Antifungal activity of camphor against four phytopathogens of Fusarium

Fusarium, one of the main fungal pathogens, can infect eld crops and cause great economic loss. This paper concerns a research on the antifungal activity of camphor. In our study, an assessment was made on the antifungal activity of camphor against four common phytopathogens: Fusarium oxysporum G5, F. solani G9, F. verticillioide, and F. graminearum. The method adopted was mycelial growth inhibition. The minimum inhibitory concentrations (MIC) of camphor against the four tested fungi were 4.0, 4.0, 4.0, and 2.0 mg/mL, and the half maximal inhibitory concentrations (IC 50 ) were 2.0, 2.0, 2.0, and 1.0 mg/mL, respectively. The paper proper also involves an investigation the, fungicidal mechanisms via cell membrane permeability, proteins and nucleic acids leakage and scanning electron microscopy. The results of preliminary antifungal mechanism revealed that camphor can cause cytomembrane destruction, enhancing the permeability of cytomembrane and releasing intracellular macromolecules, such as nucleic acids and proteins. Supposedly, the results suggested that the cytomembrane may be the target of camphor. In addition, these outcomes indicated that camphor can exhibit pronounced fungicidal activities against the four tested fungi and could be a promising alternative for the control of phytopathogenic Fusarium.


Introduction
Fusarium is one of the dominant phytopathogens causing serious crop wilt, stem rot, root rot and other soil-borne diseases (Bodah 2017). Many kinds of crops, such as corn, wheat and other cereals, are extremely susceptible to Fusarium species. As grain contaminants, they have a wide distribution and may cause farmers to sustain signi cant economic losses (Munkvold 2003;Kazan et al., 2012). Meanwhile, during their growth, they can metabolize some mycotoxins seriously harming the health of animals and humans, such as deoxynivalenol (DON) and zearalenone (ZEN) (Matny 2015). F. oxysporum, F. solani, F. verticillioide and F. graminearum are common phytopathogen species belonging in Fusarium genus, and they can cause many crop diseases. In particular, F. oxysporum, with a worldwide distribution of soilborne fungal pathogen, can infect and cause diseases to over 120 different plant species including tomatoes, bananas and cotton (Fravel et al. 2003). F. graminearum is the key pathogen causing head blight and crown rot (Liu et al. 2015;Goswami and Kistler 2004), while F. verticillioide is the main cause of maize ear rot (Chulze et al. 2000).
In the production of industrial crops, the approach to control Fusarium diseases is to choose Fusariumresistant cultivars or to apply chemical pesticides (Ferrigo et al. 2016). However, excessive and long-term application of chemically synthesized fungicides will not only cause resistance of phytopathogen, but also lead to soil and environmental pollution. Worse is that they may pose potential safety issues of food raw materials (Lee et al. 2014;Yang et al. 2018). In recent years, the discovery of e cient, green and safe natural fungicides from plants has attracted extensive attention, among which the volatile substances and alkaloids (such as matrine and oxymatrine) from plants are the hotspots (Yang and Zhao 2006;Andrade et al. 2014;Hu et al. 2014;Harkat-Madouri et al. 2015;Moss et al. 2017). Citrus essential oils consisting mainly of monoterpene hydrocarbons are widely used as fungicides in foodstuff and pharmaceutical industries (Jing et al. 2014).
Camphor (C 10 H 16 O, 1,7,7-trimethylbicyclo[2.2.1]-2-heptanone), a kind of bicyclic monoterpenoids, widely exists in some aromatic plants, such as Cinnamomum camphora, Eucalyptus globulus and Artemisia annua. It is the main component in the majority of plant essential oils (Green 1990). Previous investigations reported that camphor has been in use in medicine and cosmetics (Xiong et al. 2009). The insecticidal and insect-expelling e cacy of camphor has been widely con rmed (Moss et al. 2017;Guo et al. 2016). However, as a main component of some essential oils, whether it has promising antimicrobial activity needs further exploitation. To our knowledge, there are limited studies on the antifungal activity of camphor against the common and dominant phytopathogenic Fusarium. Therefore, the aim of this work is to evaluate the effects of camphor on the phytopathogenic fungi species of F. oxysporum G5, F. solani G9, F. verticillioide and F. graminearum on the bases of the in vitro growth capacity of fungal mycelium, changes of cytomembrane permeability, leakage of intracellular compounds, and the morphology of hypha.

