Received Date: June 15, 2015 Accepted Date: July 13, 2015 Published Date: July 20, 2015
Citation: Ladu G, Cubaiu L, d’Hallewin G, Pintore G, Petretto GL, et al. (2015) Rosmarinus officinalis L. and Myrtus communis L. Essential Oils Treatments by Vapor Contact to Control Penicillium digitatum. J Food Process Technol 6:492. doi: 10.4172/2157-7110.1000492
Copyright: © 2015 Ladu G, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Background: The antimicrobial activity of Essential Oils (EOs) has been used for centuries and nowadays the efforts to develop natural preservatives in postharvest management have augmented interest in their possible applications.
Materials and Methods: Rosmarinus officinalis L. and Myrtus communis L. EOs, and two of their components, α- and β-pinene, have been tested in vitro against Penicillium digitatum, with the aim to assess their antifungal effects when applied as fumigation. The pathogen, inoculated on PDA dishes, was treated by EO-vapors contact and the fungal growth inhibition was recorded in order to evaluate the EOs antifungal activity.
Results: The exposure to the EOs vapors shows different ability in the control of fungal growth related to the EO concentration used and the elapsed time between the fungal inoculum and EO vapor contact. The greatest antifungal activity was observed for rosemary EO, while a less control was found for the myrtle one. Treatment performed with α-pinene showed a control of the pathogen that was similar to the myrtle, whereas control with β-pinene was very poor.
Highlight: Our finding revealed that plant EOs could be successful in controlling fungal postharvest disease in a dose and compound dependent manner, but deeper researches are needed about treatment parameters because the effectiveness seems to be affected by treatment modalities.
Antifungal; Post-harvest disease; Plant extracts; SEM
Microbial spoilage is one of the several reactions that alter the safety and the organoleptic properties of perishable products. These reactions decrease the value of food because of the destruction of functional substances, the production of off-flavors and the production of food borne illness related to unsafe food intake . In the field and during postharvest several infections that occur are related to fungal diseases. Fungi are universal biological agents able to colonize fruits and vegetables, causing pathologic disorders because of their potential to synthesize a broad range of hydrolytic enzymes . The high losses in quality, nutrient content and monetary value recorded during postharvest are related to the settlement of molds toxigenic strains [3,4]. Furthermore many mold species can also synthesize mycotoxin, hazardous compounds to animal and human health. Thus inhibition of fungal growth is an effective way to prevent mycotoxin accumulation [5-7]. Furthermore consumers have become more aware of potential health risks associated to synthetic fungicide residues and are increasingly interested in commodities treated with compounds from natural sources . For these reasons new safe technologies, with no risk for consumers and low impact for the environment are needed.
Essential oils are secondary metabolites of plants, which have been traditionally used due to their antimicrobial/antifungal activity , and many of them, such as clove, oregano, thyme, basil or cinnamon are categorized as GRAS (Generally Recognized as Safe) by the USFDA (Food and Drug Administration) . Indeed, essential oils and their active compounds could potentially serve as an effective alternative to conventional antimicrobial agents. The antimicrobial activity depends on the constituents and their concentration: phenolic compounds possess the highest antimicrobial properties followed by alcohols aldehydes ketones ethers and hydrocarbons . The strong antimicrobial activity could be correlated to the high percentage of monoterpenes, phenols and ketones . It is suggested that antimicrobial activity of EOs could be the results of disturbance in several enzymatic systems involved in energy production and structural components synthesis; also they affect and disturb genetic material functionality [13,14]. Presence of phenols with hydroxyl group (-OH) are responsible of antimicrobial properties, the interaction with the membrane enzymes and protein can cause an opposite flow of protons, affecting cellular activity . Nowadays, the efforts to develop natural preservatives in postharvest management have augmented interest in their applications. The most widely used methods to determine in vitro the antimicrobial properties of EOs are agar diffusion and broth dilution, while in our study we have performed the EOs application by fumigation. Due to their bioactivity in the vapor phase  EOs could be used as fumigant during postharvest protection. The aim of the present investigation was to evaluate the in vitro fungi toxic activity of Rosmarinus officinalis L. and Myrtus communis L. EOs, and two of their ccomponents, α- and β-pinene, against Penicillium digitatum when applied as vapor contact.
Plant material and EO extraction
Aerial parts of wild Myrtus communis L. and Rosmarinus officinalis L. were collected in April (2013) from the Sassari area, Sardinia, Italy. A sample of leaves, weighing 50 g, was subjected to hydro-distillation using a Clevenger type apparatus for 2h, according to the European Pharmacopoeia protocol (2002). The EO isolation was carried out in triplicate, the obtained EOs were collected separately, dried over anhydrous sodium sulfate (Na2SO4) and stored under nitrogen atmosphere at 4ºC, in amber glass vials, until analysis.
