Roses are the most popular garden plant and cut flower, and they have been cultivated for several millennia (Debener and Linde, 2009). However, gray mold caused by the air-borne pathogen Botrytis cinerea is a devastating disease of rose worldwide, resulting in at least a 30% loss of production every year. In addition to roses, B. cinerea also infects various plant species, causing significant pre- and post-harvest damage to fruits, vegetables, and ornamentals (Ferrada et al., 2016; Jurick et al., 2017). And the process of B. cinerea infection could induce some pathogenic factors such as cell wall-degrading enzymes, reactive oxygen species (ROS), phytotoxin, oxalic acid, etc. (van Kan, 2006; Choquer et al., 2007; Williamson et al., 2007). It was reported that B. cinerea has become the second major plant pathogenic fungus after the rice blast fungus. Because of the serious damage caused by B. cinerea to plants and agricultural production, it was urgent to find fungicides, which could efficiently suppress the growth of B. cinerea (Kretschmer et al., 2009; Fernandez-Ortuno et al., 2016; Fan et al., 2017). Like B. cinerea there are many fungal pathogens and diseases severely impact crop yields, reducing the quality of agro-product, which could result in a worldwide threat to agricultural security (Fisher et al., 2012; Girard et al., 2016). Thus, the production and use of pesticides against fungal diseases has increased in recent years, which not only causes drug resistance but also produces new environmental risks (Gangemi et al., 2016). Consequently, it is urgent to investigate novel environmentally friendly fungicides that have efficient antifungal activities (Soylu et al., 2010; Fernandez-Acero et al., 2011).

Recently, the rapidly and continually developing field of nanotechnology has been widely applied to various disciplines, especially to environmental science. On the one hand, research has focused on assessing the environmental impact and ecological effects of nanoparticles (NPs), including their toxicological effects on plants, soil, and microorganisms (Hao et al., 2016; Mukherjee et al., 2016; Rui et al., 2016). On the other hand, some studies have focused on potential agricultural applications of low concentrations and low-toxicity nanomaterials. Servin et al. (2015) found that engineered nanomaterials can suppress plant disease, enhance crop yield, and play vital roles as fertilizers and pesticides. Similarly, Ditta and Arshad (2016) showed that nanomaterials can provide plants with more nutrients than conventional fertilizers. Additionally, nanomaterials can increase the storage time of vegetables and fruits, and improve animal immunity. Nanomaterials can also improve the water quality in fisheries (Peters et al., 2016). Thus, nanomaterials have huge potential to be used as novel nano-fertilizers, which can improve nutrient uptake and reduce agro-environmental pollution (Bindraban et al., 2015; Liu and Lal, 2015).

The potential applications of NPs as antifungal agents have also been investigated. For example, reduced graphene oxide (rGO) can inhibit the mycelial growth of the fungal pathogens Aspergillus niger, Aspergillus oryzae, and Fusarium oxysporum (Sawangphruk et al., 2012). Single-walled carbon nanotubes (SWCNTs), multi-walled carbon nanotubes (MWCNTs), fullerene (C₆₀), and rGO have significant antifungal activities against Fusarium graminearum and Fusarium poae (Wang et al., 2014). SWCNTs can alter the oxidative enzyme activities of the saprotrophic white-rot fungi Trametes versicolor and Phlebia tremellosa (Berry et al., 2014). The toxicity of rGO toward the white-rot fungus Phanerochaete chrysosporium was dose-dependent. A low concentration of rGO promoted the growth of the fungus, while a high concentration induces a loss of fungal activity. Scanning electron microscope (SEM) observations showed that the fibers of the fungi were disrupted (Xie et al., 2016). For metal oxide NPs, Terzi et al. (2016) reported that nano-zinc oxide (ZnO) and nano-B₂O₃ suppressed the growth of molds, while nano-CuO (copper oxide) and nano-SnO₂ prevented wood from undergoing fungal decay caused by T. versicolor. Silver (Ag) NPs can inhibit the colony formation of Bipolaris sorokiniana and Magnaporthe grisea in in vitro assays (Jo et al., 2009). De Filpo et al. (2013) reported that the photo-catalytic activities of titanium oxides (TiO₂) NPs protected wood from Hypocrea lixii and Mucor circinelloides colonization, suggesting a potential antifungla mechanism. Hollow TiO₂ NPs had the strongest antifungal activities against two potent phytopathogens, Fusarium solani and Venturia inaequalis, compared with pure and silver-doped TiO₂ NPs. The NP concentrations and visible light intensity levels contribute to the antifungal effects (Boxi et al., 2016). Zn and ZnO showed antifungal activities against fungi (Penicillium and Mucor spp.; Swain et al., 2014). Cu NPs inhibited the growth of four major plant pathogenic fungi, F. oxysporum, Curvularia lunata, Alternaria alternata, and Phoma destructiva, mainly as a result of their large surface area to volume ratio, and can be used as neo-antifungal agents, protecting crops from plant pathogenic fungi (Kanhed et al., 2014).

