IN VITRO ANTIFUNGAL AND DEMELANIZING ACTIVITY OF NEPETA RTANJENSIS ESSENTIAL OIL AGAINST THE HUMAN PATHOGEN BIPOLARIS SPICIFERA

The antifungal activity of Nepeta rtanjensis Diklić & Milojević essential oil was tested against the human pathogenic fungus Bipolaris spicifera (Bainier) Subramanian via mycelial growth assay and conidia germination assay. The minimally inhibitory concentration (MIC) of the oil was determined at 1.0 μg ml-1, while the MIC for the antifungal drug Bifonazole in a positive control was determined at 10.0 μg ml-1. The maximum of conidia germination inhibition was accomplished at 0.6 μg ml-1. In addition, at 0.6 μg ml-1 and 0.8 μg ml-1 the oil was able to cause morphophysiological changes in B. spicifera. The most significant result is the bleaching effect of the melanized conidial apparatus of the test fungi, since the melanin is the virulence factor in human pathogenic fungi. These results showed the strong antifungal properties of N. rtanjensis essential oil, supporting its possible rational use as an alternative source of new antifungal compounds.

The present research emphasizes the antifungal activity of N. rtanjensis essential oil against B. spicifera and its potential to cause the demelanizing (bleaching) of B. spicifera reproductive structures.

Essential oil
The essential oil was isolated from air-dried aerial parts of Nepeta rtanjensis, collected during the pre-flowering stage, by hydrodistilation for 2 h in a Clavenger-type apparatus.The extracted essential oil was kept in sealed glass vials at + 4˚C until further analysis.

Fungal strain used
Bipolaris spicifera (Bainier) Subram.was originally isolated from the wall of a storage room of the Serbian National Museum.Due to the high concentration of indoor air fungal spores, the room suffered from "sick building syndrome".The fungus was deposited to the Mycotheca of the Department of Algology, Mycology and Lychenology, Faculty of Biology, University of Belgrade.The fungus was maintained on a malt extract agar (MEA), and potato dextrose agar (PDA), stored at + 4˚C and subcultured once in a month.

Mycelial growth assay
Different concentrations of essential oil (0.2 -1.4 μg ml -1 ) were diluted in Petri dishes with 10 ml of MEA.For each treatment and each dose tested, three replicate Petri dishes were used.The culture medium was inoculated with 5 mm agar discs from an actively growing culture of B. spicifera.After 21 days of incubation in the dark at + 25˚C, the diameter of the colonies was recorded.Antifungal activity was expressed in terms of percentage of mycelia growth inhibition and calculated using the formula of Pandey et al., 1982): growth inhibition %= 100 (dc -dt)/dc dc = average diameter of fungal colony in control dt = average diameter of fungal colony in treatment.
Petri plates with the commercial fungicide, Bifonazole, were used as a positive control.The experiments were repeated twice.The minimum inhibitory concentration (MIC) of oil necessary for the inhibition of mycelia growth of the fungal strain was determined by the method described by Ishii (1995).

Conidia germination assay
Conidia germination assays were carried out on Petri dishes containing MEA amended with different N. rtanjensis essential oil concentrations (0.2 -1.4 μg ml -1 ).A MEA without essential oil was used as a negative control.For each treatment and each dose tested, three replicate Petri dishes were used.Petri dishes were inoculated by covering the entire surface with a suspension of 200 μl of B. spicifera conidia (10 4 ml −1 ), obtained from the sporulated mycelia of 10-day-old cultures, and incubated in the dark at +25˚ C.After 24 h, germinated and non-germinated conidia were counted under a microscope (Zeiss Axio Imager M.1, with AxioVision Release 4.6 software).At least 200 conidia were counted for each observation and scored by hemocytometer.Conidia were considered germinated when the germ tube length was at least half the length of the diameter of the conidia or longer.The experiments were repeated twice.

Light microscopy
A sample of mycelium was taken from the periphery of a colony grown on MEA enriched with different concentrations of N. rtanjensis essential oil.The samples were dyed and fixed with lactophenol -cotton blue and observed under a light microscope (Zeiss Axio Imager M.1, with AxioVision Release 4.6 software) to examine structural abnormalities.Samples from the control plate without oil were also stained and observed.

