Introduction

In order to feed the expanding global population, the traditional organic pesticides were intensively used in modern agricultural production to maintain high crop yields1. However, doing that without negative influence on environment2,3 and public health4,5,6 is still a great challenge7. Luckily, the rapid development of nanotechnology provides the alternative strategies to achieve such goals. The inorganic NPs such as Ag, CuO, MgO and ZnO have been demonstrated effective antimicrobial activities singly8,9,10,11,12,13 or combined with organic drugs14,15 in previous studies. However, to the best of our knowledge, few papers have studied the combined antimicrobial effect of inorganic NPs with organic pesticides for plant protection, let alone demonstrated synergistic effect. It is meaningful to investigate the joint effect of such combination for increasing antimicrobial activity, reducing pesticide usage and delaying the development of resistance16. More importantly, for semiconductor NPs, the combination also provides the feasibility to eliminate the pesticide residue due to the photocatalytic activity. Therefore, it prompted us to set up a novel green nanotechnology which control pathogen microorganism with a synergistic antimicrobial activity and successive photocatalytic degrade pesticide residue to protect plant efficiently and environmental friendly. Such goals led us to focus on ZnO NPs which are cheap, stable17, sensitive to pathogen fungi18,19, biocompatibility to human cells18,20,21 and have the essential excellent photocatalytic activity22.

Some reports indicated the bonding between antibiotics and NPs could result in synergistic effect14,23. From this point of view, we envisaged thiram, a widely used dithiocarbamate pesticide, could bind with Zn atoms at ZnO crystal surface to form composite antifungal system for the sulfur atoms in its structure and therefore cause more destruction effect by increasing local concentration of the antimicrobial agents14. Thus, thiram was expected to show synergistic antifungal activity with ZnO NPs. In addition, as thiram have adverse effect on human cells24,25, hepatic system26, reproductive system27 and was high toxicity to fish28 and non-target bacteria29, we employed thiram in our investigation for enhancing its pesticides efficiency and minimizing the negative influence on human health and environment.

Our following experiments firstly established a composite antifungal system that successfully showed the synergistic antifungal activity of ZnO NPs with thiram. Furthermore, the photocatalytic degradation of thiram under simulated sunlight irradiation ensured eliminating hazardous thiram residue. 0.25 g/L ZnO NPs and 0.01 g/L thiram were selected as the optimal concentrations for above two components. Infrared spectroscopy, zeta potential analysis and scanning electron microscopy indicated the formation of ZnO-thiram antifungal system, electrostatic adsorption of ZnO NPs to fungi cells and the cellular internalization of ZnO-thiram composite played important roles in enhancing antifungal activity. Oxidative stress test showed thiram could enhance the ZnO-induced oxidative damage to fungi cells that finally result in synergistic antifungal effect. These achievements offered the opportunity to establish the residue-free green nanotechnology. It is necessary to note that this green nanotechnology has both great economic returns and social benefits by tremendously decreasing the potential adverse influence on environment and public health.

Results

Preparation of composite antifungal system

Characterization of ZnO NPs used in investigation was provided in Supplementary Section 1. The sizes of ZnO NPs were around 20 nm (Supplementary Figure S1b). The surface charge of ZnO NPs was +20.7 mV and + 15.9 mV in antifungal system and deionized water respectively (Supplementary Figure S1c). Pre-treated ZnO NPs with deionized water enabled adsorption of H2O to particles surface that avoid binding unwanted organic molecule. Due to the stronger binding interaction of sulfur atom than oxygen with zinc atom, thiram molecule could replace water molecule and therefore assembled onto ZnO NPs surface to form composite antifungal system after adding thiram into ZnO NPs suspensions. Infrared Spectroscopy (IR) analysis was performed to study the interaction between the ZnO NPs and thiram. In Figure 1, the absorption located at 428 cm−1 is the characteristic ZnO absorption. Peaks of thiram at 1374, 1235 cm−1 are assigned to stretching vibration and flexural vibrations of C = S. Peak at 1374 cm−1 is assigned to the symmetric deforming vibration of –CH3. After mixing with ZnO NPs, IR spectrum of thiram samples changed at some peaks. For example, peaks at 1374 cm−1 disappeared, peaks at 848, 1374, 1505 and 1578 cm−1 were observed blue-shift for 20, 5, 27, 53 cm−1 respectively. These changes in IR spectrum indicated the weak interaction between ZnO NPs with thiram molecules. IR analysis verified our assumption that the sulfur atoms of C = S in thiram molecule may bind with Zn atom on the surface of ZnO NPs. As a result, composite antifungal systems formed, which were made of ZnO core and surrounding thiram molecules.

