The antifungal activity of nano zinc oxide (ZnO NPs) and their mode of action against two postharvest pathogenic fungi ( Botrytis cinerea and Penicillium expansum ) were investigated in this study. ZnO NPNs with size 70 ± 15 nm and concentrations 0, 3, 6 and 12 mmol l−1 were used. Traditional microbiological plating, scanning electron microscopy (SEM) and Raman spectroscopy were used to study the antifungal activities of ZnO NPs and characterize the changes in morphology and cellular composition of ZnO NPs. mycelium was treated with ZnO NP. The results showed that ZnO NPs at concentrations greater than 3 mmol l−1 could significantly inhibit the growth of B. cinerea and P. expansum . P. expansum is more sensitive to treatment with ZnO NPs than B. cinerea. SEM images and Raman spectra revealed two different antifungal activities of ZnO NPs against B. cinerea and P. expansum . ZnO NPs inhibit the growth of B. cinerea by affecting cellular functions, causing mycelial deformation. Meanwhile, the nano zinc oxide inhibited the growth of monocytes and spores of P. expansum, eventually leading to the death of the mycelium. These results suggest that ZnO NP can be used as an effective fungicide in agricultural and food safety applications.
Fungal growth is the main cause of significant economic losses during postharvest handling of fruit ( Spadaro et al. 2004 ). Botrytis cinerea and Penicillium expansum can cause severe postharvest fruit diseases including gray and green mold even when applying the most advanced postharvest technologies ( Spadaro et al. 2004 ). B. cinerea is considered one of the most important diseases of table grapes ( Latorre et al. 1994 ), while P. expansum mainly causes rot of preserved apples and pears ( Cabanas et al. 2009 ). Furthermore, P. expansum is considered to be the main producer of the mycotoxin, patulin, which is commonly found in rotting apples. The US Food and Drug Administration (FDA) limits patulin to 50 mg/l in apple juice ( Moake et al. 2006 ).
It is difficult to control fungal growth because fungi have developed resistance to many common fungicides such as benzimidazoles and dicarboximides ( Elad et al. 1992 ). To overcome this resistance, it is important to discover new antifungal agents, which can replace current control strategies. In recent years, nanoparticle (NP) materials have received increasing attention due to their unique chemical and physical properties that are significantly different from their conventional materials ( Stoimenov et al. 2002 ). Recent studies have demonstrated the antibacterial activities of various NP materials, including silver ( Kim et al., 2008a , Kim et al., 2008b , Kumar et al., 2008 ), copper (Cioffi et al., 2008). et al. 2005 ), titanium dioxide ( Kwak et al. 2001 ), and zinc oxide ( Liu et al. 2009 ).
Highly ionic nanometal oxides such as nano zinc oxide (ZnO NPs) are special in that they can be produced with high surface areas and have an unusual crystal structure ( Klabunde et al. 1996 ). Compared with organic materials, inorganic materials such as ZnO have superior strength, selectivity, and heat resistance ( Padmavathy and Vijayaraghavan 2008 ). Furthermore, zinc is an essential mineral element for human health and ZnO is a form in daily zinc supplements. ZnO NPs also have good biocompatibility with human cells ( Padmavathy and Vijayaraghavan 2008 ). The antibacterial and antifungal activity of bulk ZnO powder has been demonstrated ( Yamamoto, 2001 , Sawai and Yoshikawa, 2004). In agriculture, zinc compounds are mainly used as fungicides ( Waxman 1998 ). The 50% lethal dose (LD 50 ) of oral toxicity for ZnO was 240 mg/kg for rats ( Male 2002 ). Recent interest lies in their NP forms. It is believed that the smaller the size of ZnO, the stronger its antibacterial activity ( Yamamoto 2001 ). Preliminary studies suggest that the antibacterial activity of ZnO NPs may be related to the formation of free radicals on the surface of NPs and the destruction of lipids in bacterial cell membranes by free radicals. due to, thus leading to the leakage and decomposition of bacteria. membrane ( Brayner et al., 2006 , Reddy et al., 2007). However, to our knowledge, the effect and mode of action of ZnO NPs on the growth of fungi such as B. cinerea and P. expansum have not yet been investigated.
In this study, we investigated the antifungal activities of ZnO NPs against two important plant pathogenic fungi, B. cinerea and P. expansum . The mode of action of nano zinc oxide ZnO NPs on mycelium growth was also investigated by scanning electron microscopy (SEM) and Raman spectroscopy.
