Plasma membrane damage contributes to antifungal activity of citronellal against Penicillium digitatum

Abstract

The antifungal activity of citronellal, a typical terpenoid of plant essential oils, against Penicllium digitatum and the possible action mode involved were investigated. Results showed that the mycelial growth and spores’ germination of P. digitatum were inhibited by citronellal in a dose-dependent manner. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were determined to be 1.60 µL/mL and 3.20 µL/mL, respectively. It was found that the plasma membrane of citronellal-treated P. digitatum spores was damaged, as confirmed by the propidium iodide stain results, as well as a higher extracellular conductivity and release of cell constituents in citronellal-treated samples than those of control samples. Moreover, in vivo test results demonstrated that wax + citronellal (WC; 10 × MFC) treatment effectively reduced the incidence of green mold after 5 days of storage at 25 ± 2 °C. These findings suggested that the plasma damage mechanism contributed to the antifungal activity of citronellal against P. digitatum. In addition, citronellal was suggested to be a potential alternative to fungicidal agents in controlling green mold of citrus fruit.

Introduction

Citrus fruit are sensitive to Penicillium digitatum, P. italicum and Geotrichum citri-aurantii during postharvest handling of the fruit (Wuryatmo et al. 2014). Among them, P. digitatum is the most serious postharvest pathogen, accounting for up to 90% of product losses (Liu et al. 2010). Correspondingly, the control of postharvest diseases caused by these pathogens is vital for maintaining citrus quality and reducing economic losses. Synthetic fungicides, such as imazalil, thiabendazole, pyrimethanil, fludioxonil, are used to minimize postharvest decay effectively. However, their extensive application has led to the proliferation of resistant strains, which compromised their effectiveness, and also led to the increase of human health risks (Jhalegar et al. 2015). Therefore, there is a need to find alternatives to synthetic fungicides.

Plant essential oils might be a good choice. They have been widely reported to be an effective and safe strategy to control postharvest diseases (Pérez-Alfonso et al. 2012; Wang et al. 2012; Regnier et al. 2014; Tao et al. 2014a). Citronellal is a volatile constituent in citrus and other plant essential oils (Carrillo-Hormaza et al. 2015; Tolba et al. 2015). Its antifungal activity has been demonstrated by some researchers (Lee et al. 2008; Rammanee and Hongpattarakere 2011; Trindade et al. 2015). Previously, citronellal was found to exhibit inhibitory effect on the growth of P. digitatum, P. italicum, and P. ulaiense, with the inhibition zone of 43, 43 and 31 mm², respectively (Scora and Scora 1998). Zore et al. (2011) demonstrated that citronellal could inhibit the growth of Candida albicans by affecting membrane integrity and arresting cell cycle. The growth of nine tested fungal stains, such as P. adametzii and P. citrinum, could be completely inhibited by citronellal at a dose of 112 mg/L (Nakahara et al. 2013). In recent studies, citronellal has been observed to be one of the principal components in some plant essential oils and showed good antifungal activity against the pathogen that cause severe destruction in apple, tomato and banana (Aguiar et al. 2014; Arancibia et al. 2014). Thus, citronellal might be a potential antifungal substance in controlling postharvest disease of citrus fruit.

The objective of present work is to evaluate the effect of citronellal on P. digitatum through in vitro and in vivo experiments.

Materials and methods

Plant materials

Fruit of Newhall navel orange (Citrus sinensis Osbeck) were purchased on November 2015 from a super market near the campus of Xiangtan University, Xiangtan, China. Defect-free fruit with uniform size were chosen for the experiments.

Chemicals and microbial strains

Citronellal (95%) was purchased from TCI Shanghai (Shanghai, China). P. digitatum was provided by the Department of Biotechnology and Food Engineering, Xiangtan University, Xiangtan, China. The test strain was purified and preserved at 25 ± 2 °C on potato dextrose agar (PDA).