Figure 1
figure 1

IR spectra of ZnO (a), thiram (b), ZnO-thiram (c) and Schematic diagram of ZnO NPs combined thiram to form composite antifungal system.

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Synergistic antifungal activity of ZnO NPs with pesticide thiram

ZnO NPs and thiram displayed dose-dependent inhibition on hyphal growth of Phytophthora capsici (Figure 2a and Figure 2b). Figure 2c shows the antifungal activity of thiram in the presence of 0.25 g/L ZnO NPs. In general, ZnO NPs enhanced the antifungal activities of thiram under all tested concentrations. Calculating synergy factor (SF) by Abbortt method is a simple way to assess the interaction between the two components. The SFs for 0.25 g/L ZnO NPs with 0.00125, 0.0025, 0.005, 0.01 g/L thiram are 0.73, 1.15, 1.38 and 1.71 respectively. The SF for 0.02 g/L thiram with ZnO NPs was not calculated because Abbortt method is not suited for high control level thiram concentration16. Generally the synergy is identified when SF ≥ 1.516, so only the 0.25 g/L ZnO with 0.01 g/L thiram demonstrated synergistic interaction as its SF = 1.71. Thus, 0.01 g/L thiram was used for further investigation to evaluate the influence of ZnO NPs concentration on synergistic effect. As been shown in Figure 2d, ZnO NPs of all the concentrations greatly increased the antifungal activities of 0.01 g/L thiram. The inhibition rates were 87%, 95%, 100%, 100%, 100% for 0.05, 0.1, 0.25, 0.5, 1.0 g/L ZnO with 0.01 g/L thiram respectively. The SF were 1.95, 1.72, 1.7 and 1.5 for 0.05, 0.1, 0.25, 0.5 g/L ZnO with 0.01 g/L thiram respectively, so it can be concluded that ZnO NPs could synergistically enhance the antifungal acitivity of 0.01 g/L thiram. The SF of 1.0 g/L ZnO with 0.01 g/L thiram was not calculated due to the excessive inhibition rate of single ZnO. FICIs were also determined to verify the results derived from Abbortt method. According to the MICs of ZnO NPs, thiram and their combinations, the FICIs of 0.05, 0.1, 0.25, 0.5 g/L ZnO NPs combined with 0.01 g/L thiram were 0.275, 0.3, 0.375 and 0.5 respectively, which are lower than or equal to 0.515, suggesting synergistic antifungal effect. This result was consistent with conclusion defined by Abbortt method. According to above results, 0.25 g/L ZnO NPs was considered as the optimal dosage for getting a complete control of plant pathogen Phytophthora capsici growth with lowest ZnO NPs input. It also can be concluded that thiram concentration played a key role in defining the interaction mode of ZnO-thiram joint antifungal activity. The comprehensive investigation on antifungal activities of thiram in the presence of various ZnO NPs concentrations is demonstrated in Supplementary Section 2.

Figure 2
figure 2

Antifungal activities of ZnO NPs, thiram and ZnO-thiram.