2 .Materials and methods
2.1 . Nanoparticle materials
ZnO NP suspension with NP size 70 ± 15 nm was purchased from Alfa Aesar (Ward Hill, MA, USA). A fraction (10 ml) of the ZnO NP suspension was vacuum filtered through an aluminum oxide filter membrane with pore size 20 nm and 25 mm outer diameter (Anodisc; Whatman Inc., Clifton, NJ, USA), yielding a zero solution. have NPs. The composition of the NP-free solution was analyzed as water and dispersant, and its effect on bacterial growth was examined. Then, the initial ZnO NP suspension (12 mol l −1 ) and the NP-free solution were then diluted with potato dextrose agar (PDA, containing extract from 200 g boiled potato, 20 g glucose and 20 g agar in 1 l distilled water) to create a series of media containing ZnO NPs at concentrations of 0, 3, 6 and 12 mmol l −1 and NP-free solutions.
2.2 . Antifungal test
Two pathogenic fungi, B. cinerea and P. expansum , were obtained from the culture collection of the Food Microbiology laboratory at the University of Missouri, Columbia, MO. B. cinerea and P. expansum were cultured on PDA at 25 °C in the dark. Antifungal assays were performed using the agar dilution method ( Fraternale et al. 2003 ) with some modifications. The PDA medium was autoclaved with ZnO NPs at the concentrations of 0, 3, 6 and 12 mmol l −1 and the NP-free solution was poured into a Petri dish ( diameter 9 cm). Mushrooms were inoculated after the PDA medium solidified. A plate (1.4 cm) of filamentous material taken from the edge of 7-day-old fungal cultures was placed in the center of each Petri dish. Petri dishes with inoculum were then incubated at 25 °C. Efficacy of ZnO NP treatment was evaluated at 2, 4, 6, 9 and 12 day intervals by measuring the diameters of fungal colonies. . All tests were performed in triplicate and values are expressed in cm.
2.3 . Check the morphology of the mycelium
SEM was used to examine the morphological changes of B. cinerea and P. expansum mycelium before and after treatment with nano zinc oxide ZnO NPs. Mycelial material fragments cut from 7-day-old explants were inoculated into a PDA containing 12 mmol l -1 ZnO NP and a control (ZnO NP-free PDA), then incubated for 12 days. Then, flakes of fibrous material were cut from the edge of the fungal culture medium, and directly subjected to SEM analysis in medium mode. SEM images were taken by the FEI Quanta 600F Environmental SEM (FEI Company, Hillsboro, OR, USA) at a voltage of 7 or 10 kV and a pressure of 525 to 619 Pa.
2.4 . Raman instrumentation and data analysis
A Renishaw RM1000 Raman spectrophotometer system (Gloucestershire, UK) equipped with a Leica DMLB microscope (Wetzlar, Germany) and a 785 nm near-infrared diode laser source (up to 300 mW) was used in the study. this. The Raman scattering signal was detected using a 578 × 385 pixel CCD array detector. Raman spectra were obtained from mycelial materials isolated from B. cinerea and P. expansum cultures with or without exposure to NPN ZnO 12 mmol l -1 after incubation for 12 days. A 50× objective is used with a Raman detection range of 600 to 1800 cm -1 in extended mode. Measurements were carried out with exposure time 10 s and ∼10 mW laser power.
Data analysis was performed using Delight software version 3.2.1 (D-Squared Development Inc., LaGrande, OR, USA). Preprocessing algorithms such as smoothing and polynomial subtraction were used to remove the device noise and eliminate the base differences. Principal component analysis (PCA) was used to analyze the spectral data. PCA is a multivariate analysis method used to extract patterns or establish relationships in data sets ( Goodacre et al. 1998 ).