Notably, previous reports of the antifungal activities of nanomaterial were based on in vitro assays, in which fungi were grown on artificial media, such as potato dextrose agar. Currently, the in planta effects of nanomaterials on the growth of fungal pathogens has not been addressed. The activities of fungicides vary between in vitro and in vivo assays, as illustrated by the antifungal activities of essential oils against Sclerotinia sclerotiorum and Colletotrichum gloeosporioides (Soylu et al., 2007; Hong et al., 2015).

In this study, the antifungal activities of six nanomaterials [ferric oxide (Fe₂O₃) NPs, CuO NPs, TiO₂ NPs, MWCNTs, C₆₀, and rGO] against B. cinerea were evaluated in vitro and in vivo conditions. The distinctive antifungal effects of the six nanomaterials on the morphology, size, and location of fungal hyphae on the rose petals were characterized by using SEM.

In vitro and in planta assays are two major methods for evaluating the effects of potential antifungal agents, such as metabolites (Lecomte et al., 2012; Zhang et al., 2013), commercial fungicides (Dal Maso et al., 2014), biological control agents (Pane and Zaccardelli, 2015; Yao et al., 2016), and plant essential oils (Hong et al., 2015; Varo et al., 2017). Compared with in vitro assays, in planta experiments have more practical significance and are widely used in screening efficient fungicides and assessing antifungal activities (Dal Maso et al., 2014; Hrabětová et al., 2017; Varo et al., 2017). In our study, the inhibitory effects of NPs on fungi were determined on detached rose petals after 72 h.

In our study, three carbon nanomaterials inhibited the in planta growth of B. cinerea. The inhibitory effects of rGO on B. cinerea were obvious at both low and high concentrations, while only 200 mg/L of rGO yielding significant antifungal results. Graphene sheets may disrupt fungal structures and damage the cell wall with their sharp edges (Xie et al., 2016), producing oxidative stress (Akhavan and Ghaderi, 2012), showing further antifungal effects. These toxicological mechanisms could explain the suppressive effects of rGO on B. cinerea. After exposure to MWCNTs, the effects of fungi on rose petals were also inhibited, especially after treatment with 200 mg/L MWCNTs, while the suppressive effects of 50 mg/L MWCNTs was not significant, which was different from the antifungal effects of 50 mg/L rGO. C₆₀ significantly inhibited the growth of B. cinerea only at the 50 mg/L concentration. Similar to our results, Wang et al. (2014) found that MWCNTs and rGO could inhibit the growth of F. graminearum by suppressing spore germination at concentrations of 62.5, 125, 250, and 500 mg/L, while the inhibitory effect of C₆₀ was not obvious at the same concentrations. Carbon nanomaterials can tightly contact spores, forming CNM-spore aggregates in the co-culture system through van der Waals forces (Wang et al., 2014). These aggregates effectively suppress the activities of spores by influencing the growth of fungi and, as a result, may inhibit B. cinerea infections on roses. The aggregative effects between carbon nanomaterials and spores also indicated the crucial roles of the physical properties of nanomaterials, such as van der Waals force.