Part a, Antifungal activity of ZnO NPs alone. Part b, Antifungal activity of thiram alone. Part c, Antifungal activity of 0.25 g/L ZnO NPs with increasing thiram concentrations. (c1) 0.25 g/L ZnO (green column), 0.00125 g/L thiram (red column), 0.25 g/L ZnO with 0.00125 g/L thiram (blue column); (c2) 0.25 g/L ZnO (green column), 0.0025 g/L thiram (red column), 0.25 g/L ZnO with 0.0025 g/L thiram (blue column); (c3) 0.25 g/L ZnO (green column), 0.005 g/L thiram (red column), 0.25 g/L ZnO with 0.005 g/L thiram (blue column); (c4) 0.25 g/L ZnO (green column), 0.01 g/L thiram (red column), 0.25 g/L ZnO with 0.01 g/L thiram (blue column); (c5) 0.25 g/L ZnO (green column), 0.02 g/L thiram (red column), 0.25 g/L ZnO with 0.02 g/L thiram (blue column). Part d, Antifungal activity of 0.01 g/L thiram with increasing ZnO concentrations. (d1) 0.05 g/L ZnO (green column), 0.01 g/L thiram (red column), 0.05 g/L ZnO with 0.01 g/L thiram (blue column); (d2) 0.1 g/L ZnO (green column), 0.01 g/L thiram (red column), 0.1 g/L ZnO with 0.01 g/L thiram (blue column); (d3) 0.25 g/L ZnO (green column), 0.01 g/L thiram (red column), 0.25 g/L ZnO with 0.01 g/L thiram (blue column); (d4) 0.5 g/L ZnO (green column), 0.01 g/L thiram (red column), 0.5 g/L ZnO with 0.01 g/L thiram (blue column); (d5) 1.0 g/L ZnO (green column), 0.01 g/L thiram (red column), 1.0 g/L ZnO with 0.01 g/L thiram (blue column). Error bars are standard errors with n = 3.

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Photocatalytic degradation of pesticide thiram by ZnO NPs

For the purpose of establishing a residue-free green synergistic antifungal nanotechnology we emphasized at the beginning, the photocatalytic degradation of thiram in the presence of the ZnO NPs were investigated under simulated sunlight irradiation by a 500 W Xeon lamp. At first, we investigated the effect of catalyst concentration on degradation rates of thiram. Because 0.05, 0.1, 0.25, 0.5 g/L ZnO NPs synergistically enhanced the antifungal activity of 0.01 g/L thiram, such concentrations were used for degradation study. As been shown in Figure 3a, it is obvious that the efficiency increased with increasing ZnO NPs concentration up to 0.25 g/L. However, the degradation efficiency maintained when the catalyst amount continued increased to 0.5 g/L. These results were in accord with previous report that the efficiency became nearly flat even decreased when the catalyst concentration above a certain level30. Theoretically, increasing the concentration of the catalyst causes an increase in the degradation rate through providing more active site of the catalyst and accelerates the generation of hydroxyl radical31, whereas, above certain amount, the excessive ZnO NPs decrease light penetration and increase particles agglomeration32,33 that finally leading to decreased catalytic efficiency. These finding suggested the optimal usage of ZnO NPs was 0.25 g/L, which synergistically inhibited the fungal growth by over 90% as well as degraded thiram residue to safety level efficiently. Figure 3b shows completely photocatalytic degradation of thiram by 0.25 g/L ZnO NPs, the absorbance of thiram at 278 nm drop below zero (−0.0013) which is lower than the detection limit (0.00122), suggesting the thiram was totally degraded. The effect of thiram concentration on degradation rates is shown in Figure 3c, it was observed that the degradation efficiency decreased with increasing thiram concentration. This finding suggested a prolonged photocatalytic degradation process required for removing residue at higher thiram dosage. This effect of initial substrate concentration on degradation rates may be illustrated by the fact that a larger number of thiram molecules competed for the adsorption sites, consequently that production of oxidative radicals were insufficient and the degradation rate was reduced34.

Figure 3
figure 3

Photocatalytic degradation of thiram under continuous stimulated sunlight irradiation.

