in the development of azoxystrobin formulations.
Fig. 1

The chemical structure of azoxystrobin

Basically, two approaches can be used to increase the solubility of a chemical component: physical techniques and chemical modifications (Mirza 2017). Physical techniques primarily include high-pressure homogenization, wet media milling, and carrier co-precipitation. In chemical modification, insoluble components are grafted with hydrophilic groups or transformed into salt forms. According to the Nernst–Brunner equation, a substance’s solubility in water is negatively correlated with its particle size (Brough and Williams 2013). Hence, reducing the particle size is an effective approach to improve the solubility of hydrophobic compounds. With the development of nanotechnology, the application of nanomaterials has attracted wide attention in the field of agriculture, such as pesticide and fertilizer (Kah and Hofmann 2014). Recently, nanosuspensions with increased particle surface area have become one of the most promising formulations to enhance solubility (Yadollahi et al. 2015; Kumar Singh et al. 2016). Wet media milling has been regarded as a top-down approach for the industrial production of nanosuspensions and benefits from high efficiency, low cost, and free of organic residue (Ghosh et al. 2012; Li et al. 2016). Based on these superiorities, wet media milling provides a novel and easy method to produce poorly soluble fungicides.

Azoxystrobin inhibits mitochondrial electron transport in the respiratory chain as most strobilurin fungicides. The inhibitors accelerate electrons escaping from mitochondria, which is hastened by the generation of reactive oxygen species (ROS) (Turrens and Boveris 1980; Olsvik et al. 2010). The fungi form a set of antioxidant defense system inclusive of superoxide dismutase (SOD) and catalase (CAT) (Azevedo et al. 2007). However, excess ROS at the early stages of mitochondrial disruption can lead to fungi death (Inoue et al. 2011).

In this study, an azoxystrobin nanosuspension was prepared by wet media milling. The particle size and \(\zeta\) potential of the nanosuspension were measured by dynamic light scattering (DLS). The morphology and structure of the nanoparticles were characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The contact angle and retention volume on the leaves of cucumbers and cabbages were measured. Compared with commercially available formulations, antifungal activity of the nanosuspension was tested by potato dextrose agar (PDA) assay. The morphologies of Fusarium oxysporum were observed by optic microscopy and SEM. This study also evaluated the effects of the azoxystrobin nanosuspension on fungal protein content, SOD, and CAT.

Experimental section

Materials

Azoxystrobin (97%) was purchased from Hubei Sheng Tianheng record Biological Technology Co., Ltd. (Hubei, China). 1-Dodecanesulfonic acid sodium salt (SDS), polyvinylpyrrolidone K30 (PVP K30), hexadecyl trimethyl ammonium chloride (CTAC), and polyoxyethylene sorbitan monooleate (Tween 80) were provided by J&K Scientific Ltd. (Beijing, China). Poloxamer 188 (F68) was obtained from Sigma-Aldrich (Shanghai, China). Polycarboxylate was provided as a gift by Sinvochem S&D Co., Ltd. (Jiangsu, China). Commercially available water dispersible granules (WDG) were purchased from Jiangsu KWIN Group (WDG-A) and Jiangsu Huifeng Agrochemical Co., Ltd. (WDG-B). HPLC-grade methanol was obtained from Thermal Fisher Scientific (Tustin, CA, USA). Deionized Milli-Q water was used in all experiments (18.2 MΩ·cm, TOC ≤ 4 ppb).

Methods

Preparation of azoxystrobin nanosuspension

Azoxystrobin nanosuspension was manufactured by wet media milling. In brief, azoxystrobin powder was dispersed in an aqueous solution containing one of the six surfactants (SDS, PVP K30, F68, Tween 80, CTAC, and polycarboxylate), and the solution was subjected to mechanical stirring (Ika, mod. RW 20 digital, Germany) at 1000 rpm for 30 min. The suspension (6% azoxystrobin) was then milled with the milling media apparatus (WG-0.3 L, Vgreen nano-tech, China). The grinding chamber (0.3 L) was made of silicon carbide, and 80% of the chamber was filled with 0.3-mm zirconium oxide beads as the milling medium. The azoxystrobin crystals were fragmented into nanoparticles by the physical impact of the zirconium oxide beads at a speed of 2200 rpm.