Effect of citronellal on mycelial growth

The effects of citronellal on mycelial growth of P. digitatum were evaluated following the method of Tao et al. (2014b). The final concentrations of citronellal in the culture media were 0.00, 0.20, 0.40, 0.80, 1.60, and 3.20 μL/mL, respectively. The lowest concentration that completely prevented the growth of the fungus after 48 h of incubation at 25 ± 2 °C was regarded as the MIC (minimum inhibitory concentration). The MFC (minimum fungicidal concentration) was defined as the lowest concentration that inhibited pathogen growth after 96 h of incubation at 25 ± 2 °C.

Effect of citronellal on spores’ germination

Different concentrations of citronellal (0.00, 0.050, 0.10, 0.20, 0.40, 0.80, 1.60 μL/mL) were used to determine their effects on spores’ germination. Fungal spores were placed on glass slides in triplicate, and slides containing the spores were incubated in a moist chamber at 25 ± 2 °C for 12 h (Wang et al. 2012).

Effect of citronellal on cell morphology

Samples were taken from the periphery of the colony growing on potato dextrose broth (PDB), and then supplied with 1.60 and 3.20 μL/mL citronellal after incubation for 30 min (for mycelia) or 6 h (for spores), respectively. Samples from control plates without citronellal were also stained and observed.

Assay of plasma membrane integrity

Membrane integrity was conducted following the method of Liu et al. (2010). Aliquots of 100 μL of P. digitatum spores’ suspension collected at different culture time (0, 2, 4, and 6 h) were transferred to 20 mL PDB with 0.050 and 1.60 μL/mL citronellal, respectively. The PDB without citronellal served as the control group. P. digitatum spores’ suspension was adjusted to a final concentration of 10⁶ spores/mL. The number of spores in bright-field was defined as the total number, and membrane integrity (MI) was calculated according to the formula:

$$\text{MI}\left(\%\right)=\left[1-\left(\text{number}\text{of}\text{stained}\text{spores/number}\text{of}\text{total}\text{spores}\right)\right]\times 100$$

Measurement of extracellular conductivity

Measurement of extracellular conductivity of P. digitatum mycelia and spores was assayed following the method of Tao et al. (2014a). Citronellal at various concentrations (0, MIC and MFC) was added and incubated for 0, 30, 60, 120 min (for mycelia) and 0, 2, 4, 6 h (for spores) at 25 ± 2 °C in an environmental incubator shaker (150 rpm), respectively. The extracellular conductivity at each time point was recorded and expressed as μS/cm. Each parameter was tested in triplicate.

Determination of the release of cell constituents

The release of cell constituents into the supernatants was measured according to the method described previously (Tao et al. 2014c). The suspensions were treated with citronellal at various concentrations (0, MIC and MFC) for 0, 30, 60, 120 min (for mycelia) and 0, 2, 4, 6 h (for spores). Each parameter was tested in triplicate.

In vivo assays

In vivo assays were conducted following the method of Fan et al. (2014). After fungi were inoculated, the fruit were sprayed with wax amended with citronellal at MFC or 10 × MFC. The incidence rate of disease (measured by counting the number of green mold-inflicted lesions) was calculated as follows:

$$\text{Disease}\text{incidence}\left(\%\right)=\left(\text{number}\text{of}\text{rotten}\text{fruit/total}\text{number}\text{of}\text{total}\text{fruit}\right)\times 100$$

Statistical analysis

Each parameter was tested in triplicate. Conventional statistical methods were used to calculate means and standard deviations. Comparisons between groups were tested by one-way ANOVA analysis and LSD test (P < 0.05) using SPSS statistical software package release 16.0 (SPSS Inc., Chicago, IL, USA).

Results

Effect of citronellal on mycelial growth

As shown in Fig. 1a, the mycelial growth of P. digitatum was affected by citronellal in a dose-dependent manner (P < 0.05). High citronellal concentration (≥1.60 μL/mL) completely inhibited the mycelial growth of P. digitatum, whereas citronellal at a concentration of 0.20 μL/mL showed stimulatory effect on the mycelial growth of P. digitatum after 2 days of incubation. After 4 days of incubation, the mycelial growth of P. digitatum was still totally inhibited by 3.20 μL/mL citronellal. MIC and MFC values of citronellal for P. digitatum were 1.60 and 3.20 μL/mL, respectively.

