Introduction

Muskmelon (Cucumis melo L.) is major crops with high economic value due to their unique flavor and rich nutrition, which is widely grown in Northeast and Northwestern of China (Deng et al. 2015). However, it is quite perishable and susceptible to postharvest rot caused by phytopathogenic fungi (Yang et al. 2009). Pink rot caused by Trichothecium roseum is one of the most principal postharvest diseases of muskmelon in China (Ge et al. 2015). This disease is frequently controlled by the application of synthetic fungicides (Yang et al. 2009). Although synthetic fungicides have greatly extended the shelf life of field crops, postharvest losses still high at 50% in developing countries, with molds causing more than 70% of losses in fruits and vegetable storage (Frankova et al. 2016). Moreover, the continuous use of fungicides has attracted public concern on fungicide residues, development of fungicides resistance in pathogens, and potential harmful effects on human health and environmental safety (Tripathi and Dubey 2004; Yang et al. 2009). Therefore, alternative strategies to fungicides, that will be both safe and eco-friendly, have been widely sought (Pawlowska et al. 2012; Terry 2004; Tsuda et al. 2016).

Bio-preservation has emerged as an excellent candidate, which refers to the use of microorganisms and/or their metabolites to extend the shelf life and enhance the safety of foods (Cheong et al. 2014; Galvez et al. 2010; Plaza et al. 2016). Lactic acid bacteria (LAB), classed as generally regarded as safe (GRAS), have been used as bio-preservation organisms in foods for inhibiting the growth of fungi through the production of organic acids, fatty acids (Gerez et al. 2013), hydrogen peroxide, reuterin and bacteriocins (Yang and Chang 2010). Previous studies have exhibited the effectiveness of LAB in protecting different fruit species against various fungal diseases (Ghosh et al. 2015; Lan et al. 2012; Wang et al. 2013a). Lan et al. (2012) found that the antifungal strain Weissella cibaria 861006 inhibit the growth of Penicillium oxalicum on the surface of grapes. Moreover, Pediococcus pentosaceous 54 is shown to have protective properties against Penicillium expansum spoilage when applied in plum, pear and grape models (Crowley et al. 2013b). To our knowledge, there have been no reports on the antifungal effect of LAB against pink rot caused by Trichothecium roseum and on whether LAB influence on the activity of defense-related enzymes of muskmelon fruit.

The aims of this study were: (1) to obtain LAB with antifungal activity against T. roseum, (2) to analyze the major antifungal substances produced by LAB, and (3) to investigate the effect of cell-free supernatant produced by LAB on the activity of defense-related enzymes in muskmelon fruit.

Materials and methods

Microorganisms and culture conditions

Four LAB species, Lactobacillus plantarum C10, Lb. rhamnosus RH-11, Lb. helveticus LH-9 and Lb. sakei DL11 were provided from Microbiology Laboratory Culture Collection. All LAB strains were grown in Mann Rogosa Sharpe (MRS, Aoboxing, Beijing, China) broth at 37 °C for 24 h. Cell-free supernatant (CFS) produced by LAB was obtained by centrifugation at 6000g for 15 min at 4 °C and sterile filtration (0.45 μm, Millipore). Target bacteria T. roseum isolated from decayed muskmelon fruit, was grown on potato dextrose agar (PDA, Aoboxing, Beijing, China) at 28 °C for 7 days. The spores were collected in sterile Tween 80 at 0.05% (v/v) and counted at the microscope in a haemocytometer chamber, which concentration was used to adjust to 1 × 106 spores/ml.

Antifungal activity assays

Antifungal activity assay was performed according to the method as described by Wang et al. (2011). PDA (25 ml) containing CFS (2.5 ml) was poured into sterile plate. After solidification, this plate was inoculated with agar discs of the T. roseum (7 mm) at the center and incubated at 28 °C for 2–7 days. Each dish was diametrically monitored in perpendicular directions until the fungi growth in the control plate was almost complete. The inhibitory rate (I) was calculated as follow: I (%) = [(C − T)/(C − C0)] × 100. C means the diameter of mycelia growth in control group (mm), T means the diameter of mycelia growth in treated group (mm), and C0 means the diameter of the target fungi agar discs (7 mm). Control plates containing media mixed with sterile water (10%, v/v) were inoculated.