(a) Effect of ZnO NPs concentration on photocatalytic degradation of thiram. (b) Completely photocatalytic degradation of thiram in the presence of 0.25 g/L ZnO NPs. The absorption curve of thiram became nearly flat after 6 h irradiation. (c) Effect of initial thiram concentration on photocatalytic degradation of thiram.

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Morphological analysis of fungal growth

The interaction between ZnO NPs and fungi cells may altered the morphology of fungal samples18,19. Thus, Scanning electron microscopy (SEM) analysis was performed to investigate the influence of antifungal agents on fungal structure and provide useful information of mechanism of antifungal activity. No obvious change of fungal hyphae morphology was detected in 0.0025 g/L thiram sample (Figure 4b) compared with control (Figure 4a). Diminishing in net framework and reduced density of hyphae were observed after they were treated with 1.0 g/L ZnO NPs (Figure 4c). These result indicates intense inhibition on hyphae growth (Figure 4c). Changes in hyphae structures were found at the backside of the agar plates with some irregular objects presented on hyphae (Figure 4d). Under high magnification observation, it was observed the irregular objects connected with hyphae smoothly (Figure 4e), which inidicated these abnormal enlargements were part of hyphae rather than simply attaching on the hyphae. The same morphological changes with abnormal enlargement of hyphae were also found in ZnO-thiram samples (Figure 4f and Figure 4g). Energy-dispersive X-ray (EDX) analysis was performed at the abnormal enlargements of hyphae in pure ZnO treatments. Zincs were found on such enlargements (Figure 4h). This result implied the cellular internalization of ZnO NPs by Phytophthora capsici. Because ZnO NPs could not be digested or metabolized by fungi cells, the accumulating of ZnO NPs at someplace in hyphae therefore led to the formation of abnormal enlargements. Since the ZnO-thiram composite formed, thiram may enter fungi hyphae with the cellular internalization of ZnO NPs. In order to investigate whether thiram deposited on ZnO surface and enter fungi cells with ZnO NPs together, we also used EDX tool to analysis the abnormal enlargements on hyphae in ZnO-thiram treated sample. Demonstrated in Figure 4i, the obvious Zn-peaks were found and suggesting the uptake of ZnO NPs by fungi cells. However, the experimental thiram concentration was too low (0.0025 g/L), so that the observation of S-peak was difficult. In addition, when ZnO-thiram composites entered fungi cells, parts of thiram may consequently disassembled and took part into intracellular life activities, therefore, the signal of sulfur was further reduced. In order to figure out the problem, we tried to increase the thiram concentration to enhance the signal of sulfur in the thiram after entering the hyphae. However, due to the synergistic antifungal effect, increased thiram concentration will lead to totally inhibition on hyphae growth which made incapable to analysis hyphae with EDX tool. For this reason, we performed EDX analysis on ZnO-thiram composite directly. Seen from Figure 4j, an obvious sulfur peak appeared at 2.20, which indicated that thiram successively deposited on the ZnO surface. This was consistent with the IR analysis (Figure 1) and the both result may be confirmed.

Figure 4
figure 4

SEM study and EDX analysis of ZnO NPs and ZnO-thiram treated fungi hyphae.

SEM showed the inhibition of fungi hypha growth and abnormal enlargements (white arrows) on hypha in the presence of ZnO NPs and ZnO-thiram composites: (a) control, (b) 0.0025 g/L thiram, (c–e) 1.0 g/L ZnO, (f and g) 1.0 g/L ZnO with 0.0025 g/L thiram. EDX analysis performed on abnormal enlargements on hyphae: (h) 1.0 g/L ZnO treatment, (i) 1.0 g/L ZnO with 0.0025 g/L thiram treatment, (j) EDX analysis performed on ZnO-thiram composite directly.