Particle size and \(\zeta\) potential determination of the nanosuspension

The mean particle size, polydispersity index (PDI), and \(\zeta\) potential of the nanosuspension were analyzed by DLS with a Zetasizer Nano ZS90 (Malvern Instruments, UK) at room temperature. Each sample was measured three times for reliability.

Morphological characterization of the nanoparticles by SEM and TEM

The morphology of the azoxystrobin nanosuspension was evaluated using SEM (SU8010, Hitachi, Tokyo, Japan) at an acceleration voltage of 5 kV. The sample was placed on a clean silicon slice, dried at room temperature, and then sputtered with platinum under vacuum (EM ACE600, Leica, Germany). The size and morphology of the nanosuspension were characterized by TEM (HT7700, Hitachi, Tokyo, Japan) at an operational voltage of 80 kV. The sample was dropped on carbon-coated 300-mesh copper grids.

Powder X-ray diffraction analysis of the nanoparticles

For X-ray diffraction analysis, the water of azoxystrobin nanosuspension was removed by lyophilization (FD-81, EYELA, Tokyo, Japan). The patterns of samples were analyzed by a diffractometer (D8 ADVANCE, Bruker AXS Inc., Karlsruhe, Germany) with a Cu Kα radiation source, operated at 40-kV voltage and 40-mA current. Scans were recorded with a detector rate of 0.2°/min and 2θ range from 5 to 50°.

Wetting and spreading characteristics

Hydrophobic leaf of cabbage and hydrophilic leaf of cucumber were cultivated by light growth incubator. The contact angles of the droplets were measured with a contact angle apparatus (JC2000D2 M, Zhongchen Digital Technology Apparatus, Shanghai, China). The 7-μL diluted suspension was dropped onto the leaf using a 50-μL syringe.

The retention of the sample on the leaf was determined by the dipping method. A 15-mm-diameter leaf was perforated by a hole punch and weighed as M1. The leaf was immersed in the diluted suspension for 10 s. Until no more droplets falling, the leaf was removed and weighed as M2. The measurements were conducted at room temperature. Five tests were performed for each sample.

Azoxystrobin concentration analysis

The azoxystrobin concentration was estimated by high-performance liquid chromatography (HPLC) (Agilent 1260 series HPLC, Agilent Technologies) using a Zorbax Carbohydrate Analysis column (150 mm × 4.6 mm × 5 μm) and a 254-nm UV detector. The mobile phase consisted of methanol and water (75:25, v/v) at a flow rate of 0.8 mL/min.

In vitro dissolution

The suspensions commensurate with effective azoxystrobin were put into dialysis bags (2000 MWCO). The bags were then suspended in 100 mL of an ethanol/deionized water solution (50:50, v/v) as the release medium. The solution was shaken at 100 rpm at 28 °C using a constant temperature table (THZ-98C, Shanghai, China). A 2 mL of the outside release medium was removed at different timed intervals. Meanwhile, 2 mL of fresh mix solution was added back into the sustained release system. For quantitative analysis, the azoxystrobin concentration in the 2 mL of release medium was determined by HPLC.

Fungal antagonism assays

To examine fungal antagonism, Fusarium oxysporum was used for the antifungal activity test with PDA assay. Various azoxystrobin formulations containing the nanosuspension, WDG-A or WDG-B were precisely prepared with the following concentrations: 0.1, 0.25, 0.5, 1.0 and 5.0 μg/mL. A 5-mm-diameter mycelial disc was incubated on the test medium at 28 ± 1 °C for 2 days. The diameter of mycelium growth was determined by the criss-cross method. The toxicity regression equations and the medial lethal concentration (LC50) were calculated by probit analysis using SPSS 20 statistical software (IBM Corp., Armonk, NY, USA). Each experiment was implemented in triplicate.