Materials And Methods
Fungal strains, culture media and conditions Four plant pathogenic fungi species, F. oxysporum G5, F. solani G9 and F. verticillioide were obtained from the Laboratory of Microbial Resources and Technology, College of Life Sciences, Northwest Normal University. F. graminearum CICC 2697 was purchased from the China Center of Industrial Culture Collection. The fungi were cultured on potato dextrose agar (PDA) medium (200 g/L potato, 20 g/L dextrose and 15 g/L agar in distilled water) in a 90 mm diameter Petri dish at 28 ℃ for approximate 8 days in an incubator.

Chemicals
Camphor (analytical grade) was purchased from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). Tween-80 was supplied from Yantai Shuangshuang Chemical Co. (Yantai, China). Dimethyl sulfoxide (DMSO) was purchased from Shanghai Zhongqin Chemical Reagent Co. (Shanghai, China). All other reagents used in this work are all analytical pure except for special instructions.

Measurement of antifungal activity
The antifungal activity of camphor was detected by the method of mycelial growth inhibitory with some modi cations (Irzykowska et al. 2013). Brie y, the appropriate volume of the stock solution (sterilized by a 0.22 μm organic lter) of camphor dissolved in dimethylsulfoxide (DMSO) was thoroughly mixed with a certain amount of unsolidi ed and sterilized PDA medium, and then it was poured into a Petri dish to prepare a series of gradient concentration plates (0.125, 0.25, 0.5, 1.0, 2.0, and 4.0 mg/mL). Equal amount of DMSO mixed with PDA was adopted as the control medium. A 7-mm diameter mycelial disc of the four phytopathogenic fungi (F. oxysporum G5, F. solani G9 and F. verticillioide and F. graminearum CICC 2697) was punched from PDA culture and inoculated onto the centre of the Petri dish, respectively.
The inoculum was cultivated in a light-free incubator at 28 ℃ for 8 days. The mycelial growth momentum of the four phytopathogenic fungi was evaluated according to the cross section method once every 24 h until the 8 th day. The growth curve of the fungi was represented by a line graph. Each set of the experiments was in triplicate.

Determination of cell membrane permeability
The changes of membrane permeability of the fungal mycelium treated with camphor were detected by the method reported previously with slight modi cations and expressed as relative electric conductivity (REC) . To be brief, the vigorous mycelial disks (7 mm) of the four Fusarium strains were inoculated in 100 mL of potato dextrose (PD) liquid medium and they were kept shaking for 3 days at 200 rpm and 28 ° C, respectively. After incubation, the cultures were ltered with lter paper to obtain hypha samples under aseptic condition. The samples were thoroughly rinsed with sterile distilled water, and then the samples were prepared after being ltered again. The fresh mycelial sample (1.0 g) was added into 100 mL aqueous solution containing 1.0 and 2.0 mg/mL of camphor, and 0.2% Tween-80, respectively. The electric conductivity of the mixture was marked as L 1 . The mixture without addition of camphor was used as a control. The electric conductivity was assayed with a conductivity meter (AZ-8362, Taiwan, China) respectively at 1, 2, 4, 8, 12, 24, and 48 h and marked as L 2 . In addition, the electric conductivity of the control prepared with boiled water for 30 min was remarked as L 0 . The permeability of fungal cytomembrane was calculated and expressed as the following equation (1): Relative electric conductivity (REC, %) = 100% × (L 2 -L 1 )/L 0 (1) Detection of intracellular macromolecules leakage As described in section 2.4, the leakage of intracellular macromolecules (nucleic acid and protein) from the fungal mycelium was detected according to Ma et al. (2017). After the mixtures were incubated at 28 ℃ for 48 h, 10 mL of the culture was collected and centrifuged at 5000 rpm for 10 min and the absorbances of nucleic acids and proteins in supernatant were determined by a UV-3600 spectrophotometer (Shimadzu, Japan) at 260 nm and 280 nm, respectively (Luo et al. 2014).
Scanning electron microscopic observation of mycelia morphology After treatment with camphor for 48h, the hyphae were immobilized with 2.5% glutaraldehyde buffer, and refrigerated overnight at 4 ℃. They were then rinsed with 0.1mol/L PBS for three times, and the samples were dehydrated and replaced in anhydrous ethanol solution with concentrations of 30%, 50%, 70%, 80%, 90%, 100% in turn. The dehydration time was 10min and 15min each time. Then it was solidi ed overnight in a freezer. Finally, the samples were sprayed with gold and their morphology was observed using a JSM-5600LV scanning electron microscope (SEM, JEOL, Japan).