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Detection of oxidative stress in synergistic antifungal behavior of ZnO NPs with thiram

Some researches indicated the generation of reactive oxygen species (ROS) as the main mechanism responsible for the antimicrobial activity of nano ZnO13,18,35,36. The generation of ROS such as hydroxyl radical (·OH), superoxide (O2·−) and hydrogen peroxide (H2O2)37,38, induce oxidative stress which can damage cell membranes, nucleic acids and cellular proteins, may lead to cell death41. In our experiment, the oxidative stress was detected in carrot broth containing ZnO NPs, results are showed in Supplementary Section 3 and it suggested the level of oxidative stress correlated to the concentration of ZnO NPs.

In order to investigate the oxidative damage toward fungi cells induced by ZnO NPs, histidine was used in our investigation for its antioxidant property. Histidine was reported to protect living cells against oxidative damage through scavenging free radicals39,40. Optimal usage of histidine was determined according to condition experiment (Supplementary Section 4). In our investigation (Figure 5a), histidine reduced the inhibition rate of fungal growth of both ZnO and ZnO-thiram samples. However, the protective effect of histidine to fungi was more significant in ZnO samples than in ZnO-thiram with reductions of antifungal activities by 24% and 13% respectively. These findings confirmed the oxidative damage to fungi hyphae from ZnO NPs in another aspect. It also can be concluded that the combination of ZnO with thiram enhanced the oxidative stress and damage to fungi cells.

Figure 5
figure 5

Oxidative stress study of ZnO and ZnO-thiram treated samples.

(a): Effect of antioxditant histidine on antifungal activity of 1.0 g/L ZnO, 1.0 g/L ZnO with 0.0025 g/L thiram, 0.0025 g/L thiram. Reductions of antifungal activities were 24% and 13% respectively for ZnO and ZnO-thiram samples. However, no protective effect was observed in thiram sample. (b): SOD activities of fungi cells in the presence of 0.0025 g/L thiram, 1.0 g/L ZnO and 1.0 g/L ZnO with 0.0025 g/L thiram. It shows ZnO-thiram treated sample exhibits a higher SOD activity than ZnO treated alone. The above two test suggest the ZnO-induced oxidative damage were enhanced by thiram. Error bars are standard errors with n = 3.

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As living cell possess a cellular defense system including antioxidant enzymes such as SOD to scavenge and prevent damages caused by ROS18, the SOD activities generally reflected the oxidative stress endured by living cell. Thus, SOD assay was introduced to detect the response of fungi cells exposed to oxidative stress and investigate the mechanism of synergistic antifungal activity of ZnO-thiram composite antifungal system. Interestingly, the SOD activities increased in a dose-dependent manner was observed (Supplementary Figure S5), 41.0, 58.5 and 100.7 U/mg protein of SOD activity were detected in 0.5, 1.0 and 2.0 g/L ZnO NPs samples respectively, in contrast, the control set was 28.1 U/mg protein of SOD activity. This finding suggested that ZnO NPs induced the formation of ROS and imposed oxidative stress to fungi cells. Similar results was reported by Patra et al18 that ZnO NPs induce the increase of SOD activity in two fungi strains. Further experiments were conducted to investigate the influence of thiram and ZnO-thiram combination. It was found 0.0025 g/L thiram did not induce an increasing of SOD activity, instead, it slightly reduced SOD activity compared to control (Figure 5b). It was speculated that the toxicity of thiram to Phytophthora capsici was not based on formation of oxidative stress. However, investigation demonstrated that when 0.0025 g/L thiram was introduced into 1.0 g/L ZnO NPs, the SOD activity level (91.0 U/mg protein) was higher than 1.0 g/L ZnO NPs treatment alone. This result confirmed antioxidant test that ZnO-thiram treatment stimulated an enhanced oxidative stress on fungi cells compared to ZnO NPs alone.