Gas chromatography-mass spectrometry analysis
The GC-MS analysis was carried out using a Hewlett Packard 5890 GC (Palo Alto, USA) equipped with a Hewlett Packard 5971 (Palo Alto, USA) mass selective detector (MSD, operating in the EI mode at 70 eV). The GC capillary column was a HP5 (30 m × 0.25 mm, film thickness 0.17 μm), the following temperature program was used: 60ºC hold for 3 min, then increased to 210ºC at a rate of 4ºC/min, then held at 210ºC for 15 min, then increased to 300ºC at a rate of 10ºC/min, and finally held at 300ºC for 15 min. Helium was used as carrier gas at a constant flow of 1 mL/min for both columns. Identification of the individual components was performed by comparison with the coinjected pure compounds or, when pure standard was not available, by matching the MS fragmentation patterns and retention indices with the built in libraries or literature data and commercial mass spectral libraries (NIST/EPA/NIH 1999; HP1607 purchased from Agilent Technologies).
Gas chromatography – flame ionization detector analysis
The GC analysis of the EOs was carried out using an Agilent 6890 N instrument equipped with a FID and an HP-5 capillary column (30 m × 0.25 mm, film thickness 0.17 μm). The column temperature program was the same described in GC-MS section. Injector and detector temperatures were 280°C. Helium was used as carrier gas at a flow rate of 1 mL/min. The relative proportions percentages of the HS constituents were obtained by FID peak area normalization without the use of any correction factor.
A hydrocarbon mixture of n-alkanes (C9-C22) was analyzed separately under the same chromatographic conditions used on the HP-5MS capillary columns to calculate the retention indexes with the generalized equation by Vandel Dool, Kartz .
As regards α- and β-pinene, these compounds were purchased from Sigma Aldrich.
Antifungal activity assay
The plant EOs as well as the α- and β-pinene activity was evaluated against a wild strain of Penicillium digitatum (Ispa-Pd1) isolated from orange fruit. PDA dishes were inoculated with 20 μL of a conidial suspension at the concentration of 1×104 conidia/mL. The dishes were then treated by vapor contact, after 0 (t 0), 24 (t 24) and 48 (t 48) hours from inoculation, with 0, 50 or 100 ppm of each compound.
Fumigations were performed at 20°C inside suitable boxes (Figure 1), equipped with a circulation fan (12 V - 0, 16 A) and a rubber septum for EOs applications. In order to obtain a gradual and controlled evaporation a heating system was constructed in our laboratory. Heat was delivered by a couple of electric resistances (10 ohms each) placed inside the chambers, connected to a stabilized power supply located outside. The power supply was provided with a voltage regulator (1-15 V of output) that allows controlling the temperature of the resistances. The established amounts of the compounds were injected through the rubber septum into a heatproof glass vessel, placed on the electric resistances. Once each dose was injected inside the vessels, the circulation fans were turned on for 45 min to circulate the compound within the chambers. The experimental design was used to examine the inhibiting impact on the pathogen of the two concentrations of each compound used. After treatments the dishes were incubated for 7 days at 25°C and the radial growths were daily recorded. Moreover, at the fourth day after treatment, some plugs from the PDA plates were taken for Scan Electron Microscopy (SEM) observations.
Data were reported as mean ± SD of the radial growth of nine dishes, for each concentration and compound. Analysis of Variance (ANOVA) of all data from analytical determinations was performed using the MSTAT-C software (Michigan State University, East Lansing, 1995).
The EOs of rosemary and myrtle used in this study were obtained by steam distillation of wild plants collected in Sardinia, Italy. The qualitative analysis was performed by Gas Chromatography-Mass Spectrometry (GC-MS), whereas their percentage composition (semiquantitative analysis) was achieved by GC-FID technique. The chemical composition of rosemary and myrtle EOs are reported in Tables 1 and 2, respectively and data are consistent with those reported in previous papers [18-20]. The GC-MS was also used to perform a dynamic study of the system, aimed to determine the atmosphere composition inside the boxes during the experiment (data not shown).
