**3. Fungicide activity**

#### **3.1** *Aspergillus fumigatus*

*Aspergillus* genus is a famous taxonomic group of molds. Some of its species are very important animal and human pathogens. The disease in humans is caused mainly by *Aspergillus fumigatus*, *Aspergillus flavus* and *Aspergillus niger*. Other species, for example,

Regarding the addition of growth regulators and their effect on the development of the *explant*s, it was found that, in general, the number of shoots was favored in medium supplemented with BAP (MS medium + 1 mg/L BAP) and without auxins (2.23 shoots/*explant*) (Table 3). The length of shoots was higher in the media with the highest concentration of BAP but with the addition of the auxin NAA, MS + 1.5 mg/L BAP + 0.2 mg/L NAA (16.07 mm). It is explained, among other things, by the presence of auxin which

> **MS + 1BAP**

Shoot number 1.33 2.23 2.08 2.16 2.15 Shoot length (mm) 9.45 13.58 15.16 11.62 16.07 Root number 3.13 0 5.94 5.79 11.26 Root length (mm) 3.50 0 8.86 12.88 35.40 Number of internodes 0.78 0.78 1.0 0.89 0.91 *Explants* with *calli* (%) 0 0 0.27 0.44 0.45 *Calli* diameter (mm) 0 0 1.23 3.35 3.41 Table 3. Number and length of shoots and roots, number of internodes and percentage and

In rooting, the results were not as expected because, as has been said, the auxin IBA, is related to the root development, so was expect that MS medium + BAP + 0.2 IBA had had a higher rate of rooting, as results obtained by Dhandapani et al. (2008). However, in our study, MS medium supplied with 1.5 BAP + 0.2 NAA revealed the largest number and length of roots, 11.3 roots/*explant* with 35.4 mm. Similar results were verified by Echeverrigaray and colleagues (2005) for thyme cultivars. With regard to the number of internodes, the medium with the addition of IBA (MS + 1 mg/L BAP + 0.2 mg/L IBA)

This result was not expected since the auxin IBA is more related with rooting, as reported by Dhandapani et al. (2008) also in *Catharanthus roseus*. The culture media with the lowest values for the internodes was MS medium supplemented with NAA (MS + 1 mg/L BAP + 0.2 mg/L NAA). The culture medium MS + 1.5 mg/L BAP + 0.2 mg/L NAA proved to be the best in the number and diameter of *calli* (3.41 mm), this result was expected, since Paramageetham et al. (2007) in *Centella asiática* L. had found that the formation of *calli* occurred in response to an interaction between auxin, its concentration and type of *explant*.

*Aspergillus* genus is a famous taxonomic group of molds. Some of its species are very important animal and human pathogens. The disease in humans is caused mainly by *Aspergillus fumigatus*, *Aspergillus flavus* and *Aspergillus niger*. Other species, for example,

diameter of *calli per explant,* during eight weeks of *in vitro* culture.

**MS + 1BAP+0.2 IBA** 

**MS + 1BAP+0.2 NAA** 

**MS + 1.5BAP+0.2 NAA** 

promotes cell elongation.

**Culture media MS** 

originated the highest values.

**3. Fungicide activity 3.1** *Aspergillus fumigatus* *Aspergillus terreus* or *Aspergillus nidulans* are quantitatively less prevalent (Karthaus, 2010). *A. fumigatus* is one of the most ubiquitous of the airborne saprophytic fungi that is pathogenic to plants, animals and humans. Inhalation of conidia by immunocompetent individuals rarely has any adverse effect (Latgé, 1999). However, apart from the production of mycotoxins, *A. fumigatus* is a dangerous human pathogenic, which is able to cause very serious human and animal mycoses with a high frequency of resistance to chemical antifungal drugs (Verweij et al., 2009; Lass-Florl et al., 2010; Xu et al., 2010; Zabka et al., 2011). Dramatic increases in the incidence of aspergillosis caused primarily by *A. fumigatus* have occurred in recent years. A high infection-associated death rate of up to and over 50% is attributed even today to these fungi (Karthaus, 2010). *A. fumigatus* has become the most important airborne pathogen in developed countries, causing a significant mortality in invasive aspergillosis (Latgé, 1999; Chamilos & Kontoyiannis, 2005). Patients who have been treated with steroid therapy or those with chronic obstructive pulmonary disease or severe hepatic failure are at high risk for developing pulmonary aspergillosis (Meersseman et al., 2007; Morace & Bhorgi, 2010).