Discussion

To the best of our knowledge, it is the first time to report synergistic antifungal effect of semiconductor NPs with pesticide. Synergy between antibiotics may generate from several pathways15,23. According to IR analysis (Figure 1), we demonstrated the formation of composite antifungal system by weak interaction between ZnO NPs and thiram. This led to the increasing thiram concentration at active sites where antifungal agents contacted with pathogen fungi and therefore caused more destruction. Generally, the fungi surface is negative charged at biological pH41,42 due to the arrangement of the carboxyl and phosphate groups on the cell walls. Thus, positive charged ZnO NPs (Supplementary Figure S1c) were considered to have close physical interaction with fungi cells by direct electrostatic adsorption. As a result, cell membrane damage43 and cellular internalization were promoted. The cellular internalization of ZnO NPs was confirmed by SEM and EDX analysis (Figure 4). The penetration of ZnO NPs could cause more ZnO-induced oxidative damage intracellular than outside the cells. Meanwhile, as the ZnO-thiram composites formed, the uptake of ZnO NPs could help thiram entering the fungi cells that promote to exert thiram toxicity. When ZnO-thiram composite enter fungi cells, it is important to investigate their joint effect on cellular life activities for further understanding the mechanism of synergy. Recent studies have shown ZnO NPs induced the generation of ROS which cause oxidative stress18,36. Though excessive ROS-generation imposed unacceptable oxidative stress to cells that result in cell damage, small amount ROS can be tolerated by most cell types38. However, when ROS defense system be weakened or inactivated, the same amount ZnO NPs could exhibit higher toxicity to fungi cells. The mode of thiram action was thought to be a complex process, one of its fungicide effects was considered as the depletion of glutathione (GSH)44 and intracellular inactivation of glutathione reductase45 due to the disulfide bridge in the thiram structure. GSH plays an important role in cellular life-activity, it has been reported to protect cells against the destructive effects of oxidative stress caused by ROS46. Decrease in levels of GSH resulted in more oxidative damage to the fungi cells by the same amount ZnO NPs. GSH was also claimed to modulate critical cellular processes such as transport of amino acids, stabilization of cell membranes46, regulation of gene expression and apoptosis47. Lack of GSH could lead to disturbance in such cellular processes and finally fungi cells were more vulnerable when exposed to ZnO NPs. Interestingly, as the synergy could only be identified with 0.01 g/L thiram rather than with lower thiram concentration (Figure 2c), we suggest it is another proof concerning the role of GSH depletion in synergistic antifungal activity of thiram with ZnO NPs. The depletion of GSH may be insufficient to reduce the antioxidant capability of fungi at lower thiram concentration samples, whereas higher concentration thiram damaged the cellular antioxidant system adequately that provided the opportunity for ZnO NPs to develop more oxidative damage to fungi cells. As a result, ZnO NPs exhibited synergistic antifungal activity with thiram (Figure 2c and Figure 2d), correspondingly, the protection effect of histidine decreased (Figure 5a) and the SOD activity of fungi cells increased in ZnO-thiram samples (Figure 5b).

In conclusion, as been shown in Figure 6, we speculated that the formation of ZnO-thiram composite antifungal system, electrostatic adsorption of ZnO-thiram groups to fungi cells and the cellular internalization ZnO NPs played important roles in synergistic antifungal activities which facilitate ZnO NPs, thiram and their combination interacted with fungi hyphae. As a result, antioxidant capability of fungi cells was weakened and led to more damage under ZnO-induced oxidative stress, finally the synergistic antifungal activity was obtained against pathogen fungi Phytophthora capsici.

Figure 6
figure 6

Schematic diagram of residue-free green nanotechnology enhanced the pesticide efficiency and ensured food safety and public health.

Figure 6 and drawings within it were created by author J.Z.X.