Fungal hyphae microscopy analysis

Fusarium oxysporum was cultured with PDA, and then a 10-mm-diameter mycelial disc was incubated in the potato dextrose broth (PDB) medium at 28 ± 1 °C for 2 days. To evaluate the effect of the azoxystrobin nanosuspension on Fusarium oxysporum, the azoxystrobin nanosuspension with a final concentration of 5.0 μg/mL was added to the PDB medium. The hyphae from control and azoxystrobin-treated groups were collected after 24 h. Bright field microscopy images were observed using an inverted fluorescence microscope (Olympus IX71, Tokyo, Japan) with a 40-time objective lens. Furthermore, the hyphae were fixed with a 2.5% glutaraldehyde solution at 4 °C overnight. The fixed hyphae were washed with phosphate-buffered saline (PBS) solution (pH 7.4) three times for 10 min. Afterward, the washed hyphae were dehydrated with 50, 70, 90, and 100% ethanol for 10 min and then were further dehydrated with absolute ethanol for 20 min. The dehydrated hyphae were dropped onto clean silicon slices and sputtered with platinum under vacuum (EM ACE600, Leica, Germany). The morphology of the hyphae was recorded with SEM (SU8010, Hitachi, Tokyo, Japan) at an acceleration voltage of 5 kV.

SOD and CAT assays

The Fusarium oxysporum exposed to azoxystrobin nanosuspension (5 μg/mL) was collected to extract protein at different times after exposure for 1, 6, 18, 24, and 48 h, respectively. Protein was extracted by milling hyphae with a glass homogenizer in an ice bath. The homogenate was centrifuged (SORVALL ST16R, Thermo Scientific, MA, USA) at 10,000 rpm for 20 min at 4 °C. The supernatant was collected and used for the analysis of antioxidant enzyme activities. The concentration of soluble protein was measured by the Bradford method (1976) using bovine serum albumin (BSA) as a standard protein. SOD and CAT activities were determined by UV spectrophotometry (UV-2600, Shimadzu, Japan) using commercial assay kits (Jiancheng Institute, Nanjing, China).

Statistical analysis

Data analysis was performed using SPSS 20 statistical software (IBM Corp., Armonk, NY, USA). Data were presented as the mean ± standard deviation (SD). Quantitative data of contact angle and retention volume were evaluated with a one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) method. A probability less than 0.05 was considered statistically significant. The other quantitative data were examined using a one-way ANOVA followed by the Student-Newman-Keuls (SNK) method.

Results and discussion

Preparation of the azoxystrobin nanosuspension

The influence of six surfactants on the nanosuspension was examined using mean particle size and PDI as measuring indices. Nanosuspensions containing 6% (w/w) azoxystrobin and 0.9% surfactant (w/w) were prepared by wet media milling at 2200 rpm for 20 min. As shown in Table 1, surfactants had significant effects on the mean particle size and the dispersity of the suspension. Among the six investigated surfactants, SDS and PVP K30 were screened out, which could reduce the mean particle size of the suspension less than 400 nm and PDI no more than 0.25. A PDI less than 0.25 suggested that the suspension had a narrow distribution (Ibrahim et al. 2014; Ninjbadgar et al. 2015). During the milling process, nanoparticle agglomeration was mainly caused by van der Waals forces and electrostatic interactions (Rance et al. 2010; Moerz and Huber 2015). For anionic surfactant, SDS could provide sufficient electric charge to induce electrostatic repulsion for the suspended particles (Teeranachaideekul et al. 2008). For covering the surface of the particles, nonionic surfactant PVP K30 could prevent nanosuspensions from aggregating by the steric hindrance effect (Palermo et al. 2012). Appropriate surfactants were crucial to inhibit particle agglomeration and to keep the system stable (Elsayed et al. 2014). To combine electrostatic repulsion and steric stabilization, SDS and PVP K30 (1:1, w/w) were mixed to prepare the azoxystrobin nanosuspension. As shown in Fig. 2, particle size and PDI did not decrease further with extended grinding time (George and Ghosh 2013). In contrast, a wet grinding time of more than 90 min caused the particle size to increase and the PDI to broaden. A potential reason for this effect was that high shear mechanical force could lead to secondary aggregation during milling, depending on the temperature (Kargosha and Shirazi 2015).
Table 1

