MATERIALS AND METHODS

The experiments were carry out during 2011, after having collected oils and fungal strains necessary to run the test at the laboratory of the Plant pathology research center in Rome, Italy (CRA-PAV).

Oils: Stocks of the following medicinal oils were purchased; Thyme Oil (TO), Laurel Oil (LO), Peppermint Oil (PO), Oregano Oil (OO), Rosemary Oil (RO) (Esperis s.p.a., Milan, Italy), Tea Tree oil (TTO) (Sovimpex, Marseille, France) and Clove Oil (CO) (Cedax Srl, Forlì-Cesena, Italy). The main active components of the oils used for in vitro trials are: terpinen-4-ol (40.7%) and γ-terpinene (20.3%) for TTO, thymol (41%) for TO, eugenol (16,3%) for CO, menthol (63,4%) for PO, 1,8 cineole (41,8%) for RO, thymol and carvacrol (67%) for OO, 1,8 cineole (40%) for LO. The concentrations were determined by the respective producer Companies, except for LO that was analysed by an accredited laboratory (Agri-bio-eco s.r.l., Pomezia, Italy).

Fungi: A total of 6 fungi (P. pinodella, P. pinodes, D. phaseolorum var. caulivora, P. longicolla, A. lentis and C. gloeosporioides), maintained on solid PDA medium (potato dextrose agar), were used for antifungal activity evaluation of TTO, TO, CO, LO, RO, PO and OO. The strain 1 of P. pinodella was provided from A.T.C.C. 16235-USA, while the others belong to CRA-PAV collection and were isolated from lentil seed (A. lentis), chickling vetch seed (P. pinodes and P. pinodella) soybean seeds (P. longicolla, D. phaseolorum var. caulivora) and from Banksia serrata leaves (C. gloeosporioides).

In-vitro activity: Mycelial growth was determined on solid PDA medium amended with different oils at the following concentrations (v/v): 0% (control), 0.01, 0.025, 0.05 and 0.1% for TO, CO and OO, 0% (control), 0.25, 0.5, 1 and 1.5% for LO and RO, 0% (control), 0.1, 0.25, 0.5 and 1% for tea tree oil and finally 0% (control), 0.025, 0.05, 0.1 and 0.25% for PO, in presence of Tween 20 (0.5% v/v) as emulsifying agent. The experiments were conducted twice in duplicate in 90 mm Petri dishes containing the medium modified and inoculated with 6 mm plugs of PDA from actively growing cultures; after inoculations dishes were kept in the dark at 24°C. Radial growth of colonies was measured the seventh day after inoculation at two perpendicular points along the diameter of the plate and the means was used to calculate the mean daily radius of the fungal colony. Percentage inhibition of mycelial growth (MGI) by the oils was calculated using the equation:

where, C and T are the average of the colonies radius values in control and treatments, respectively, measured for each fungus at the seventh day after inoculation, obtained in both experiments. The Minimum Inhibitory Concentration (MIC), that produced a 90% reduction of growth compared with the growth of the oil-free control, was computed for each combination of fungus/compound at the seventh day after inoculation by second-degree polynomial regression.

Statistical analysis: Analysis of variance (ANOVA) was performed on the data obtained using CoStat-Statistics Software version 6.4. The significance of the differences among treated samples was evaluated using the Student-Newman-Keuls test (SNK) for multiple comparisons (significance level 0.05).

RESULTS AND DISCUSSION

The antifungal activity of the oils was evaluated considering the percentage of MGI (Table 1) and the MIC (Table 2-3), computed for each combination fungus/compound. All the essential oils were significantly (p≤0.05) active against all the fungal isolates tested at variable degree. About TO and CO, the concentration of 0.1% was able to completely inhibit the growth of all the fungi tested.

Table 1:	In vitro activity of oil as percentage of mycelial growth inhibition of fungus at different oil concentrations
Hundred value refers to complete inhibition with no mycelial growth, SD: Standard deviation, a-d: Significance differences by Student-Newman-Keuls test (p≤0.05), Value followed by the same letter within the same row do not differ significantly

Table 2:	Minimum inhibitory concentration of oils at the 7th day after inoculation
TO: Thyme, CO: Clove, PO: Peppermint and OO: Oregano

Table 3:	Minimum inhibitory concentration of oils at the 7th day after inoculation
TTO: Tree oil, RO: Rosemary oil and LO: Laurel oil

For some fungal strains, even the lower concentration of 0.05% was sufficient to ensure a complete inhibition statistically comparable (p<0.05) to the higher concentration, in particular: P. pinodella strain 1, P. pinodes, P. longicolla strain 2 for each oils, D. phaseolorum var. Caulivora, P. pinodella strain 2 and A. lentis for CO. PO and OO gave steady results as they induced the total inhibition of growth for all fungi tested, at the concentration of 0.1 and 0.05%, respectively. TTO provided a total inhibition of fungal growth at concentrations much higher, achieving maximum results against all isolates at the concentration of 0.5%. For some fungi like P. pinodella, P. pinodes, P. longicolla strain 2 and A. lentis even the lower concentration of 0.25% was effective (Table 1).