Effect of CFS on the spore and mycelial morphology of T. roseum

MRS agar (15 ml) was poured into a petri dish. CFS was inoculated as two 3 cm long lines and incubated at 37 °C for 72 h. This plate was then overlaid with PDA (15 ml) containing 1 × 106 spores/ml and incubated at 28 °C for 6 days. The mycelia were harvested from the cultures grown on PDA. Spores were prepared by washing with 0.85% NaCl containing Tween 80 (0.1% v/v) and then centrifuged at 6000g for 15 min. The sediments were collected for SEM.

The mycelia and spores were rinsed with phosphate buffer (0.1 mol/l, pH 7.4), and then fixed with 2.5% glutaraldehyde at 4 °C for 24 h. The sediments were dehydrated by a graded series of ethanol (50, 70, 80, 90, 95 and 100%) for 20 min at each step. After dehydration, these samples were dried with vacuum freezing dryer (Free Zone 2.5 L, Labconco, USA). Finally, all samples were coated with gold–palladium and observed using S-4800 SEM (Hitachi, Japan).

Quantification of organic acids and phenyllactic acid (PLA)

Lactic acid, acetic acid and propionic acid in the CFS were quantified by Agilent 6980N gas chromatograph system (Agilent, USA) equipped with CNW CD-ACID WAX column (30 m × 0.25 mm × 0.25 μm). The column temperature programme was: initial temperature of 110 °C, then raised to 150 °C at 10 °C/min and temperature held for 5 min, and finally increased to 230 °C at 10 °C/min, and held for 15 min. The injector temperature and FID detector temperature were 280 and 300 °C, respectively. The flow rate of hydrogen and nitrogen carrier gas was 30 ml/min, and the flow rate of air was 300 ml/min. One microliter sample was injected in the split mode (15:1).

PLA was determined by Agilent 6980 N gas chromatograph system (Agilent, USA) equipped with RTX-5 column (30 m × 0.25 mm × 0.25 μm). The column temperature was initially held at 180 °C for 20 min, and then raised to 280 °C at 20 °C/min, held for 10 min. The injector temperature and FID detector temperature were 280 and 300 °C, respectively. The flow rate of hydrogen and nitrogen carrier gas was 30 ml/min, and the flow rate of air was 300 ml/min. One microliter sample was injected in the split mode (20:1).

Determination of the antifungal activities of acids mixtures

Acids mixtures included lactic acid (Sigma L6661, USA), acetic acid (Sigma 695092, USA), propionic acid (Sigma 402907, USA) and PLA (Sigma P7251, USA). The individual acid and acids mixtures were prepared according to the data obtained after quantification by gas chromatograph analysis. The pH of all samples assayed (CFS, individual acids and acids mixtures) was adjusted to pH 4.0. The antifungal activities of all samples were assayed using previously described method.

Effect of pH on antifungal activity of CFS

Aliquots of CFS were adjusted to pH 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 and 7.0 respectively, by adding the appropriate volumes of 4 N HCl and 4 N NaOH. The antifungal activity was tested by previously described method.

Effect of LAB on muskmelon fruit decay

Muskmelon fruit was selected for uniformity of size, absence of defects, and washed with running water. After drying in air, the fruit were disinfected with 2% (v/v) sodium hypochlorite for 5 min, and washed with sterile water, air-dried, then sterilized with 70% ethanol. Three wounds (3 mm deep × 3 mm wide) were punched in each fruit using a sterile needle, and then 20 μl of CFS and a ring fungal mycelium were inoculated in each wound and incubated at 25 °C.

