THE INHIBITORY EFFECT OF TROPOLONE AND HINOKITIOL ON THE MYCELIUM GROWTH OF PHOMA NARCISSI IN VITRO

Tropolone and hinokitiol (β-thujaplicin) that are present in heartwood of several Cupressaceae trees are known for their antibacterial, antifungal and insecticidal properties. In the present studies it was showed that tropolone and hinokitiol greatly inhibited in vitro , on PDA medium, the mycelium growth of Phoma narcissi , a pathogen of Hippeastrum and other species of family Amaryllidaceae. Total inhibition of the mycelium growth of Phoma narcissi took place at a tropolone concentration of 6.0 µg·cm -3 and at a hinokitiol concentration of 50.0 µg·cm -3 . Fungicidal doses of tropolone and hinokitiol for the mycelium growth of Phoma narcissi were also documented. The results presented in this paper are discussed with data available in literature on the antifungal action of tropolone and hinokitiol on other species of pathogenic fungi.


INTRODUCTION
Hinokitiol (β-thujaplicin) is a tropolone-related compound (Fig. 1) that is present in the heartwood of several Cupressaceae trees, such as Chamaecyparis obtusa Sieb.In the present studies we showed a strong inhibitory effect of tropolone and hinokitiol on the mycelium growth of Phoma narcissi, a pathogen of Hippeastrum and other species of Amaryllidaceae (S a n i e w s k a , 1998).

MATERIAL AND METHODS
The stock culture of Phoma narcissi (Aderh.)Boerema, de Gruyter et Noordel.was maintained on potato-dextrose-agar (PDA-Merck), slants at 25 o C in the dark.
The effect of tropolone and hinokitiol (β-thujaplicin) (purchased from Sigma-Aldrich Chemicals) on the mycelium growth of Phoma narcissi on potato-dextrose--agar medium was investigated.These compounds were used at the following final concentrations: tropolone -2.0, 4.0, 6.0, 8.0, 10.0 and 50.0 μg•cm-³ and hinokitiol -10.0, 15.0, 25.0, 50.0, 75.0 and 100.0 μg•cm-³ in PDA medium.Hinokitiol was dissolved in 50% of ethanol and tropolone was dissolved in distilled and sterilized water, and then they were added to PDA medium after sterilization at a temperature of about 50 o C. Five mm diameter plugs were taken from 7-day-old culture of Phoma narcissi, and placed in the middle of 90 mm Petri dishes containing PDA medium supplemented earlier with the tested compounds.The control plates contained the culture growing on pure PDA, without any additions and supplemented with ethanol at an appropriate concentration.Five Perti dishes were used as an experimental unit and the trial was repeated twice.The incubation was conducted in darkness at 25 o C.After 2, 4 and 6 days of incubation, the diameter of the fungal colonies was measured in two perpendicular directions.
Additionally, the mycelial plugs from which the colonies did not develop were transferred into the plates containing clean PDA and observed during the 6-day--incubation.
The data were subjected to an analysis of variance and Duncan's multiple range test at 5% of significance was used for means separation.