The effect of surfactant on the mean particle size and PDI of the nanosuspension

Surfactant

Mean size (d.nm)

PDI

Tween 80

2054.0 ± 216.20

0.52 ± 0.07

F68

1233.3 ± 39.11

0.59 ± 0.07

SDS

307.5 ± 5.09

0.18 ± 0.01

Polycarboxylate

854.7 ± 60.25

0.45 ± 0.07

PVP K30

344.9 ± 5.39

0.07 ± 0.02

CTAC

661.0 ± 8.63

0.16 ± 0.03

Fig. 2

The effect of milling time on the characteristics of the nanosuspension. (a) mean particle size; (b) PDI

The particle size and \(\zeta\) potential

The mean particle size, D90 and PDI of the aqueous diluted nanosuspension measured by DLS were 238.1 ± 1.5 nm, 463.7 ± 17.7 nm, and 0.17 ± 0.02, respectively. A suspension with an absolute \(\zeta\) potential higher than 30 mV implicated a stable system by electrostatic repulsion (Pamies et al. 2014). As shown in Fig. 3b, the \(\zeta\) potential of the aqueous diluted nanosuspension was − 31.8 ± 0.3 mV. The highly negative \(\zeta\) potential value can be attributed to anionic SDS and indicates excellent stability.
Fig. 3

The characteristic of nanosuspension by DLS. a hydrodynamic particle size; b \(\zeta\) potential

Morphology

To evaluate the shape of the nanoparticles, samples were observed by SEM and TEM. Based on Fig. 4a, c, the particles exhibited irregular bulk shape. The sizes of particles were from 80 to 530 nm, with an average diameter of around 200 nm, which was approximately 16% smaller than that measured by DLS. The reason may lie in the disparate particle morphology (Cho et al. 2004). Electron microscopy images showed the veritable size of a single particle in a dried state (Mcallister et al. 2016). In contrast, the size measured by dynamic light scattering technique was a hydrated state value of spherical particles, reflecting a single particle or aggregates (Zhong and Jin 2009; Singh et al. 2017).
Fig. 4

The morphologies of the nanosuspension characterized by electron microscopy. a SEM image; b statistical particle size based on SEM; c TEM image; d statistical particle size based on TEM

Crystalline state

As shown in the X-ray diffraction spectrum (Fig. 5), the characteristic peaks of lyophilized azoxystrobin nanoparticles were at 11.0, 13.8, 17.7, 19.1 and 22.2°. The characteristic diffraction peaks of the nanoparticles could be primarily attributed to azoxystrobin as it accounted for 80% of the ingredients in the lyophilized powders. As previously reported, the wet milling process induced violent collisions between the milling media and the substance, which can destroy or disorder the crystal structure (Lee et al. 2015). During the grinding period, the high rate of rotation of the zirconium oxide media could bring out massive heat and mechanical shearing force, which probably enhanced lattice vibrations and changed the crystalline state into an amorphous one (George and Ghosh 2013; Ito et al. 2016). In other words, wet media milling may cause changes in the physical structure of the organic components such as crystal defects or crystalline transition. XRD analysis revealed that lyophilized nanoparticles principally maintained a crystalline state, only a little of amorphous content.
Fig. 5