Effect of CFS on induction of defense-related enzyme activitives

For enzyme extraction, approximate 1.5 g of sample was taken from 3 to 8 mm below the peel. Each sample was packed with silver paper and frozen in liquid nitrogen, and kept at − 80 °C until the crude enzyme extraction.

All enzymes extraction procedures were conducted at 4 °C. For phenylalnine ammonialyase (PLA), the reaction mixtures contained 1.5 g of frozen sample, 2.5 ml of cold borate buffer (0.1 N, pH 8.8), 1 mmol/l EDTA, 10% (w/v) polyvinylpolypyrrolidone (PVPP), and 0.05 mM β-mercaptoethanol. For peroxidase (POD) and polyphenoloxidase (PPO), 1.5 g of frozen sample was homogenized in 2.5 ml of cold sodium phosphate buffer (50 mM, pH 7.5), containing 1 mmol/l phenylmethylsulfonyl fluoride (PMSF), 1 mmol/l polyethyleneglycol (PEG), 10% (w/v) PVPP, and 0.1% (v/v) Triton X-100. The mixtures were centrifuged at 12,000g for 30 min and supernatant was used for the enzyme assay.

Supernatant (500 μl) was incubated with 3 ml l-phenylalanine (7 mM) at 37 °C for 30 min. The enzyme activity was measured at an absorbance of 290 nm using ultraviolet–visible spectrophotometer (PerkinElmer, USA). The PAL activity was expressed as U/mg protein, where one unit was defined as 0.01 ΔOD290 per minute.

The reaction mixtures of PPO contained 200 μl of supernatant, 200 μl of cold PBS (50 mM, pH 7.0) and 0.5 ml of 50 mM pyrocatechin. The reaction mixtures of POD contained 200 μl of supernatant, 2.5 ml of 25 mM guaiacol and 200 μl of 25 mM H2O2. The enzyme activity was monitored at 240 nm for 2 min at 24 °C. The PPO and POD activity was expressed as U/mg protein, where 1 U was defined as 0.01 ΔOD290 per minute.

Effect of CFS on the contents of total phenolics and falconoids

The froze sample (1.5 g) was homogenized in 2.5 ml of 1% HCl methanol solvent, then centrifuged at 12,000g for 10 min at 4 °C and the supernatant was used for the assay. The total phenol and falconoid content were expressed as ΔOD280/g FW and ΔOD325/g FW, respectively.

Statistical analysis

All experiments were performed as three independent replicates and expressed as mean ± standard deviation. Statistical analyses were performed using the Origin Pro 8.0 (Origin Lab Corporation, USA) and SPSS 19.0 software (SPSS, Inc., Chicago, IL, USA).

Results

Antifungal activity of LAB

The inhibitory rate of strain C10, RH-11, LH-9 and DL11 reached 93.93, 89.69, 83.93 and 78.29%; respectively (Fig. 1).The maximum inhibitory rate was exhibited by the CFS of Lb. plantarum C10. Therefore, Lb. plantarum C10 was selected for further analysis.

Fig. 1
figure 1

The inhibitory rate of CFS produced by four LAB strains against T. roseum. C10: Lb. plantarum C10, RH-11: Lb. rhamnosus RH-11, LH-9: Lb. helveticus LH-9, DL11: Lb. sakei DL11

Full size image

Effect of CFS on the spore and mycelial morphology of T. roseum

SEM imaging showed that the spores of T. roseum appeared to be intact and grew normally in control group (Fig. 2a). When treated with CFS, the seriously damaged and deformed structures of spores were observed, accompanied with intracellular components leakage (Fig. 2b). The mycelial surface of T. roseum in control group was smooth with no breakage (Fig. 2c). By contrast, the mycelial surface of T. roseum treated with CFS was collapsed and appeared abnormally club-shaped inflated ends (Fig. 2d). SEM observations indicated that CFS seriously damaged the structures of spores and mycelia, accompanied with cytoplasmic leakage.