3.1 . Antifungal effects of nano zinc oxide ZnO . NPs
The ZnO NP suspensions were examined by transmission electron microscopy (TEM) ( Figure 1 ). The shape of the ZnO NPs is mainly rod-like. Dimensions in accordance with product description, length 70 ± 15 nm. The ZnO NP-free solution was obtained by filtration and its composition included water and dispersant ( Liu et al. 2009 ). The ZnO NP-free solution did not affect fungal growth and normal colony formation was observed (data not shown). Figure 2 , Figure 3 show the effect of nano zinc oxide ZnO NPs on the growth of B. cinerea and P. expansum cultured on PDAs containing different concentrations of ZnO NPs (0, 3, 6, 12 mmol). l −1 ) and incubated at 25 °C for 12 days. In general, the use of ZnO NP suspension was effective in inhibiting fungal growth for both B. cinerea and P. expansum . The mean growth of B. cinerea was inhibited from 63% to 80% of the colony growth diameter after 12 days of incubation when the concentration of ZnO NPs increased from 3 to 12 mmol l −1 ( Figure 2). ). For P. expansum , the reduction rate of fungal growth varied from 61% to 91% as the concentration of ZnO NPs increased from 3 to 12 mmol l −1 with almost complete inhibition at 6 mmol l −1 ( Figure 3 ). Significant differences were found for different concentrations of ZnO NP treatment ( P < 0.05). These results indicate that nano zinc oxide ZnO NPs at concentrations greater than 3 mmol l −1 can significantly inhibit the growth of B. cinerea and P. expansum ; and zinc oxide nanoparticles were effective against P. expansum than B. cinerea .
Hình 1 . Hình ảnh TEM của huyền phù hạt nano kẽm oxit (ZnO NP)
Figure 2 . Antifungal activity of ZnO NPs nano zinc oxide against Botrytis cinerea on PDA in the presence of different concentrations of ZnO NPs. Data are displayed with the mean and standard error values of bacteria count . Each point represents the mean of three measurements.
Figure 3 . Antifungal activity of ZnO NPs nano zinc oxide against Penicillium expansum on PDA in the presence of different concentrations of ZnO NPs. Data are displayed with the mean and standard error values of bacteria count . Each point represents the mean of three measurements.
3.2 . Morphological analysis of fungal growth
To investigate the mechanism by which ZnO NPs affect the growth of B. cinerea and P. expansum , SEM analysis was used to examine the structural change of fungal samples after ZnO NP treatment. PDA medium containing 12 mmol l -1 ZnO NPs was prepared, and fungal samples were then inoculated in PDA dishes and incubated at 25 °C for 12 days. Figures 4 A and B show images of mycelium obtained from the edge of B. cinerea culture in the control (untreated sample), showing the mycelium with a typical “mesh” structure and smooth surface . After treatment with 12 mmol l −1 ZnO NPNs, the mycelium lost its smoothness and formed abnormal bulges on the surface of the mycelium ( Figures 4C and D), indicating that the nano zinc oxide inhibited growth of B. cinerea by deforming the structure of the mycelium. SEM observations of the outer mycelium from P. expansum colonies revealed branched spores with a chain and circular structure ( Figures 5 A and B ). After treatment with 12 mmol l -1 ZnO NPs for 12 days, the sporulation of P. expansum was completely inhibited and spore growth of P. expansum was prevented ( Figures 5C and D). These results showed that ZnO NPs distort and damage the spores of P. expansum . As a result, fungal growth is greatly inhibited.
Figure 4 . SEM image of Botrytis cinerea without (A and B) or with (C and D) ZnO NP suspension treatment.
Figure 5 . SEM images of Penicillium expansum without (A and B) or with (C and D) ZnO NP nano zinc oxide suspension treatment.
3.3 . Studying the mode of action of nano zinc oxide ZnO NPs by Raman . spectroscopy
Raman spectroscopy was used to further study the mode of action of zinc oxide nanoparticles and their antifungal mechanism. B. cinerea and P. expansum were incubated on PDA with or without ZnO NPN at 25 °C for 12 days. Then, fungal colonies were examined by Raman spectroscopy. The spectra of B. cinerea and P. expansum exhibit distinctive absorption bands between 600 and 1800 cm -1 , revealing fluctuating information about carbohydrates, proteins, lipids, and nucleic acids. The main Raman peaks obtained from mycelium and their band designations are shown in Table 1 ( Schuster et al., 2000 , Maquelin et al., 2002, Chan et al., 2007 ). Figure 6 shows the mean spectra of B. cinerea cultured on PDA with or without 12 mol l −1 ZnO NPs. For example, bands about 743, 772, 938 and 1082 cm −1 are assigned to nucleic acids; bands about 848, 868, 895, 965 and 1048 cm −1 are assigned to carbohydrates; bands around 1005, 1254 and 1668 cm −1 are assigned to the protein; and bands around 1416 and 1460 cm −1 are indicated for lipids. It was observed that the intensity of nucleic acids and carbohydrate bands was significantly increased in mycelium treated with ZnO NPs; while no obvious change of Raman signal of proteins and lipids was observed. Figure 7 shows the mean spectrum of P. expansum grown on PDA with or without NPN ZnO 12 mmol l −1. For example, a range of about 1198 cm −1 is assigned to carbohydrates; bands about 1368 and 1454 cm −1 are assigned to lipids; and a band around 1603 is assigned to the protein. No obvious Raman peaks were observed from the mycelium of P. expansum after ZnO treatment. PCA was performed on Raman spectra of B. cinerea and P. expansum incubated on PDA with or without NPN ZnO 12 mmol l −1. A two-dimensional (2D) PCA plot with the first two PC points is shown in Figure 8 . Clear separation was observed in the data cluster of control B. cinerea (B_C), B. cinerea treated with NPN ZnO (B_Z), control P. expansum (P_C) and P. expansum treated with ZnO NPs (P_Z).