Similar to carbon nanomaterials, Fe₂O₃ and CuO NPs also showed in planta antifungal effects on rose petals, which implied potential applications to protect flowers from fungal infection. Interestingly, the inhibitory effects of CuO and Fe₂O₃ NPs were significant only at the 50 mg/L concentration, but were not obvious at 200 mg/L. The antifungal effects of CuO NPs mainly resulted from the release of Cu²⁺, which is a major toxicity mechanism of Cu-based nanomaterials, such as Cu, CuO, and Cu₂O NPs (Kanhed et al., 2014; Zarzuela et al., 2017). As in our results, CuO NPs also inhibited the decay of wood by Gloeophyllum trabeum and T. versicolor, which indicated the potential application of CuO NPs as fungicides (Terzi et al., 2016). Similarly, Fe₂O₃ NPs suppressed the infection of B. cinerea significantly at the 50 mg/L concentration, while the antifungal effects were not obvious after exposure to 200 mg/L Fe₂O₃ NPs. TiO₂ NPs can induce cell damage in Pichia pastoris by impairing the ROS-associated scavenging system, especially the cycle and regulation of glutathione, which resulted in the accumulation of ROS (Liu et al., 2016). However, in our study, the antifungal effects of TiO₂ NPs were not obvious at either 50 or 200 mg/L concentrations. Considering the similar size of TiO₂ NPs, these different results may reflect the different sensitivities of P. pastoris and B. cinerea. The various sensitivities of different fungi to metal NPs emphasize the need for screening antifungal NPs in planta. Besides the efficient antifungal effects of NPs investigated in our study, other NPs also showed antifungal property against B. cinerea. For example, Ag NPs could significantly inhibit the growth of B. cinerea at concentration of 25 mg/L, and the inhibition effect was dose-dependent (Derbalah et al., 2012). Additionally, Ag NPs could also suppress other fungi such as A. alternata, S. sclerotiorum, Macrophomina phaseolina, Rhizoctonia solani, and C. lunata (Kovačec et al., 2017). ZnO NPs was another antifungal metal NP, it was reported that ZnO NPs could significantly decrease the growing activity of B. cinerea at concentrations greater than 3 mmol/L via affecting cellular functions especially deforming the fungal hyphae (He et al., 2011). Thus, nanomaterials could become an idealistic substitute for the conventional fungicide.

SEM observation after 72-h incubation further indicated the inhibition effects of NPs on fungi growth. It was obvious that after NP treatment, the number of mycelia was decreased comparing with control, meanwhile the growth state of mycelia was not active in the treatment of NPs, as the diameter of mycelia was considerably decreased compared with control (Figure 5). The decrease of mycelia number could be ascribed to the inhibition effects of the carbon nanomaterials such as MWCNTs, SWCNTs, GO, and rGO which could suppress spore germination by blocking the water channels of spores (Wang et al., 2014). Meanwhile metal dissolution should be another possible factor that participated in inhibiting fungi growth in rose petal (Zarzuela et al., 2017).

To date, several mechanisms have been proposed to illustrate the inhibited effect of NPs on various fungi. On the one hand, the closely adherence of NPs to the cell surface could induced a serious changes, such as physical cutting, water channel blocking, membrane permeability increasing, and even the penetration of NPs into fungus cells, which could result in cell death (Wang et al., 2014). On the other hand, the existence of NPs could induce ROS accumulation via impairing the ROS-scavenging system of fungi (Liu et al., 2016). Furthermore, the ions release of metal-based NPs was another mechanism of NP toxicity to fungi (Zarzuela et al., 2017).

In summary, the results of our study indicated the efficient antifungal activities of carbon nanomaterials and metal NPs on B. cinerea, pointed out the potential for the use of nanomaterial against pathogenic fungi on flowers.

Taken together, as potential fungistats, C₆₀ and CuO NPs could effectively suppress B. cinerea infections and improved flower quality at relatively low concentrations (50 mg/L), which could reduce the environmental risks resulting from NP release and reduce the production costs of novel NP fungistats. Our study showed the huge potential of NP applications in the field of horticulture because of their antifungal properties. In our study, we tried to increase the NP efficiency on inhibiting fungi growth at a relative low exposure dose to minimize the NP residue, meanwhile previous studies also pointed out NPs such as MWCNTs and Fe₂O₃ NPs have relatively lower toxicity, and could stimulate the growth and development of plants (Khodakovskaya et al., 2013; Rui et al., 2016). Considering the complex toxicity mechanisms of different NPs, any NP applications should be carefully evaluated for the potential environmental risks.

YR and ZZ proposed the present study. YH, ZZ, CM, BX, and YR designed the experiments. YH, XC, ZeZ, AA, NZ, JZ, SW, ZX, and TH performed the experiments and analyzed the data. YH, ZZ, and YR wrote the paper. All authors have read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.