Scanning electron microscopy (SEM)
Treated and control B. spicifera colonies were used for SEM observations.5 x 10 mm segments were cut from the culture growing on the MEA and placed in vials containing 3% glutaraldehyde in 0.05 M phosphate buffer (pH 6.8) at 4˚C.Samples were kept in this solution for 48 h and then washed with distilled water and dehydrated in an ethanol.Then the samples were dried in liquid carbon dioxide and placed in desiccators until further use.The fungal materials were deposited on adhesive tape fixed to specimen tabs and then ion sputter coated with gold.Microstructure characterization of the samples was carried out with a JEOL JSM 6460 LV instrument equipped with an OXFORD INSTRUMENTS EDS analyzer.

Statistical analysis
One way ANOVA was performed for mycelial growth assay and conidia germination assay.A P value less than 0.05 was considered statistically significant.

RESULTS
Nepeta rtanjensis essential oil showed a strong antifungal and bleaching activity against mycelial growth and conidia germination of Bipolaris spicifera (Table 1).The radial growth of the fungal colony was sig- nificantly reduced in response to different concentrations of essential oil.At 1.0 μg ml -1 the inhibition of fungal development reached its maximum (100% of radial growth inhibition) and this concentration was regarded as the MIC for the test fungus (P<0.05).

DISCUSSION
Nepeta rtanjensis essential oil showed the ability to interfere with all stages in the reproduction cycle of the human pathogenic fungus Bipolaris spicifera: conidia germination, mycelial growth and intensity of sporulation which is demonstrated with radial mycelial growth inhibition, inhibition of conidia germination and low conidia production in the treated samples.The high fungicidal activity of the tested oil was proven with a MIC value (1.0 μg ml -1 ) and the MIC value for the referent antifungal agent Bifonazole was much higher (10.0 μg ml -1 ).Espinel-Ingrof et al. (2002) tested the optimal conditions for determining the MICs for uncommon molds, including B. spicifera, and reported that the MICs for amphotericin B ranged from 0.06 to 8 μg ml -1 , for itraconazole from 0.03 to 8 μg ml -1 , for posaconazole from 0.007 to 8 μg ml -1 , for ravuconazole from 0.2 to 8 μg ml -1 and for voriconazole from 0.12 to 8 μg ml -1 .Essential oils are known to cause morphophysiological changes in fungi through a lack of sporulation, depigmentation and aberrant development of conidiophores (eg.Sharma and Tripathi, 2008;Moreira et al., 2010).The most significant documented morphophysiological changes in our investigation included demelanization (bleaching) and an aberrant conidial apparatus of B. spicifera.Further investigations are required to determinate whether depigmentation is a result of an inhibition of a melanin biosynthesis or melanin bleaching phenomenon.Reinoculation of hyaline B. spicifera on sterile Petri dishes with MEA suggested that depigmentation was reversible.Essential oils are known to interfere with the cell metabolism of fungi.Many components of essential oils can act as regulators of intermediary metabolism; they can change the membrane structure and interfere with the nutrient uptake from the medium; they can affect enzyme synthesis at nuclear or ribosomal level, or they can substitute the limiting factor in intermediary metabolism (Fries, 1973).Previous investigations of N. rtanjensis essential oil's chemical composition showed the presence of phenolic and terpenoid compounds with 4aα, 7α, 7aβ-nepetalactone as the major component (Ljaljević-Grbić et al., 2008).According to Mossier et al. (1993) phenolic derivates are well-known causes of depigmentation.Experiments with other human pathogenic fungi suggested that melanins are not essential for fungal growth but appear to be important as a virulence factor.The mechanisms by which melanins enhance virulence in fungi are yet to be determined, but it has been reported that pigmented cells of Aspergillus fumigatus (Tsai et al., 1997), Exophiala dermatidis (Dixon et al., 1992) and Cryptococcus neoformans (Kwon Chung et al., 1982) are more virulent than hyaline cells.According to Polak (1999), the loss of the production of melanin significantly reduces the pathogenic power of Dematiaceae ( 24).The present study showed the in vitro antifungal activity of N. rtanjensis essential oil against B. spicifera.In vitro data may be helpful in determining the potential usefulness of the plants for treatment of Bipolaris infections.

Fig. 1 .
Fig. 1.The influence of Nepeta rtanjensis essential oil on Bipolaris spicifera mycelia growth.A. a) Negative control; b) Positive control; c) 0.6 μg ml -1 ; d) 0.8 μg ml -1 ; B. the detail of Ad; C. Normal conidia production in control culture; D. The lowest conidia production in culture with Bifonazole added; E. The pore conidia production in culture with 0.8 μg ml -1 essential oil added (C,D,E, 50x magnification).