Table 1 . The banding of Raman peaks in the range 600–1800 cm -1 ( Schuster et al., 2000 , Maquelin et al., 2002 , Chan et al., 2007 ).
Figure 6 . Raman spectra of Botrytis cinerea with (A) or without (B) upon treatment of ZnO NP nano zinc oxide suspension. Measurements are taken from 600 to 1800 cm -1 under 10 s and laser power ∼10 mW.
Figure 7 . Raman spectra of Penicillium expanded with (A) or without (B) upon treatment of ZnO NP nano zinc oxide suspension. Measurements are taken from 600 to 1800 cm -1 under 10 s and laser power ∼10 mW.
Figure 8 . PCA plot based on Raman spectra of fungal samples: Botrytis cinerea with ZnO NP (B_Z) and control (B_C) zinc oxide nano suspension treatment; Penicillium extended with ZnO NP suspension treatment (P_Z) and control (P_C).
Nano zinc oxide usually exist in agglomerated form during its manufacture (Zhang et al. 2007 ). Two methods, ultrasound and addition of dispersant, are commonly used to break up NP aggregates. Commonly used dispersants include polyvinylpyrrolidone, polyethylene glycol, and others ( Brayner et al. 2006 ). Figure 1 shows the zinc oxide nanoparticles used in this study ( Liu et al. 2009 ). Information on dispersant used in this proprietary commercial NP preparation is not available from the manufacturer. ZnO NPs were uniformly dispersed in the PDA, which was demonstrated by the consistent inhibitory results obtained in this study.
Significant antifungal activity against B. cinerea and P. expansum was found using nano zinc oxide ZnO NPs as low as 3 mmol l −1. When the concentration of ZnO NP zinc oxide nanosheets increased from 3 to 12 mmol l −1 , the efficiency of ZnO NP treatment was enhanced. Compared with B. cinerea , P. expansum is more sensitive to ZnO NP zinc oxide nanotreatment. However, the different antifungal effects may be due to the different growth morphologies of these two fungi. P. expansum tends to grow more densely on the surface of the agar plate than B. cinerea, so it is more exposed to ZnO NPs than B. cinerea. Another possible reason for the difference could be the innate tolerance of each fungus to ZnO NPs. Sawai and Yoshikawa (2004) reported the minimum inhibitory concentration of bulk ZnO powder for Saccharomyces cerevisiae , Candida albicans , Aspergillus niger, and Rhizopus stolonifer being above 100 mg ml −1 (∼1.2 mol l −1 ) by indirect conductivity test ( Sawai and Yoshikawa 2004 ). The ZnO NPNs in our study showed a significant enhancement in antibacterial activity due to their unique properties such as large surface area. However, Kasemets et al. (2009) found that nano and large amounts of ZnO were equally toxic to S. cerevisiae .
SEM has been successfully used to evaluate the morphological changes of microbial cells induced by nano zinc oxide ZnO NPs ( Brayner et al., 2006 , Zhang et al., 2007 ) and the mycelium treated with chemical agents. other substances ( Sharma and Sharma, 2008 , Yen et al., 2008 ). Some studies suggest that ZnO NPs can induce structural changes of microbial cell membranes, causing cytoplasmic leakage and ultimately bacterial cell death ( Sawai and Yoshikawa, 2004 , Brayner et al., 2006 ). In this study, P. expansum produced spores while biomass B. cinerea mainly consisted of mycelium. ZnO NP treatment inhibited the growth of spore cells and distorts the native cells of P. expansum . Compared with P. expansum , B. cinerea seems to be more resistant to ZnO NPs. The fine structure of the mycelium B. cinerea was preserved, although the surface of the hyphae was deformed. Therefore, ZnO NPs can exhibit different antifungal activities against P. expansum and B. cinerea .