Statistical analysis
All cultivation and determination were performed in triplicate. The results from each experimental group were expressed as mean ± standard deviation. SPSS software (17.0, USA) was employed to analyze the signi cant difference between the groups at the level of 0.05.

E cacy of antifungal activity
The antifungal activities of camphor against the four plant pathogens Fusarium are shown in Table 1, Fig. 1 and Fig. 2. As can be seen from Fig. 1, signi cant differences begin to appear among the groups from the third day. With the extension of culture time and the increase of camphor concentration, the inhibitory effect of camphor on the growth of four tested fungi species gradually increases. When the concentration of camphor added was 2 mg/mL, none of the four tested Fusarium strains grow normally. Moreover, the sensitivity of different strains to camphor is different. For example, as can be seen from the growth graphs, F. graminearum shows strongest sensitivity to camphor. The results show that camphor has a strong inhibitory effect on the growth of the four tested plant pathogens, and the effect is concentration-dependent. Fig. 2 shows the growth status of the four plant pathogens Fusarium after 8 days of cultivation. F. oxysporum G5 F. solani G9 and F. verticillioide do not show growth on PDA + 4 mg/mL camphor. F. graminearum fails to grow normally when the camphor dosage is 2 mg/mL. Therefore, the minimum inhibitory concentration (MIC) values of camphor against F. oxysporum G5, F. solani G9, F. verticillioide and F. graminearum are determined to be 4.0, 4.0, 4.0, and 2.0 mg/mL, respectively. The half maximal inhibitory concentration (IC 50 ) is calculated as 2.0, 2.0, 2.0, and 1.0 mg/mL, respectively. These results conform to those of the previous study reported by Gazdağlı et al. (2018). They found that the MIC and the IC 50 of camphor against on F. culmorum 9F and F. graminearum H11 were 2 mg/mL and 1 mg/mL, respectively. Table 1 shows the absolute inhibition rate of camphor in vivo against the tested fungi. According to the Table 1, when the concentration of camphor is 1.00 mg/mL, the absolute inhibition rate of F. graminearum reaches 89.41%, much higher than those of other groups. In general, camphor has the strongest inhibitory effect on F. graminearum. When the concentration of camphor is 2.00 mg/mL, the absolute inhibition rates of F. oxysporum G5, F. solani G9, F. verticillioide and F. graminearum reach 83.65%, 91.98%, 82.61%, and 95.84%, respectively, suggesting that camphor exhibit pronounced fungicidal activities against the four tested fungi.