RESULTS AND DISCUSSION
Tropolone applied to the PDA medium at a concentration of 2.0 and 4.0 μg•cm-³ inhibited the mycelium growth of Phoma narcissi in 81.4 and 97.2%, respectively, after 7 days incubation, but tropolone at a concentration of 6.0 μg•cm-³ or higher totally inhibited the mycelium growth of the pathogen (Fig. 2 and 3).It should be mentioned that the disks of mycelium Phoma narcissi incubated during 7 days on PDA supplemented with tropolone at a concentration of 6.0 and 8.0 μg•cm-³ and transferred to clean PDA started the mycelium growth but in a weaker degree, proportionally to the tropolone concentration; 10 μg•cm-³ of tropolone or higher concentrations were fungicidal and the lack of growth of the mycelium Phoma narcissi was observed (data not presented).
Hinokitiol applied to the PDA medium at a concentration of 10.0, 15.0 and 25.0 μg•cm-³ limited the mycelium growth of Phoma narcissi in 50.5, 61.5 and 88.4%, respectively, after 7 days incubation and a concentration of the compound at 50.0 μg•cm-³ and higher totally inhibited the mycelium growth of the pathogen (Fig. 4).It was documented that hinokitiol at a concentration of 100.0 μg•cm-³ had fungicidal activity (data not presented).On PDA the mycelium of Phoma narcissi was smoky-grey, whereas its reverse was grey-olivaceous, locally dark-grey, with an olivaceous-black center; probably the grey colour of the mycelium is caused by the presence of melanins.The addition of hinokitiol at a low concentration to PDA caused that the mycelium of Phoma narcissi was light-grey or beige on both sides (Fig. 5); it is suggested that hinokitiol as an inhibitor of catechol oxidase (K h a n and A n d r a w i s, 1985; Va l e r o et al. 1991) limited the formation and accumulation of melanins in the mycelium of the pathogen.
Thus, in the case of Phoma narcissi tropolone had a much stronger inhibitory effect on the mycelium growth than hinokitiol (Fig. 2 and 4).
M o r t i a et al. ( 2003) showed clear antifungal activity of tropolone on seven species of fungi tested Fig. 2. Inhibitory effect of tropolone on the mycelium growth of Phoma narcissi; surface of the mycelium growth in the control on PDA after 2, 4 and 7 days of incubation is 7.1; 24.6 and 59.7 cm 2 , respectively.
Values followed by the same letter do not differ at 5% level of significance (Duncan's test).compounds, showed antifungal activity on seven species of plant pathogenic fungi.
It is well known that food packages can be made antimicrobial active by the incorporation and immobilization of antimicrobial agents or by surface modification and surface coating.S u p p a k u l et al. ( 2003) present active packaging technologies with an emphasis on antimicrobial packaging and its applications, and hinokitiol is considered as one of safe antimicrobial agents.
et Z u c c ., Thuja plicata D. Don (A r i m a et al. 2003; Ya m a n o et al. 2005), Thujopsis dolabrata Sieb.et Zucc.hondai Makino (M o r i t a et al. 2003), Hiba arboruitae (F a l l i k and G r i n b e r g , 1992), Cupressus lusitanica (Z h a o and S a k a i, 2003).Tropolone and hinokitiol are known to have insecticidal and antimicrobial activity (T r u s t and C o o m b s , 1973; M o r i t a et al. 2003; A r i m a et al. 2003; Ya m a n o et al. 2005; B a y a et al. 2001).F a l l i k and G r i n b e r g (1972) showed that hinokitiol inhibited in vitro spore germination and mycelial growth of Botrytis cinerea, Alternaria alternata, Rhizopus stolonifer and Mucor spp.and M o r i t a et al. (2003) documented antifungal activity of tropolone and hinokitiol against Pythium aphanidermatum, Thanatephorus cucumeris, Fusarium solani, Botryotinia fuckeliana, Phomopsis obscurans, Colletotrichum orbiculare and Colletotrichum lagenarium.Earlier in 1989 it was reported in two patents that hinokitiol had strong antifungal activity against Helicobasidium mompa and Rosellinia necatrix (cited after M o r i t a et al. 2003).The mechanism of antimicrobial and insecticidal activity of tropolone and hinokitiol is unknown but it was documented that tropolone greatly inhibited polyphenol oxidase (K h a n and A n d r a w i s , 1985; Va l e r o et al. 1991), and tropolone and hinokitiol showed inhibitory activity toward metalloproteases such as carboxypeptidase A and collagenase (M o r i t a et al. 2003).

Fig. 3 .
Fig. 3. Inhibitory effect of tropolone at a concentration of 2.0 μg•cm ³ on the mycelium growth of Phoma narcissi after 7 days incubation; on left control, PDA, on right PDA + tropolone.