Fig. 2
figure 2

SEM micrographs of mycelia and spores of T. roseum treated with the CFS of Lb. plantarum C10. a the spores without treated with the CFS of Lb. plantarum C10; b the spores treated with the CFS of Lb. plantarum C10; c the mycelia without treated with the CFS of Lb. plantarum C10; d the mycelia treated with the CFS of Lb. plantarum C10

Full size image

Characterization of the antifungal activity of acids mixtures

To determine if the antifungal activity was due to organic acid production, quantification of organic acid in the CFS of Lb. plantarum C10 was performed by gas chromatograph analysis. Lactic acid was produced in the highest quantity (61.71 μl/ml) and acetic acid (5.83 μl/ml) and PLA (5.62 μl/ml) were produced in small quantities. Propionic acid (0.09 μl/ml) was produced in trivial amounts.

The inhibitory rate of lactic acid, acetic acid, propionic acid and PLA reached 90.12, 37.54, 3.64 and 9.08%, respectively. Acids mixtures were reconstituted in accordance with the content of each acid existed in the CFS, which showed strongly antifungal activity and inhibitory rate reached 98.18%. The acids mixtures had higher activity than the CFS (93.93%). This result indicated that the acid compounds produced by Lb. plantarum C10 were the major antifungal substances.

Effect of pH on antifungal activity of CFS

The maximum antifungal activity was observed at pH 3.5. However, the activity was completely disappeared at pH 7.0 (Fig. 3). This result further indicated that the acid compounds produced by Lb. plantarum C10 were the major antifungal substances.

Fig. 3
figure 3

Effect of pH on the antifungal activity of CFS produced by Lb. plantarum C10

Full size image

Effect of LAB treatment on fruit decay

The lesion area of fruits inoculated with T. roseum and CFS during storage for 7 days is presented in Fig. 4. By day 7, browning and rotting of fruit was observed on positive and negative control groups, and the lesion area of fruit achieved 6259.97 and 6288.05 mm2, respectively. By contrast, the evidently reduced account of T. roseum contamination was observed on the treated group, and the growth zone of T. roseum decreased 19.58 and 13.73%, respectively. This result indicated that CFS significantly reduced contamination zone of T. roseum in muskmelons.

Fig. 4
figure 4

The growth zone of T. roseum in muskmelon during storage for 7 days. C10: muskmelon fruit treated with CFS produced by Lb. plantarum C10; NC untreated muskmelon fruit used as negative control, PC muskmelon fruit treated with MRS used as positive control

Full size image

Effect of CFS on induction of defense-related enzyme activities

Changes in PAL, POD and PPO activities in response to the CFS are shown in Fig. 5. The activity of PAL was continuously induced by the CFS treatment and reached a peak 36.93 U/mg protein on the sixth days, then declined until the end of storage (Fig. 5a). PAL activity in fruit treated with CFS was 12.0 and 11.6% higher than that in the negative and positive control groups (Fig. 5a). This result suggested that PAL activity in muskmelon was induced by the CFS treatment.

Fig. 5
figure 5

Effects of the CFS produced by Lb. plantarum C10 on the activity of PAL (a), POD (b), PPO (c) and the contents of total phenolics (d) and flavonoids (e) in muskmelon fruit. C10: muskmelon fruit treated with CFS produced by Lb. plantarum C10; NC untreated muskmelon fruit used as negative control, PC muskmelon fruit treated with MRS used as positive control

Full size image

The same trend was observed in POD activity. The activity was markedly elevated from 0 to 6 days and then slightly decreased from 6 to 7 days (Fig. 5b). The activity of POD in fruit treated with CFS achieved 2.43 U/mg protein on the sixth days, which was 18.5 and 15.6% higher than that in the negative and positive control groups (Fig. 5b). This result suggested that POD activity in muskmelon was induced by the CFS treatment.