XRD patterns of lyophilized azoxystrobin nanoparticles and related ingredients

In vitro dissolution of various azoxystrobin formulations

The release kinetics of azoxystrobin in different formulations were shown in Fig. 6. After fitting the accumulated release data with multiple kinetic models, the results demonstrated that the release kinetic profiles were well corresponding with the first-order kinetics, with R2 value over 0.99. As seen from the accumulated release trend, 50% release from the nanosuspension, WDG-A and WDG-B was observed at 0.99, 3.93, and 4.33 h, respectively. In previous research describing the impact of nanosizing on dissolution rates, griseofulvin nanocrystals took less than 2 min to release 80% of the nanocrystals, while a micronized suspension required more than 20 min (Murdande et al. 2015). A similar phenomenon was previously observed using paliperidone palmitate nanosuspensions with various particle size (Leng et al. 2014). Likewise, the azoxystrobin nanosuspension showed an enhancing dissolution rate compared to the commercial WDG formulation. Based on the Ostwald-Freundlich equation, particle size had a significant effect on the dissolution rate of poorly soluble compounds (Murdande et al. 2015). Furthermore, increased surface area most likely improved dissolution rates according to the Noyes-Whitney equation. The improved dissolution rate of the azoxystrobin nanosuspension was a result of the substantially reduced particle size.
Fig. 6

Cumulative release profiles of azoxystrobin derived from nanosuspension (NS), WDG-A and WDG-B

Stability of the nanosuspension

As crucial indices to evaluate stability, the particle size and distribution of the nanosuspension were determined by DLS (Ali et al. 2009). To estimate the effect of temperature on azoxystrobin nanosuspension stability, the mean particle size and distribution changes were recorded after storage at 4, 25 and 54 °C, respectively. As shown in Fig. 7a, the mean particle size of the nanosuspension increased from 238 to 288 nm over 14 days of storage at 25 °C. As seen from Fig. 7b, a similar particle size variation was observed at 4 °C for 7 days. The initial mean particle size increased by 30% during storage for 14 days at 54 °C (Fig. 7c). A reasonable explanation was that nanoparticle agglomeration likely occurred during high-temperature storage (Yue et al. 2015). PDI reflected the distribution width of particle size and was another reference index to characterize suspension stability (Holzapfel et al. 2005). The PDIs of the nanosuspensions stored at different temperatures were all less than 0.25, indicating a narrow particle distribution (Fig. 7).
Fig. 7

The effect of temperature on particle size and PDI. a 25 °C; b 4 °C; c 54 °C

Wetting and spreading characteristics

To evaluate the wettability behavior of the nanosuspension on cucumber and cabbage leaves, contact angles and retention volumes were measured. The contact angle was a vital criterion to assess the hydrophilic and hydrophobic properties of suspensions (Sun et al. 2016). As shown in Fig. 8, the contact angle of water on the cucumber leaf surface was 75° (less than 90°), reflecting the hydrophilic characteristic of cucumber leaf (Lin et al. 2016). On the contrary, the contact angle of water on a cabbage leaf was 128°, implying a hydrophobic characteristic. On cucumber leaves, the contact angles of the nanosuspension, WDG-A, and WDG-B were 63° ± 5°, 73° ± 2°, and 71° ± 3°, respectively. Meanwhile, the contact angles on cabbage surfaces were 112° ± 4°, 117° ± 5°, and 121° ± 4°, respectively. The smaller contact angle of the droplet indicated that the liquid was easier to spread and wet on the leaf surface (Zeng et al. 2015). Significantly different contact angles were observed when comparing the nanosuspension to WDG-A and WDG-B. The azoxystrobin nanosuspension possessed smaller contact angles, which led to an enhancing wettability and spreading performance on cucumber and cabbage leaves. Retention volumes on cucumber and cabbage leaves were presented in Fig. 9. On cucumber leaves, the retention volumes of nanosuspension, WDG-A, and WDG-B were 25.04 ± 2.47, 21.34 ± 0.95 and 21.93 ± 2.48 mg/cm2, respectively. On cabbage leaves, the retention volumes of nanosuspension, WDG-A, and WDG-B were 24.75 ± 2.39, 16.12 ± 1.93 and 20.63 ± 1.36 mg/cm2, respectively. The retention volumes of the nanosuspension on cucumber and cabbage surfaces were higher than the commercially available WDG formulation, which may bring about the reduction in the pesticide dosage.
Fig. 8