|α-pinene||30,89||0,15||938||MS, RI, STD|
|verbenene||0,97||0,11||963||MS, RI, STD|
|α-pinene||2,61||0,21||974||MS, RI, STD|
|myrcene||1,79||0,07||993||MS, RI, STD|
|α-phellandrene||0,42||0,00||999||MS, RI, STD|
|α-terpinene||0,47||0,02||1014||MS, RI, STD|
|p-cymene||1,78||0,05||1026||MS, RI, STD|
|3-carene||0,45||0,03||1031||MS, RI, STD|
|limonene||4,19||0,08||1035||MS, RI, STD|
|cineole 1,8||13,26||0,03||1040||MS, RI, STD|
|ocimene beta (z)||0,17||0,00||1050||MS, RI|
|α–terpinene||0,78||0,02||1087||MS, RI, STD|
|terpinolene||1,01||0,00||1108||MS, RI, STD|
|linalool||0,88||0,04||1138||MS, RI, STD|
|camphor||7,19||0,34||1142||MS, RI, STD|
|pinocamphone trans||0,16||0,00||1163||MS, RI|
|borneol||3,80||0,02||1171||MS, RI, STD|
|terpinen 4-ol||0,81||0,02||1192||MS, RI, STD|
|α-terpineol||0,61||0,75||1197||MS, RI, STD|
|myrtenol||0,69||0,63||1197||MS, RI, STD|
|verbenone||2,78||3,65||1201||MS, RI, STD|
|trans carveol||2,86||3,92||1215||MS, RI|
|bornyl acetate||8,94||0,38||1280||MS, RI, STD|
|pinocarvyl acetate||0,08||0,01||1300||MS, RI|
|α-caryophyllene||1,52||0,05||1420||MS, RI, STD|
|α–humulene||0,15||0,01||1453||MS, RI, STD|
Results are expressed as mean of Fid peak area normalization ± standard deviation; RI: calculated linear Retention Index, ID: Identification Method.
Table 1: Percentage composition of Rosmarinus officinalis EO.
|propyl butanoate||2,97||0,67||918||MS, RI|
|α-pinene||35,01||2,81||938||MS, RI, STD|
|β-pinene||0,41||0,12||974||MS, RI, STD|
|myrcene||0,38||0,10||993||MS, RI, STD|
|propanoic acid 2 methyl 2 methylpropyl||0,62||0,03||1005||MS, RI|
|butanoic acid 2 methyl 2 methylpropyl||0,36||0,03||1019||MS, RI|
|p-cymene||0,13||0,09||1026||MS, RI, STD|
|cineole 1,8||28,37||2,68||1040||MS, RI, STD|
|ocimene beta (z)||0,23||0,23||1050||MS, RI|
|ocimene beta (E)||0,33||0,07||1058||MS, RI|
|γ-terpinene||0,18||0,11||1087||MS, RI, STD|
|terpinolene||0,23||0,01||1108||MS, RI, STD|
|linalool||15,81||2,78||1138||MS, RI, STD|
|pinocarveol cis||0,08||0,03||1177||MS, RI|
|terpinen 4-ol||0,20||0,04||1192||MS, RI, STD|
|α-terpineol||2,57||0,51||1197||MS, RI, STD|
|linalyl acetate||2,38||0,58||1258||MS, RI, STD|
|pinocarvyl acetate||0,06||0,04||1300||MS, RI|
|myrtenil acetate||0,08||0,08||1343||MS, RI|
|terpinil acetate||0,86||0,15||1350||MS, RI, STD|
|neryl acetate||1,08||1,46||1367||MS, RI, STD|
|geranyl acetate||2,59||1,87||1387||MS, RI, STD|
|methyl eugenol||1,16||0,46||1408||MS, RI|
|α-humulene||0,25||0,09||1453||MS, RI, STD|
|caryophyllene oxide||0,24||0,11||1584||MS, RI, STD|
|humulene epoxide||0,57||0,22||1610||MS, RI|
|Leptospremone (not ident isom)||0,11||0,07||1621||MS, RI|
Results are expressed as mean of Fid peak area normalization ± standard deviation; RI: calculated linear Retention Index, ID: identification method.
Table 2: Percentage composition of Myrtus communis EO.
The antifungal activity of the compounds was screened by means of vapor contact as described under Material and Methods. Pure essential oils of rosemary and myrtle, and two of their components, α-pinene and β-pinene, were evaluated for their in vitro activity against the postharvest fruit-decaying agent P. digitatum.
The radial growth dynamics, recorded for seven days from the treatment are reported in Figure 2. In particular, when the treatment was performed right after the inoculum (t 0), rosemary EO reduced the colony diameter by 50% when the fumigation was carried out at the concentration of 50 ppm and by 57% at 100 ppm. As regard the treatment performed after 24 h from the inoculation, a completely inhibition was obtained with a fumigation of 50 ppm. The treatment carried out after 48 hours, at a concentration of 50 ppm, was able to delay the pathogen growth for 5 days and by the end of the experiment the colony diameter was reduced by 70%, whereas a completely inhibition was observed at 100 ppm (Figure 2a).