Fig. 1.

a Effect of citronellal at different concentrations on mycelial growth of P. digitatum incubated at 25 ± 2 °C for 5 days; b effect of different citronellal concentrations on the germination rate of P. digitatum spores incubated at 25 ± 2 °C for 12 h; c light microscopy image of P. digitatum mycelia after 30 min of incubation (A, B, and C) or spores after 6 h of incubation (D, E, and F) with or without citronellal. Data presented are the means of pooled data. Error bars indicate the standard deviations of the means (n = 3). Symbols with different letters at each time point indicate significant differences according to Duncan’s multiple range test (P < 0.05)

Effect of citronellal on spores’ germination

The percentage of germinated spores of P. digitatum is recorded in Fig. 1b. Citronellal at a concentration of 0.050 μL/mL stimulated the germination of spores. In contrast, the spores’ germination was visibly inhibited by citronellal at concentrations higher than 0.20 μL/mL. The germination rates for 0.40, 0.80, 1.60 μL/mL citronellal-treated samples were 53.0, 41.7 and 3.7%, respectively, which was significant lower than those of control (63.3%, P < 0.05).

Light microscopy

Light microscopy of the control P. digitatum hyphae showed normal morphology (Fig. 1c-A). Treatment with citronellal at MIC (Fig. 1c-B) or MFC (Fig. 1c-C) apparently altered the morphology of P. digitatum hyphae, including irregular hyphae, loss of linearity, and formation of warty surfaces. The control P. digitatum spores were normal and homogenous shape (Fig. 1c-D). By contrast, the MIC-treated spores showed a sunken surface and malformation (Fig. 1c-E), whereas the spores treated with MFC of citronellal were anomalous shape, with unordered structure and rough surface (Fig. 1c-F).

Effect of citronellal on plasma membrane integrity of P. digitatum spores

The plasma membranes of P. digitatum spores were markedly damaged by citronellal (P < 0.05) (Fig. 2a). The MI values of P. digitatum spores declined obviously with the increase of incubation time, in contrast to a relatively high level in those of control spores. After 6 h of incubation, the MI values of citronellal-treated and control P. digitatum spores were 58.4 and 90.2%, respectively.

Fig. 2.

a Effect of citronellal (1.60 μL/mL) on the plasma membrane integrity of P. digitatum spores(A, B, C, and D stand for the PI staining results at 6 h of exposure, whereas E stands for the percentage of plasma membrane integrity; b Effect of citronellal on the extracellular conductivity; c The 260 nm-absorbing material release of mycelia and spores. (Filed square) control; (open circle) citronellal at MIC; (filed triangle) citronellal at MFC. Data presented are the means of pooled data. Error bars indicate the SDs of the means (n = 3). Symbols with different letters at each time point indicate significant differences according to Duncan’s multiple range test (P < 0.05)

Extracellular conductivity

The extracellular conductivity in mycelia or spores suspensions was increased by the citronellal treatments (Fig. 2b-A). The extracellular conductivity in spores with MIC of citronellal for 6 h was 162.0 ± 1.0 μS/cm, which was significantly higher (P < 0.05) than those of control (133.3 ± 3.2 μS/cm) but significantly lower (P < 0.05) than those with MFC of citronellal (169.2 ± 2.0 μS/cm) (Fig. 2b-B)

Release of cell constituents

During the early exposure of MIC or MFC before 30 min (mycelia) and 6 h (spores), the release of cell constituents significantly increased (P < 0.05). The OD_260nm value in P. digitatum mycelial suspensions with MIC of citronellal for 30 min was 0.36 ± 0.03, which was significantly higher (P < 0.05) than those of the control (0.16 ± 0.02) but significantly lower (P < 0.05) than those with MFC of citronellal (0.56 ± 0.03) (Fig. 2c-A). The OD_260nm value of P. digitatum spores suspensions treated with MIC and MFC of citronellal maintained a smooth ascending trend after 2 h of exposure, whereas those with MFC continuously increased after 4 h of exposure, and reached to the highest absorbance of 0.45 ± 0.02 and 0.65 ± 0.17 at 6 h of exposure, respectively (Fig. 2c-D).

