**4. The antifungal compounds from** *Syzygium aromaticum*

Clove bud and leaf contain secondary metabolites that strongly inhibit the growth of *P. palmivora* and *F. oxysporum*. Under continuous extraction with hexane followed by ethyl acetate and methanol, the antifungal compounds mainly present in the hexane extract suggesting that the active compound is nonpolar compound. Upon extraction of plant material with hexane, most volatile oil components will also present in the extract. Clove oil also demonstrates strong antifungal activity. These findings lead to the hypothesis that the antifungal compound of clove is also the component of clove oil. The major component of clove oil is eugenol. At least two compounds from the extract and the volatile oil of clove are responsible for the antiphytophthora and antifusarium activity. The two compounds were identified as eugenol and eugenol acetate. The activity of eugenol is higher than eugenol acetate.

Observation under scanning electron microscope showed that the hypha of *F. oxysporum* shrank after treated with eugenol (**Figure 1**). Higher magnification showed that the surface of hypha is no longer smooth and the cells may be leaking [38]. A number of mechanisms have been proposed to explain how eugenol acts as antifungal agent. Eugenol alters the membrane and cell wall [39] and induces leakage of protein and lipid from the cells due to the leakage of cell walls [40]. Extensive lesion of the cell membrane reduces quantity of ergosterol [41].

compared to the clove bud. However, the oil content of clove leaf is relatively high compared to the other leaves. The oil content of clove leaves is approximately 2% with the major components which are eugenol 60% and caryophyllene 21**%** [37]. Clove leaves are considered to be a potential source of secondary metabolite for antifusarium and antiphytophthora. Volatile oil from clove leaves can be obtained from leaves that have already fallen on the ground; therefore, it can be collected throughout the year without disturbing the growth of the tree. In addition, the availability of clove leaves does not depend on the season and can be collected at any time.

Note: Extract was made by maceration in methanol: +: 1–25% inhibition; ++: 26–50% inhibition; +++: 51–75% inhibition;

**No. Plant Plant organ Concentration**

70 Fusarium - Plant Diseases, Pathogen Diversity, Genetic Diversity, Resistance and Molecular Markers

 *Ageratum conyzoides* Linn. Aerial part — — — *Eclipta alba* Aerial part — — — *Cassia alata* L. Leaves — — — *Piper betle* L. Leaves — ++ +++ *Piper crocatum* Leaves — — — *Cymbopogon nardus* L. Leaves — — — *Cinnamomum burmannii* Leaves — — — *Syzygium aromaticum* L. Leaves — + ++++ *Syzygium aromaticum* L. Flower bud +++ ++++ ++++ *Garcinia mangostana* Fruit cortex — — — *Allium sativum* L. Bulb — — — *Alpinia galanga* L. Rhizome — — — *Curcuma domestica* Val. Rhizome ++ ++ ++ *Curcuma xanthorrhiza* Roxb. Rhizome — — + *Curcuma zedoaria* (Berg.) Roscoe. Rhizome — — — *Zingiber officinale* Rosc. Rhizome — — + *Acorus calamus* Rhizome — ++ ++

**2.5% 5% 10%**

Clove bud and leaf contain secondary metabolites that strongly inhibit the growth of *P. palmivora* and *F. oxysporum*. Under continuous extraction with hexane followed by ethyl acetate and methanol, the antifungal compounds mainly present in the hexane extract

**4. The antifungal compounds from** *Syzygium aromaticum*

++++: 76–100% inhibition.

**Table 2.** Activity of antifungal plant on *Fusarium oxysporum*.

**Figure 1.** Scanning electron microscopy of normal *Fusarium oxysporum* (a) treated with eugenol (b) at 350× magnification (above) and at 2000× magnification (below).

It was proposed that the inhibition of ergosterol synthesis leads to the damage of cell membrane functionality and integrity [42]. However, the effect of eugenol is not reversed by osmotic support, indicating that its effect does not affect the cell wall synthesis and assembly. Furthermore, eugenol does not bind ergosterol, the main sterol of fungal membrane [43].

**5. Structure requirement of eugenol derivatives for antifungal activity**

Eugenol derivatives had been synthesized and their antifungal activities evaluated [43]. Some structures and their antifungal activities are shown in **Figure 2**. It seems that the aromatic, ortho-oxygenation, and the double bond at the terminal of side chain are essential for

[A] MIC =125 ppm [B] MIC= 31 ppm

[C] MIC= 62 ppm [D] MIC > 250 ppm

[E] MIC= 62 ppm [F] MIC > 250 ppm

H3CO

HO

[G] MIC=125ppm [H] MIC > 250 ppm

**Figure 2.** Derivatives of eugenol and their antifungal activities.

O

O

H3CO

HO NO2

Plant Secondary Metabolites for Antifusarium and Antiphytophthora

http://dx.doi.org/10.5772/intechopen.71552

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NO2

Eugenol is suggested to block aromatic and branched chain amino acid synthesis across the cytoplasmic membrane. Eugenol inhibits growth of yeast strain carrying a mutation in gene encoding an enzyme, a tryptophan, phenylalanine, tyrosine, and isoleucine biosynthesis pathway, in a medium supplemented with the related amino acid [44].

There are two approaches to obtain antifusarium from clove. Firstly, the secondary metabolites from clove leaves or buds can be extracted using nonpolar solvent such as hexane, petroleum ether, gasoline, or kerosene. Subsequently, the solvent is removed through evaporation leaving the concentrated extract containing antifusarium and antiphytophthora compounds. Hexane and petroleum ether have relatively low boiling point; therefore, it is easy to evaporate, and while the boiling point of gasoline and kerosene is higher than 100°C, higher temperature or lower pressure is needed to evaporate. By using extraction combined with distillation to recover the solvent, more efficient production system can be developed. Secondly, since eugenol is a component of volatile oil, the oil of clove leaves can be obtained through steam distillation by which the oil will evaporate together with steam, and upon condensation the oil will separate from water and the oil can be collected. To obtain pure eugenol, further separation processes will be needed, such as liquid–liquid extraction, vacuum fraction distillation, and chromatographic techniques.

There are some other plant metabolites having antifungal activity, and the effect is stronger than eugenol. Thymol and other components of volatile oil had been compared, and the results are as shown in **Table 3** [45]. Thymol is the component of volatile oil from *Thymus vulgaris*, herbal medicine commonly used in cough mixture. Most of these compounds demonstrate minimal inhibition concentration (MIC) above 50 ppm. The natural antifungal that demonstrates antifungal activity similar to that of commercially distributed is xanthorrhizol with MIC lower than 10 ppm [17]. Xanthorrhizol is the major component of volatile oil isolated from the rhizome of *C. xanthorrhiza*.


**Table 3.** Relative antifungal activity of plant component compared to thymol.