The myrtle EO was less effective against the development of P. digitatum (Figure 2b). The ability to completely inhibit the fungal growth is confirmed when the vapor contact is applied after 24 h from the inoculation, while a reduction of the colony diameter was recorded for the treatments carried out at 0 and 48 hours, with a control percentage of only 30% for both EO concentrations.
The effect observed with fumigations carried out with α-pinene (Figure 2c), in general was similar to that of myrtle EO, even if a better control of the pathogen development was observed with 100 ppm 0 hours after the inoculum. Finally as regard treatments carried out with β-pinene (Figure 2d) a poor control activity was observed. Only in the treatment carried out after 24 h at 100 ppm a completely inhibition of the pathogen development was recorded.
The antifungal activity, on the growth of P. digitatum, among different EOs used is presented in Table 3. Statistical differences in the radial growth (p<0.05) were found among them at t 0 with a concentration of 100 ppm, and at t 48 with 50 and 100 ppm.
|Time (h)||Conc. (ppm)||EOs|
Means followed by different letters in each row indicate significant differences according to Duncan’s multiple range test at p<0.05
Table 3: Radial growth of Penicillium digitatum after seven days.
The data obtained with α- and β-pinene highlight the synergism that occurs between the pools of compounds of each plant EO with respect to the antimicrobial activity of the single component. Indeed, the major components of EOs are very important for their biological activity, but the minor components also play a significant role, as they can strengthen the effect of major components .
The effectiveness of EOs fumigations against P. digitatum appears dose dependent and related to the elapsed time between fungal inoculation and treatment, probably related to a variable sensitivity of the pathogen during the different physiological phase.
The results obtained are in general consistent with previous literature data where the antifungal effectiveness of plant EOs was assessed [22-27]. Pintore et al.  and Angioni et al.  reported the weak antifungal activity of Sardinian rosemary, on the contrary in our work it show the greatest effectiveness, it is may be due to the treatment done by vapor contact and by the chemical composition of rosemary EO used in this study. Environmental factors were considered to play a key role in the chemical composition of plant EOs , that also depends on growing conditions season or vegetative period of plant [30-34].
Different modes of action are involved in the antimicrobial activity of EOs and to date they are not completely understood but there are some features on which some authors agree to explain the inhibitory activity: the hydrophobicity of these compounds leads them to cross cell membrane and interact with cell compounds [35,36]. Some hydrophobic compounds could change the microbial membrane permeability and the loss of differential permeability is generally considered the cause of cell death [2,37]. Phenolic compounds are known to alter microbial cell permeability, allowing the loss of macromolecules from the interior, they could interact with membrane proteins causing a deformation of structure and functionality . All the actions culminate with the inhibition of germination, suppression of mycelia growth and germ tube elongation, therefore it has been suggested that the action of plant compounds might be related to the perception/transduction of signals involved in the switch from vegetative to reproductive development.
Scan electron microscopy observations were performed in order to obtain more information on the ultra-structural alterations of P. digitatum cells, treated with the plant EOs. SEM examinations revealed considerable morphological alterations (Figure 3), explained by the EO modification induced on the fungal morphogenesis and growth, through the interference of their components with the enzymes involved in cell wall synthesis, leading to changes in the hyphae integrity .
Rosemary oil vapor treated samples (Figures 3b-3d) showed the collapse of whole hyphae and complete inhibition of conidia production as compared with untreated control (Figure 3a), and they could be due to the loss of integrity of the cell wall and plasma membrane permeability [40,41].
There is evidence that plant compounds are able to affect several intracellular functions with consequent disruption of the normal mycelium development.
EOs from plants have antimicrobial activity against a variety of food borne fungi. In this study we tested the effect of Rosmarinus officinalis L. and Myrtus communis L. EOs on mycelia growth of green mold when applied as vapor contact in suitable box designed for this purpose. Results revealed that rosemary could be effective in controlling P. digitatum, while as light activity was observed with myrtle. Further researches are needed about treatment modalities and concentration because the effectiveness seems to be affected by these parameters.
Based on our findings, this work could be an important tool for the assessment of EOs inhibitory potential on the fungal growth, when the treatments are carried out by vapor contact, since that the most widely used methods, for determining the antimicrobial properties of EOs, are not ideals because of the complexity, volatility and water insolubility of these compounds.
Our results confirms that plants essential oils are one of the encouraging safe and environmentally-friendly candidates for future use as substitutes to conventional synthetic fungicides, for managing plants pathogens interactions and food contaminants and decays.
Thanks to Salvatore Marceddu and Antonello Petretto for technical assistance.