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Using the synergistic antifungal ZnO-thiram concentration ratio, we successfully demonstrated a completely degradation of pesticide thiram by ZnO NPs in an aqueous solution system. This entirely removal of organic pollutants by semiconductor NPs was consistent with previous studies of photocatalytic degradation of organic pollutant48,49. Generally, photocatalytic experiments were conducted in an aqueous solution system. According to the photocatalytic degradation theory, the deposited thiram could be degraded after attacking by photo-generated electron−hole pairs, then released active sites on ZnO were available for free thiram molecules. Therefore, the free thiram continued depositing/absorbing on ZnO NPs surface and consequently being degraded. Moreover, the photo-generated holes can react with OH− or H2O and oxidize them into ·OH radicals; meanwhile the photo-generated electrons could react with O2 adsorbed on the catalyst surface or dissolved in water and reduce it to superoxide radical anion O2·−. These highly oxidative radicals could oxidize free thiram existed in the system. As a result, all thiram molecules, whether deposited or free, were degraded completely. It is also worthy to mention that even in practical pesticides application, due to the moisture in the air and the transpiration effect of plant, the micro aqueous solution system could formed on leafs and provide photocatalytic reaction system for thiram degradation. So thiram could also be totally degraded in practical systems.

Combining the synergistic antifungal activity and successive photocatalytic degradation of thiram, we established a residue-free green synergistic antifungal nanotechnology for pesticide thiram by ZnO NPs. The schematic diagram of the nanotechnology is depicted in Figure 6. Owing to the synergistic antifungal activity, this nanotechnology firstly offers the opportunity to reduce thiram usage from 0.04 g/L to 0.01 g/L in the presence of ZnO NPs without compromise in pathogen control. Furthermore, the complete photocatalytic degradation enables the ZnO NPs to remove the excess thiram after antifungal process and consequently restrict the thiram residue under maximum residue limits set up by government administrations. Such significant decline in thiram usage and successive degradation of thiram to safety level benefit the human social greatly by providing economic advantage as well as avoiding the negative impacts on public health and environment. This paper displayed notable effects of our residue-free green nanotechnology, however, more work still need to be done for further deeper understanding the synergistic mechanism and broadening the application range with other pesticides.

Methods

Materials and Characterization

ZnO NPs purchased from Tianxin Zinc Industry Co., Ltd. (Baoji, Shanxi, China) were characterized by X-ray diffraction (XRD) and transmission electron microscopy (TEM). Before antifungal test, ZnO NPs were pre-treated with deionized water. Zeta potential of ZnO NPs was examined by Zetasizer Nano (Malvern, UK). Thiram used in this study was purchased from Sinopharm Chemical Reagent Co, Ltd. Shanghai China (analytical grade). Thiram was added to ZnO NPs water suspension with vigorous shaking to form the composite antifungal system. The assembling of thiram on the surface of ZnO NPs by weak binding was characterized by infrared spectroscopy (Nicolet 6700, Thermo, USA). Carrot agar (CA) medium (juice of 200 g fresh carrot and 13 g agar in 1000 mL distill water) were prepared for fungal cultivation and antifungal test.

Antifungal test

Pathogenic fungi Phytophthora capsici used in this study was obtained from China Agricultural University. Antifungal tests were performed by the mycelial growth rate method in presence of various concentrations of inhibitor. ZnO NPs, thiram and ZnO-thiram combinations were added to melted CA medium at about 45 ± 5°C for evaluating antifungal activities. The mediums were shaken vigorously and then poured into the Petri dishes (9 cm diameter). The fungi were inoculated after the CA medium solidified. Agar plugs (5 mm) obtained from the edge of 7-day-old fungal cultures were placed in the center of each Petri dish. All samples were then incubated at 25°C in the dark. The diameters of fungal colonies were measured after 4 day incubation. All of the samples were prepared in triplicate and all experiments were repeated twice or more. Antifungal efficacy were calculated as followed method:

Antifungal efficacy = (Dc – Dt) × 100%/Dc

where Dt = diameter of colonies in test plate; Dc = diameter of colonies in control plate.