The contact angles of azoxystrobin formulations on cucumber and cabbage surfaces (one-way ANOVA, followed by LSD test, *p < 0.05, **p < 0.01)

Fig. 9

The retention volumes of azoxystrobin formulations on cucumber and cabbage surfaces (one-way ANOVA, followed by LSD test, *p < 0.05, **p < 0.01)

Antifungal activity evaluation

In this study, the PDA assay was evaluated the antifungal activity of three azoxystrobin formulations. The LC50 values were used as a measurement antifungal activity of azoxystrobin against Fusarium oxysporum. As shown in Table 2, the azoxystrobin nanosuspension exhibited the highest toxicity with an LC50 of 1.4243 μg/mL, followed by WDG-A and WDG-B with LC50 values of 2.4668 and 2.4221 μg/mL, respectively. The lower LC50 value of the azoxystrobin nanosuspension showed that its toxicity index was around 1.7-fold that of the others. As reported, the antifungal activity of azoxystrobin was attributed to its ability to bind the Q0 site of complex III in the fungus mitochondria, which resulted in the inhibition of mitochondrial respiration and blocked the synthesis of ATP (Gao et al. 2002). It was well known that the small size of nanoparticles increases their ability to penetrate cell membranes, giving them access to their target site (Verma et al. 2008). In conclusion, the antifungal activity of the azoxystrobin nanosuspension was superior to the conventional WDG formulations.
Table 2

Indoor toxicity of three azoxystrobin formulations against Fusarium oxysporum

Formulation

Regressive equation

R 2

LC50 (μg/mL)

Toxicity index

Nanosuspension

y = 5.4764 + 0.1929 x

0.9971

1.4243

1.7319

WDG-A

y = 5.4025 + 0.2440 x

0.9937

2.4668

1

WDG-B

y = 5.4433 + 0.1465 x

0.9993

2.4221

1.0185

Effect on mycelial morphology

The Fusarium oxysporum optical morphology is illustrated in Fig. 10. Figure 10a reflected the control group, which maintained intact, chiseled septa, and a strong budding reproductive capacity. In contrast, Fig. 10b displayed that the hyphae tended to intertwine when exposed to the azoxystrobin nanosuspension. In comparison to the control group, the azoxystrobin nanosuspension caused deformation in the fungi, with the hyphae being thinner and the septa being poorly defined. A similar phenomenon was previously presented in the E. salmonicolor with different concentrations of ZnO nanoparticles (Arciniegas-Grijalba et al. 2017). The Fusarium oxysporum microstructure was shown in Fig. 11. Figure 11a displayed normal mycelia with smooth walls and intact surfaces without shrinkage. However, the mycelia treated with the azoxystrobin nanosuspension became severely shrunk and crumpled (Fig. 11b). In a previous study, Chlorella vulgaris cells were also apparently depressed or shrunk in 300 or 600 μg/L azoxystrobin treatments (Liu et al. 2015). When the azoxystrobin nanosuspension penetrated the Fusarium oxysporum cell wall and impacted on the mitochondria target site, it can disturb the cell wall and enhance its permeability. From the mycelial morphological examinations, azoxystrobin nanosuspension indeed inhibited the growth of Fusarium oxysporum.
Fig. 10

Optical microscopy images of Fusarium oxysporum hyphal structures. a control; b treatment with azoxystrobin nanosuspension

Fig. 11

SEM images of Fusarium oxysporum hyphal structures. a control; b treatment with azoxystrobin nanosuspension