Oscillometric techniques, such as Raman spectroscopy, are useful for monitoring subtle changes in microbial cells because the technique is quick, non-destructive, and requires minimal sample preparation. ( De Gussem et al. 2006 ). Raman spectroscopy has been widely applied to the monitoring and characterization of bacteria and fungi ( De Gussem et al., 2005 , Sengupta et al., 2006 , Chan et al., 2007 , Szeghalmi et al., 2007 ), and to investigate the mode of action of antibacterial agents ( Lopez-Diez et al., 2005 , Neugebauer et al., 2006 , Sideroudi et al., 2006). Raman scattering changes in a characteristic molecular oscillation pattern ( Kneipp et al. 1999 ). Thus, the “fingerprint-like” spectrum carries overall and specific information about various chemical and biochemical compounds in complex systems ( Naumann 2000 ). Raman spectroscopy has been successfully used in studying the mechanism of antibacterial action of antibiotics. Neugebauer et al. (2006) reported that ciprofloxacin, a fluoroquinolone drug, affects the growth of Bacillus pumilus by interacting with the gyrase–DNA complex. This result is inferred from changes in the Raman bands of nucleic acids and the building blocks of proteins. The same mechanism of amikacin against Pseudomonas aeruginosa was also reported by Lopez-Diez et al. (2005) using Raman spectroscopy.
The mechanism of inhibitory action of ZnO NPs on microorganisms is not fully understood. Several studies have reported that the integration of ZnO NPs into bacterial cells can induce the continuous release of membrane lipids and proteins, altering the membrane permeability of bacterial cells ( Amro et al. et al., 2000 , Brayner et al., 2006 ). As shown in the Raman spectra of B. cinerea , the intensity of nucleic acids and carbohydrate bands was significantly increased by ZnO NP treatment while no obvious changes were observed in the Raman signals of proteins and lipids. The difference between B. cinerea signals with or without ZnO NP treatment was further demonstrated by PCA to be statistically significant. These results suggest that ZnO NPs zinc oxide nanoparticles can influence cellular functions and ultimately cause an increase in nucleic acid content. The increase in nucleic acids may be due to the stress response of the mycelium. The increase in carbohydrates may be due to a self-defense mechanism against ZnO NPs ( Alvarez-Peral et al. 2002 ). Others have also reported observations of an increase in carbohydrates in mushrooms treated with NPs ( Kim et al., 2008a , Kim et al., 2008b ). The deformed structures of mycelium cells in Figures 4C and D may be due to excessive accumulation of nucleic acids and carbohydrates. These data suggest a different mechanism for the inhibitory effect of ZnO NPs on fungi than those previously reported for bacteria ( Liu et al. 2009 ). On the other hand, no obvious Raman peak was observed from the mycelium of P. expansum after ZnO treatment. The bands assigned to carbohydrates, lipids and proteins from P. expansum were significantly reduced by the treatment of ZnO NPs, indicating that the growth of P. expansum was completely inhibited. The inhibited fungi did not have enough substrate to generate Raman signals so that no obvious Raman signal was detected. These results are in agreement with the microplating and SEM results (Figures 5 C and D) that the growth of P. expansum was greatly inhibited.
In conclusion, ∼70 nm NPN ZnO zinc oxide nanosheets have significant antifungal properties against B. cinerea and P. expansum , and the inhibitory effect increases as the concentration of ZnO NPNs increases. ZnO NPs at concentrations greater than 3 mmol l −1 could significantly inhibit the growth of B. cinerea and P. expansum. Zinc oxide nanoparticles were more effective against P. expansum B. cinerea . The data obtained from SEM and Raman spectroscopy indicate that different mechanisms of ZnO NPs against two different fungal species may exist. ZnO NPs inhibit the growth of B. cinerea by interfering with cell function and causing distortion in mycelium. Meanwhile, ZnO NPs with a concentration higher than 6 mmol l -1 resulted in complete inhibition of the growth of P. expansum by suppressing the growth of fellow cells and sporangia. These results suggest that ZnO NP can be used as an effective fungicide in agricultural and food safety applications. Further studies are needed to investigate the feasibility of incorporating ZnO NPs into films and other packaging materials, and the use of ZnO NPs to address food safety issues in a safe manner. fully and responsibly.
LiliHe1 YangLiu1 Azlin Mustapha MengshiLin