The combined antimicrobial effects of pesticides with ZnO NPs could be evaluated by two methods. One is to calculate the expected efficacy of ZnO NPs and thiram mixtures by Abbortt formula first:

%Cexp = A+B-(A × B/100)

in which A and B are the control efficacy given by the single pesticide50. Then, increased control efficacy of pesticides mixtures (synergy factor, SF) was calculated using formula below16:

SF = %Cobs/%Cexp

Where %Cobs = observed efficacy. SF ≥ 1.5 showed synergistic effect, SF ≤ 0.5 demonstrated antagonism. Others were addictive effect. Another is the fractional inhibitory concentration index (FICI). The FICIs were calculated by the following equation according to minimum inhibitory concentration (MIC). FICI = FICA + FICB = MICcomb·A/MICalone·A + MICcomb·B/MICalone·B, where MICalone·A and MICalone·B are the MIC values of drugs A and B when acting alone and MICcomb·A and MICcomb·B are the concentrations of drugs A and B at combinations. The interpretation of the FICI was as follows: a FICI ≤ 0.5 demonstrated synergy; a FICI between 0.5 and 4 indicated no interaction; and a FICI ≥ 4 showed antagonism15.

Photocatalytic degradation procedure

All photocatalytic experiments were conducted using an SGY-II photochemical reactor purchased from Nanjing Stonetech. EEC Ltd., Nanjing, China. Series of 40 mL thiram aqueous solutions of desired concentration were filled in each quartz tubes (50 mL) and required amount ZnO NPs were added. Before irradiation, quartz tubes with sample solutions were subjected to sonication for 20 min to disaggregate ZnO NPs and then placed inside the reactor for 30 min standing to achieve adsorption equilibrium. Magnetic stirrers were located at the base of reactor that a homogenous ZnO NPs suspension could be maintained throughout the reaction. Thiram solutions were illuminated with a 500 W Xeon lamp without filters. Samples were collected before and at regular intervals during the irradiation. Analysis were performed, after separation of the photocatalyst particles by centrifugation (3000 rpm, twice), with a UV-Vis spectrophotometer (UV-Vis 8453, Agilent, USA), measuring the absorbance at maximum wavelength 278 nm. The detection limit of the UV-Vis spectrophotometer for our experiment was also determined.

Morphological test of fungal hyphae

Scanning electron microscopy (SEM) was used to examine morphological changes of Phytophthora capsici hyphae with or without ZnO NPs treatment. Pieces of fungi hyphae material cut from 7-day-old cultures were inoculated onto the CA medium containing 1.0 g/L ZnO NPs, 0.0025 g/L thiram, 1.0 g/L ZnO NPs + 0.0025 g/L thiram and control treatment (ZnO-free and thiram-free) followed by incubation for 4 days. Then, agar plugs contained fungi hyphae were cut from the edge of the fungal cultures and directly subjected to SEM analysis under the environmental mode. SEM images were taken by FEI Quanta 200F Environmental SEM at a voltage of 20 kV and a pressure 350 Pa.

Inhibitory effect of antioxidant on ZnO NPs antifungal activity

Histidine was reported as a well-known antioxidant which could scavenge hydroxyl radicals and singlet oxygen. In order to determine the Oxidative stress caused by ZnO NPs, histidine were add to CA media which contain 1.0 g/L ZnO, 0.0025 g/L thiram and 1.0 g/L ZnO + 0.0025 g/L thiram respectively. The fungi were inoculated after the CA media solidified. Inhibition effect of histidine on ZnO antifungal activities were measured after 4-day cultivation.

Superoxide Dismutase (SOD) Catalase activity assay

For SOD assay, fungi mycellial balls were isolated and washed with 0.85% saline solution after 48 h inoculation. The samples were subjected to sonication for 5 min to rupture the fungal cell wall. Homogenate were taken to determine the activities of SOD using assay kits (Nanjing Jiancheng Bioengineering). Assay was based on determining the inhibition of nitroblue tetrazolium (NBT) reduction due to superoxide anion produced by a xanthine/xanthine oxidase system. One enzyme unit of SOD was defined as that amount of protein (in mg) causing a 50% inhibition of the formation of formazan dye.