Effect on protein content, SOD, and CAT

Compared to the control group, the protein content of Fusarium oxysporum decreased until 18 h after exposure to the azoxystrobin nanosuspension (Fig. 12a). During the first 6 h of azoxystrobin nanosuspension exposure, the protein content increased. As a result of that, an oxidative stress response was generated and induced corresponding protein (Han et al. 2013; Hernández-Gea et al. 2013; Fucho et al. 2017). SOD enzymes, such as Mn-SOD in mitochondria and CuZn-SOD in cytosol, can eliminate superoxide anion-free radicals and protect organisms from oxidative damage (Mansuroğlu et al. 2015). As shown in Fig. 12b, the total SOD activity of the exposed group was less than that of the control group after 24 h. The decreasing SOD activity suggested that the defense system of Fusarium oxysporum was compromised. Azoxystrobin was a well-known mitochondrial respiration inhibitor. Therefore, the activity of Mn-SOD was investigated. Compared to that of the control group, a significant reduction in Mn-SOD activity was detected in azoxystrobin nanosuspension treatment (Fig. 12c). As seen from Fig. 12b, c, total SOD was principally derived from CuZn-SOD in the cytosol. CAT was a vital enzyme in antioxidant systems as protecting organisms from ROS oxidative damage. The scavenge of H2O2 was mainly catalyzed by CAT(Kaneko and Ishii 2009). The CAT activity of the exposed group was approximately 2.53-fold higher than that of the control group after 1 h (Fig. 12d) and then gradually decreased over time. These results implicated that H2O2 was enriched in fungi of the exposed group and led to oxidative damage. With prolonged exposure time, the decreasing activities of SOD and CAT indicated that the azoxystrobin nanosuspension could reduce the antioxidant defenses of Fusarium oxysporum.
Fig. 12

Effect of the azoxystrobin nanosuspension on total protein content and anti-oxidase activities. a total protein; b SOD; c Mn-SOD; d CAT

Conclusions

For scale-up manufacture without organic residues, wet media milling technique has become more prevalent to enhance bioavailability of poorly soluble substance. In this study, the anionic surfactant SDS and polymeric PVP K30 were optimized from six conventional surfactants for the preparation of azoxystrobin nanosuspensions by wet media milling. The average diameter of the nanosuspension was approximately 200 nm in the dried state and the \(\zeta\) potential was  –31.8 mV. Compared to WDG-A and WDG-B, the nanosuspension possessed the greater retention and the smaller contact angle on hydrophobic cabbage and hydrophilic cucumber leaves. Furthermore, the toxicity index of the azoxystrobin nanosuspension was approximately 1.7-fold that of the other formulations against Fusarium oxysporum. Morphological alterations of Fusarium oxysporum were observed by optical microscopy and SEM. The hyphal deformations disclosed that the azoxystrobin nanosuspension can disturb cell walls and enhance cell wall permeability. Antioxidant enzyme activities were affected by the azoxystrobin nanosuspension and Fusarium oxysporum was more susceptible to oxidative damage. In conclusion, these findings reveal that the nanosuspension produced by wet media milling is a desirable nanoformulation for azoxystrobin to improve the antifungal activity.

Notes

Acknowledgements

This study was financially supported by the Major National Scientific Research Program of China (No. 2014CB932200), the National Key Research and Development Program of China (2017YFD0201207, 2016YFD0200502), and the Agricultural Science and Technology Innovation Program (CAAS-XTCX2016004).

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Junwei Yao
    • 1
    • 2
  • Bo Cui
    • 1
  • Xiang Zhao
    • 1
  • Yan Wang
    • 1
  • Zhanghua Zeng
    • 1
  • Changjiao Sun
    • 1
  • Dongsheng Yang
    • 1
  • Guoqiang Liu
    • 1
  • Jinming Gao
    • 2
  • Haixin Cui
    • 1
  1. 1.Institute of Environment and Sustainable Development in AgricultureChinese Academy of Agricultural SciencesBeijingChina
  2. 2.Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of Chemistry & PharmacyNorthwest A&F UniversityYanglingChina

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