Changes in PPO activity of all groups showed the same trend in which the activity increased initially and then decreased (Fig. 5c). The PPO activity in CFS-treated fruit reached a peak 3.02 U/mg protein on the sixth days, which was 24.3 and 19.8% higher than that in the negative and positive control groups (Fig. 5c). The result indicated that inoculated with CFS markedly induced PPO activity in muskmelon.

Effect of CFS treatment on the contents of total phenolics and flavonoids

Total phenolics content increased to a maximum on the third days and then declined steadily until the end of storage (Fig. 5d). Accumulation of phenolics compounds in fruit by abiotic or biotic elicitors has been considered as a defence mechanism (Wang et al. 2013b). The content of total phenolics treated with CFS reached a peak on the third days, which was 6.3 and 5.0% higher than that in the negative and positive control groups (Fig. 5d). This result indicated that CFS treatment strongly induced accumulation of the total phenolics.

The content of flavonoids declined at 1d and progressively increased to a peak on third days, and then decreased steadily until the end of storage (Fig. 5e). The decrease of flavonoids content at the beginning might be due to the fact of difference in treatment (Sivankalyani et al. 2016). The flavonoids content treated with CFS reached a peak on third days, which was 11.8% higher than that in control groups (Fig. 5e). Accumulation of flavonoids in fruit by biotic elicitors has been regarded as plant defense reactions. This result indicated that the CFS treatment evidently triggered the accumulation of flavonoids in fruit.

Discussion

T. roseum is responsible for major postharvest losses of muskmelon and accounts for up to 10% of the total fungal decay during fruit storage (Yi et al. 2008). The use of LAB bio-preservatives to control plant diseases has been explored as an alternative to the use of synthetic fungicides (Pawlowska et al. 2012; Tripathi and Dubey 2004). Multiple studies have highlighted the antifungal properties of LAB in order to reduce fungal spoilage in fruits (Crowley et al. 2013a). Wang et al. (2013a) demonstrated that the shuffled mutant strain Lb. plantarum F3C2 inhibit the growth of Penicillium digitatum KM08 on the surface of kumquats. In addition, Lb. plantarum IMAU10014 was found to have a high ability to prevent Botrytis cinerea growth on tomato leaves, and its activity substance include proteinaceous substance and other metabolites (Wang et al. 2011). In this study, the CFS from Lb. plantarum C10 exhibited strong antifungal activity towards T. roseum and its inhibitory rate reached 93.93%.

It is well known that the role of organic acids as antifungal substances has been widely reported in the literature (Lavermicocca et al. 2000; Prema et al. 2008). Acetic acid and propionic acid are commonly used by manufactures as preservatives in a variety of foods (Gerez et al. 2013). In this study, the contents of lactic acid, acetic acid, propionic acid and PLA in the CFS of Lb. plantarum C10 were detected using gas chromatograph analysis. The inhibitory rate of acids mixtures reached 98.18%, which was higher than the CFS. In addition, the antifungal activity of CFS was lost after neutralization treatment, which confirmed the acidic nature of the antifungal substances. These results demonstrated that the organic acids produced by Lb. plantarum C10 were the main antifungal substances. Various organic acids produced by LAB have been implemented as fungal inhibitors, where synergistic effects are believed to be involved. For example, a mixture of lactic acid, acetic acid and PLA was identified as being responsible for antifungal activity of Lb. plantarum CRL 759, and its antifungal activity was lost after neutralization the pH of the CFS (Gerez et al. 2013).

The antimicrobial effects of organic acids were attributed to reduction of pH to a level below the range of fungi growth (Crowley et al. 2013b). In addition to the effect on pH, undissociated acid could cause the collapse of electrochemical proton gradients, resulting in bacteriostasis and death of susceptible bacteria (Li et al. 2014). The antifungal mechanism of organic acids is quite complicated and still not fully understood (Torres et al. 2011). In this study, we preliminarily studied the effects of the CFS on the mycelia and spores morphology of T. roseum by SEM analysis. The result indicated that the CFS seriously damaged the structures of spores and mycelia, resulted in cytoplasmic leakage. Similar result have been reported for Lb. casei AST18, the mycelia and spores of Penicillum chrysogenum treated with CFS of Lb. casei AST18 were significantly damaged and appeared cytoplasmic leakage (Li et al. 2014). To obtain more detailed information about the antifungal mechanism of LAB, more assays needed to be performed in the future, such as ultrastructure changes, DNA damage and generation of reactive oxygen species.