Analysis of antifungal mechanism
Relative electric conductivity (REC) was detected to re ect the variation of fungal cytomembrane permeability. As shown in Fig. 3, the REC of camphor-treated hyphae suspension increases with dosage and time compared with that of untreated hyphae. After 8h of culture, the electric conductivity of camphor-treated F. oxysporum G5 began to increase rapidly. After incubation for more than 12 hours, the relative conductivity of F. oxysporum G5 in the treatment groups (1.00 and 2.00 mg/mL) began to exceed that in the control (Fig. 3A). 48 h after being treated with camphor of different concentrations :1.00 and 2.00 mg/mL, the relative conductivity of F. solani G9 reached 28.9% and 67.5%, respectively, much higher than that of the control group (Fig. 3B). Fig. 3C re ects that the relative conductivity of mycelium suspension (F. verticillioide) increases with the increase of camphor concentration and the prolongation of treatment time, showing a positive correlation. 48 h after the treatment, the relative conductivity of the control, 1 mg/mL and 2 mg/mL groups (F. verticillioide) became 16.9%, 24.9% and 35.1%, respectively. The results in Fig. 3D indict that during the incubation process, the relative conductivity of the control (F. graminearum) obviously remains unchanged, but with the addition of camphor, the relative conductivity increases signi cantly.
To explore the mechanism of camphor inhibiting Fusarium growth, observation was conducted on the morphology of the four Fusarium strains by SEM (Fig. 4). As shown in Fig. 4 A, C, E, G, without the treatment of camphor, the mycelium surfaces of F. oxysporum G5, F. solani G9, F. verticillioide and F. graminearum untreated with camphor are relative smooth and complete. Whereas, 48 h after treatment with camphor, the morphology of mycelia changes in different degrees mainly in that the mycelia becomes folded or fractured (Fig. 4 B , D, F and H), indicating that camphor seriously interfers with cell wall synthesis and suppresses the growth of mycelia.
In order to investigate the effect of camphor on the membrane permeability of the Fusarium strains, determination was performed on the contents of nucleic acids and proteins in mixtures treated with camphor for 48 h (Fig. 5). Compared with the control group, after the treatment with 1 and 2 mg/mL of camphor, there is great increase in the absorption values at the characteristic wavelength of 260 nm (nucleic acid characteristic absorption peak) and 280 nm (protein characteristic absorption peak). This indicates that camphor has increased the permeability of cells, resulting in the leak of a large amount of intracellular nucleic acids and proteins. It was also found that the release degree of biomolecules varies with different strains. When the concentration of camphor was 2 mg/mL, F. graminearum showed the largest release of nucleic acids and proteins.
As to nucleic acid release (Fig. 5A), F. solani G9 ranks the rst,, followed by F. verticillioide and F. oxysporum G5, and nally F. graminearum. In the matter of protein leakage (Fig. 5B), the rst place goes to F. solani G9 with a signi cantly higher leakage than the control, followed by F. verticillioide and F. graminearum, and ultimately F. oxysporum G5. Compared with the control, at 260 nm (nucleic acids) and 280 nm (proteins), the absorbance values of F. oxysporum G5, F. solani G9, F. verticillioide and F. graminearum in suspensions treated with camphor at 1.0 mg/mL increase 1.43, 2.25, 1.92, 1.25 folds and 1.44, 3.71, 2.61, 1.25 folds respectively, and at 2.0 mg/mL level, they increase 2.01, 6.34, 2.54, 1.71 folds and 2.00, 7.94, 3.91, 2.10 folds, respectively. The results obviously imply that camphor disruptes the intact cytomembrane structure of F. solani G9, while slightly affecting F. oxysporum G5 and F. graminearum affected slightly, so it can be concluded that the impact of camphor on the permeability and structure of Fusarium cytomembrane also varies with species.
To sum up, camphor can inhibit the growth of Fusarium through various ways at different levels. It is speculated that the antifungal mechanism of camphor to Fusarium mainly involves its interference with the normal gene expression and protein synthesis in fungal cells. In this way, it can cause damage to the structural integrity of the fungal cells and cytomembrane permeability, and make mycelium fold up or break at morphology level, as well as help the release of intracellular substances and the increase of REC at physiological level. Our study also suggests that camphor, as the main active ingredient of natural plant essential oils, has the potential to be developed as a fungicide for plant protection and industrial crop production.