The results regarding LO showed a different effectiveness among two different strains of P. longicolla, as they showed a different behavior in presence of the same oil, demonstrating a possible variability intra-species: for P. longicolla strain 1, the percentage of maximum inhibition (100%) was 1.5%, while for P. longicolla strain 2 was 1%, but no statistical differences were found up to 0.25% of LO; for all other isolates the optimum concentration was 0.5% (Table 1).

The results about RO were more contrasting among the different isolates: P. pinodella strain 2 underwent to a complete inhibition of fungal growth at 0.25% concentration; the optimum concentration rose to 0.5% for A. lentis and P. pinodes and to 1% for D. phaseolorum var. caulivora, P. pinodella strain 1, P. longicolla strain 2 and C. gloeosporioides; finally the growth of P. longicolla strain 1 was totally inhibited at the highest concentration tested of 1.5% (Table 1).

In general, data obtained at the lower concentrations possessed higher variability other than an unsatisfying antifungal activity; as the concentrations (and consequently the effectiveness) raise, the standard deviations became lower, obtaining an higher robustness of the data. Comparing MIC values, the oils tested can be split into two distinct groups on the basis of their activity. TO, CO, PO and OO presented a comparable effectiveness, with very low MIC values (Table 2), while TTO, LO and RO showed MIC values around 10 times higher than the previous (Table 3). However, the oils activity was proved to be dependent on the fungal specie and, in the case of LO and RO, even on the strain, as reported above. The highest MIC value was 1.25% of LO for P. longicolla strain 1 while the lowest one was 0.03% of TO for D. phaseolorum var. caulivora.

Broadly, our results demonstrated that all the oils possess a remarkable antifungal activity, with TO, CO, PO and OO being more effective at the same concentration than TTO, LO and RO in mycelial growth inhibition and this occurred with all the fungi tested. Previous results (Dzamic et al., 2008) indicate that the antimicrobial activity of essential oils is affected by their composition and the higher antifungal effect could be attributed to the higher amount of compounds with the phenol structure such as thymol, carvacrol, eugenol and menthol, that are indeed the main components of TO, CO, PO and OO.

The oils tested posses different commercial values and different volumes are needed to achieve the highest MIC concentration effective against fungi. In order to provide an effective and affordable solution that combines the best effectiveness and the lowest price, the cost of 1 liter treatment solution was calculated for each oil considering the concentration of the active component, the efficacy (MIC values) and the value (Table 4): CO and PO seem to give the best perspective considering the volume needed for one litre solution treatment, followed by TO and OO; LO is the oil with less perspective because of the high cost.

Table 4:	Cost of 1 litre solution treatment, calculated on the basis of its cost and the volume needed to achieve the highest MIC concentration
*€: Euro currency

Many other studies were focused on the antifungal activities of essential oils against plant pathogenic fungi of agricultural interest. In a recent paper (Riccioni and Orzali, 2011) the antifungal activity of thyme and tea tree oil was showed against seven seed borne pathogens of other crops: F. graminearum, F. culmorum, A. radicina, A. dauci, A. rabiei, C. lindemuthianum and D. avenae. Velluti et al. (2004) tested cinnamon, clove, oregano, palmarosa and lemongrass oils against F. graminearum, one of the main causal agents of the root and foot rot and fusarium ear blight in wheat; Faria et al. (2006) investigated the activity of Ocimum gratissimum essential oil against some phytopatogenic fungi like Botryosphaeria rhodina, Rhizoctonia sp. and Alternaria sp. Terzi et al. (2007) reported the in vitro antifungal activity of the tea tree essential oil and its major components against some cereals pathogen fungi as Blumeria graminis f. sp. hordei, F. culmorum, F. graminearum, Pyrenophora graminea. Simic et al. (2004) tested the chemical composition of some lauraceae essential oils and their antifungal activities against 17 micromycetes.

CONCLUSION

Results achieved in this study were consistent with the previous ones, confirming the essential oils antifungal broad-spectrum efficacy and showing promising prospects for the utilization of natural plant essential oils in crop production systems as alternative safe natural antifungal agents, on the basis of their efficacy, non-toxicity to humans, systemicity, biodegradability, easy availability and cost. Seed treatment against seed-borne fungi could be one of this potentially use.

However, because the in vitro effects did not always provide a good criterion for their in-vivo performances, further investigations are needed as well as field experiments to verify their possible phytotoxicity on plant/seed material and their effectiveness in field conditions as seed treatment.

ACKNOWLEDGMENT

The authors thank the Ministero delle Politiche Agricole Alimentari e Forestali for financial support the “Piano Sementiero Nazionale per l’Agricoltura Biologica” coordinated by INRAN-ENSE.