In vivo experiments

The ability of citronellal treatment to inhibit the disease development of citrus fruit inoculated with P. digitatum is presented in Table 1. The control fruit began to decay after 2 days of storage, whereas the incidence of fruit decay significantly (P < 0.05) delayed in citronellal-treated fruit in a dose-dependent manner. As incubation time was prolonged up to 4 days after inoculation, despite that the incidence of decay in control fruit was 100%, those in citronellal-treated (1 × and 10 × MFC) fruit were 100 and 5%, respectively.

Table 1.

Decay incidences in inoculated fruit treated with wax and wax + citronellal (WC; 1 × and 10 × MFC) treatment during storage at 25 ± 2 °C for 5 days and 85–90% relative humidity

Treatment	Disease incidence (%)
	Inoculation period (days)
	1	2	3	4	5
Wax	0a	25a	80a	100a	100a
WC(1 × MFC)	0a	10b	65b	100a	100a
WC(10 × MFC)	0a	0c	0c	5b	30b

Data presented are the means of pooled data (n = 10). Columns with different letters at each time point indicate significant differences according to Duncan’s multiple range test (P < 0.05)

Discussion

In the present study, citronellal exhibited a pronounced antifungal activity against the mycelial growth of P. digitatum, with MIC and MFC values being 1.60 μL/mL and 3.20 μL/mL, respectively. The MIC value was higher than those for citronellol, geraniol, neral, geranial and myrtaceae essential oils, but lower than those for citral, octanal, α-terpineol (Lee et al. 2008; Zhou et al. 2014). In addition, the MFC value was lower than those for citral, and α-terpineol (Zhou et al. 2014). The germination of P. digitatum spores was also visibly inhibited by citronellal at concentrations higher than 0.20 μL/mL. This result was in agreement with previous reports describing the antifungal activity of citronellal (Nakahara et al. 2013; Aguiar et al. 2014). Interestingly, citronellal at a concentration of 0.050 μL/mL could stimulate the germination of P. digitatum spores. This phenomenon was also observed in some previous studies. For example, Droby et al. (2008) reported that limonene, α-pinene, β-pinene and myrcene were stimulatory to P. digitatum and P. italicum at relatively low concentrations. In our previous studies, the spores’ germination and mycelial growth of P. italicum and P. digitatum were found to be mildly stimulated by the ‘Shatangju’ (C. reticulata Blanco) essential oil, β-myrcene, terpinen-4-ol, and citronellal (Wang et al. 2012; Tao et al. 2014a).

The permeability and integrity of cell plasma membrane serve important functions in maintaining fungal viability (Paul et al. 2011; Tao et al. 2014a). Previous studies have shown that the lipophilicity of essential oils enables them to preferentially partition from an aqueous phase into membrane structures of the fungi, resulting in membrane expansion, increased membrane fluidity and permeability, disturbance of membrane-embedded proteins, inhibition of respiration, alteration of ion transport processes in fungi and induced the leakage of other cellular contents (Tao et al. 2014b; Tyagi and Malik 2010; Zhou et al. 2014). In the current research, the results of PI staining and microscopy showed that the membrane integrity of P. digitatum spores obviously declined after citronellal treatment (Fig. 2a). In addition, exposure of P. digitatum to citronellal caused fast the loss of 260 nm absorbing materials and the increase of extracellular conductivity. These findings indicate that citronellal can induce damage to cell plasma membrane integrity of P. digitatum cells, which is in agreement with other studies (Zore et al. 2011; Tao et al. 2014c).

The in vivo results showed that citronellal elicited an evident inhibitory effect on green mold rot in navel orange fruit. These results confirmed those observed in the in vitro assay and also agreed with reports on other essential oils or their volatile compounds in controlling the diseases in citrus fruit (Fan et al. 2014; Pérez-Alfonso et al. 2012; Regnier et al. 2014; Tao et al. 2014b).

Conclusion

Our present research indicates that citronellal can inhibit the mycelial growth and spores’ germination of P. digitatum by a plasma damage mechanism. In addition, citronellal combined with wax treatment can reduce the incidence rate of postharvest green mold in citrus fruit. These results suggest that citronellal might be developed to be an alternative as natural fungicidal agents in controlling the postharvest disease of citrus fruit.