In recent years, the induction of defense responses in postharvest fruits by various biocontrol agents has become an increasingly attractive option for preventing pathogens (Lu et al. 2013; Wang et al. 2013c; Zhang et al. 2016). Fruits are able to protect themselves upon various phytopathogen attacks by producing a wide spectrum of defence enzymes that improve both cellular protection and disease resistance (Konappa et al. 2016). Defense-related enzymes, including PAL, POD and PPO, are commonly considered important in induced resistance of fruits (Passardi et al. 2004). PAL is a rate-limiting enzyme in the phenylpropanoid pathway, and an increase in PAL activity is associated with biosynthesis of active metabolites such as salicylic acid, phytoalexins, lignins, flavonids and phenolics (Zhang et al. 2011). Induction of PAL activity increased significantly in fruit surface-wounds following treatment with the CFS. This result is in agreement with previous finding that PAL activity in tomato was enhanced by treatment with Lb. paracasei (Konappa et al. 2016).

POD, a biochemical indicator of disease resistance, is a prerequisite for ligin synthesis to reinforce the cell wall and may also alter the antioxidant ability of fruits to cope with pathogens (Brisson et al. 1995). An increase in POD could enhance resistance against pathogen spreading and progress in intercellular spaces of parenchyma cells by accelerating the reinforcement of cell walls. Besides POD, rapidly elevated level of PPO is also important for resistant pathogens. PPO can produce antimicrobial quinones through oxidizing polyphenolic compounds (Mohammadi and Kazemi 2002). Compared with the control group, the activities of POD and PPO in CFS-treated muskmelon fruit were obviously enhanced in this study. Similarly, Lu et al. (2013) reported that Rhodosporidium paludigenum reduces the disease incidence caused by Penicillium digitatum in citrus fruit and enhances activities of defensive enzymes (PAL, PPO and POD).

Accumulation of phenolics compounds at infection sites may limit the development of the pathogens and lead to rapid cell death. Flavonoids are produced in the phenylpropanoid pathway, which are directly involved in fruit protection against pathogens defense responses. The concurrent increases in phenolics, flavonoids and activities of defense-related enzymes are consistent with the findings of Konappa et al. (2016), in which Lb. paracasei enhanced activities of POD, PPO and PAL and promoted the accumulation of phenolics.

Lb. plantarum C10 treatment significantly reduced the decay incidence and lesion area of muskmelon fruit infected with T. roseum. Similar result has been recorded for Meyerozyma guilliermondii, treatment with M. guilliermondii obviously reduced the decay lesion diameter of pears caused by P. expansum (Yan et al. 2018). These results suggested that the CFS produced by Lb. plantarum C10, as a biopreservative, effectively reduced pink rot incidence by enhancing the activities of defense-related enzymes.

Conclusion

CFS produced by Lb. plantarum C10 strongly inhibited the growth of T. roseum and inhibitory rate reached 93.93%. Acid compounds produced by Lb. plantarum C10 were the major antifungal substances and exhibited a narrow pH range from 3.5 to 6.5. The antifungal mode of the CFS was to damage the structures of spores and mycelia. When CFS from Lb. plantarum C10 was applied to muskmelon, the growth zone of T. roseum decreased 19.58% compared to the control, the increased activities of PAL, POD and PPO, and the accumulation of total phenolics and flavanoids were observed. Therefore, Lb. plantarum C10 can be used as a biocontrol agent to control the T. roseum caused muskmelon fruit rot.