Discussion
It is known that essential oils extracted from aromatic plants are widely used as fungicides (Arif et al. 2009;Gazdağlı et al. 2018;Yörük 2018). Camphor is the main active compound in essential oil. It is found in many aromatic plants, such as Cinnamonum camphora, Piper capense, Salvia o cinalis, Eucalyptus globulus and Artemisia annua (Guo et al. 2014;Fu et al. 2015;Soidrou et al. 2013;Wijesundara and Rupasinghe 2018;Marinas et al. 2015;Harkat-Madouri et al. 2015). According to the previous reports and our ndings, camphor is one of the main bioactive components in plant essential oils and an important effective antimicrobial substance, playing an important role in inhibiting pathogenic microorganisms including fungi. Therefore, it is speculated that the strong antimicrobial activity of some essential oil extracted from aromatic plants is related to its camphor content.
Generally, REC is adopted to evaluate the changes in cytomembrane permeability of microorganisms and other types of cells. In previous work, it was reported that monocaprin affects the REC of Saccharomyces cerevisiae, Aspergillus niger and Penicillium citrinum and that the permeability of P. citrinum may be more easily disturbed (Ma et al. 2018). The results imply that the sensitivity of fungi strains to fungistat varies with species and genus. When the camphor concentration is set as 1.00 and 2.00 mg/mL, the nal REC (48h after treatment) of F. oxysporum G5, F. solani G9, F. verticillioide and F. graminearum reaches 15.8%, 28.9%, 24.9%, 82.2; and 18.9%, 67.5%, 35.1%, 93.0%, respectively. F. graminearum displays the highest rise in REC among the four tested Fusarium strains. These data suggest that the permeability of fungal cytomembrane may be more easily disturbed by camphor, resulting in the release of intracellular ions and charged biomolecules (Molatová et al. 2010;Ma et al. 2018).
The natural compounds extracted from plants seem to increase the permeability of microbial cell membranes, resulting in a leakage of cellular content (Burt 2004). Excessive leakage of intracellular ions and macromolecules, caused by increased membrane permeability, can result in cell death (Labbe and Saleh 2008). Storia et al. (2011) proposed that microbial cell wall and membrane are the targets of the antimicrobial activities of many natural plant compounds, such as carvacrol. The treatment of foodborne contaminated microorganisms with carvacrol could change the cell morphology and structure of some G + and Gfood-related bacteria. Previous study has also revealed that β-carboline oxadiazole derivatives could change the normal cell activities of Rhizoctonia solani, the dominant pathogenic fungus causing rice sheath blight (Zhang et al. 2018). Such adverse effects mainly embrace the decrease of mitochondrial membrane potential, the accumulation of reactive oxygen species, the blocked DNA synthesis and the destruction to cell structure. Thus, there could be a similar action mechanism of camphor against the four tested phytopathogenic Fusarium strains.
Cell membranes play an important role in maintaining the normal physiological and metabolic activities of cells. Many fungicides inhibit fungal growth by interfering with and destroying the formation and integrity of cell membranes (Avis 2007). When the cell membrane is destroyed, macromolecules are left out (Chavan and Tupe 2014). In the previous study, it was found that the Mentha piperita essential oil (MPE) changes the surface properties and permeability of Fusarium sporotrichioides hyphae (Rachitha et al. 2017). The increase of the concentration of MPE can trigger corresponding changes to cells, such as intracellular contents leakage, mycelia distortion, pH change, etc. The leakage of nucleic acid and protein manifests that camphor treatment can disturb the normal metabolism of Fusarium, and destroy the cell structure, thus inhibiting the growth of mycelia. As an important volatile component in natural plant extract, the potential antifungal mechanism of camphor could damage the fungus cell and disturb the cellular metabolism (Marilena et al. 2001).
The ndings from Gazdağlı et al. (2018) revealed the antifungal mechanism of camphor on F. graminearum and F. culmorum through gene expression level. The analysis of qPCR shows that camphor treatment down-regulates the tri5 (deoxynivalenol production) expression, while up-regulates the expression of some genes related to essential cellular activity directly determining the fungal life cycle, such as hog1, mst20, CAT, POD, mgv1, and stuA genes. The similar ndings from Yörük (2018) show that tetraconazole (TCZ, an important antifungal agent) could ght against F. graminearum at genomic, epigenetics, transcriptomics and apoptotic levels. Increasing TCZ concentration could enhance the expression of genes related to apoptosis (Hog1) and oxidative stress (POD), whereas down-regulating the expression of tri5.
This study shows that camphor has strong antifungal activity against F. oxysporum G5, F. solani G9, F. verticillioide and F. graminearum, and that the absolute inhibition rate of the four phytopathogenic fungi could be increased by more than 80% by adding 2 mg/mL camphor in PDA media. The preliminary study on the mechanism exhibits that camphor could participate in and obstruct the formation of cell wall and cytomembrane of the phytopathogens. The involvement of camphor makes fungi release intracellular ions, nucleic acids and proteins necessary for normal cell activity, ultimately inhibiting the growth of fungi. In addition, the essential oils extracted from some plant with strong antimicrobial activity may be related to their camphor content. Camphor may serve as a potential alternative fungicide for its friendliness to environment and humans. In the future, further studies will be conducted on the molecular regulation mechanism and transcriptomics of camphor inhibiting the growth of important plant pathogens.