The treatment of aspergillosis is most problematic and questionable owing to toxicity and side effects of the used medicines on the base of synthetic fungicides (Karthaus, 2010). Fungal infections remain a significant cause of morbidity and mortality despite advances in medicine and the emergence of new antifungal agents. Drug resistance in fungi is increasing and is becoming a serious concern and the high use and misuse of antifungal as probably the main cause of this situation. Therefore, there is an urgent need to search for effective new antifungal agents in treatment of infectious diseases at present (Xing et al., 2011).

Currently, there are four classes of antifungal agents with activity against *Aspergillus*: the polyenes, such as amphotericin B its lipid formulations, and nystatin (including liposomal nystatin); the triazoles, including itraconazole, the voriconazole and the investigational posaconazole, the echinocandins, such as caspofungin, the micafungin, and the anidulafungin; and the allylamines such as terbinafine (Groll & Kolve, 2004; Chamilos & Kontoyiannis, 2005; Meersseman et al., 2007; Shi et al., 2010). Clinical resistance of invasive aspergillosis to amphotericin B based therapy is observed frequently in clinical practice (Chamilos & Kontoyiannis, 2005). However, they are intrinsically resistant to fluconazole (Moghaddam et al., 2010).

In other way, pathogenic and toxinogenic fungi are one of the major economic problems of crop and food production (Zabka et al., 2011). In terms of food safety, species of *Fusarium, Aspergillus* and *Penicillium* genera are considered the most significant because they produce the great majority of known mycotoxins (Palumbo et al., 2008; Zabka et al., 2011). There has been increasing concern of the consumers about foods free or with lower level of chemical preservatives because these could be toxic for humans (Bedin et al., 1999; Souza et al., 2005). This resulted in increasing search for new technologies for use in food conservation systems which include alternatives antimicrobials (Brull & Coote, 1999; Souza et al., 2005).

#### **3.2 Aromatic plants and antifungal activity**

The spread of multidrug-resistant strains of fungus and the reduced number of drugs available led to a search for therapeutic alternatives, namely among medicinal plants and compounds isolated from them used for their antifungal properties. In these natural sources, a series of molecules with antifungal activity have been found, which are of great importance to

*In Vitro* Multiplication of Aromatic and Medicinal Plants and Fungicide Activity 131

were dispensed into sterilized Petri dishes (9 cm). After solidification, a mycelial disk of 4 mm diameter of the test *Aspergillus fumigatus* taken from 4 days -old fungi culture, was

The mycelial disks on PDA without any test constituents were performed in the same way and used as control. Radial growth of colonies was measured at two points along the diameter of the plate and the mean of these two readings was taken as the diameter of the fungal colony. After incubation at 25°C in darkness, growth zones were measured at the third, fifth and the seventh day. The growth of the colonies in control sets was compared with that of various treatments and the difference was converted into percent inhibition [(*C* - *T*) x 100/*C*] where *C* and *T* are the radial diameters of the colony in control and treatment, respectively. The percentage of *A. fumigatus* growth inhibition is expressed as a mean of three replicate tests for each treatment. The complete antifungal analysis was carried out

The analyses were performed using SPSS® (Statistical Package for the Social Sciences) version 19.0. The one-way analysis of variance (ANOVA) followed by Tukey's Test with P =

Effect of four different concentrations (5 mg/mL, 10 mg/mL, 20 mg/mL and 25 mg/mL) of *Thymus* and *Mentha* extract plants was tested against *Aspergillus fumigatus*. Antifungal activity was assayed and data on effect of plant extracts on the growth of *Aspergillus fumigatus* in the third, fifth and seventh day is presented in Table 4. The data revealed that reduction in growth

*rotundifolia* 7.0a 3.9 \_\_ \_\_\_ 1.2 7.4 \_\_\_ \_\_\_ 3.9 9.9 \_\_\_ \_\_\_

The results indicated that *Thymus mastichina* exhibited antifungal activity against the tested *Aspergillus fumigatus* at two different concentrations of 20 mg/mL and 25 mg/mL. The highest antifungal activity was exhibited at 25 mg/mL in *Thymus*. The percent of inhibition were statistically significant with different concentrations in *Thymus*. The lowest concentration of *Thymus mastichina* did not show any activity against *A. fumigates* in the 3

Table 4. Inhibition effect of plant extracts on *Aspergillus fumigatus* in four different

days, while the other two higher concentrations showed good antifungal activity.

**% Inhibition of** *Aspergillus fumigatus*

Third day Fifth day Seventh day

Concentrations of aqueous plant extracts in PDA (mg/mL) 5 10 20 25 5 10 20 25 5 10 20 25

\_ 4.6 16.7 \_\_\_ 0.9 7.2 18.9

placed at the center of the medium.

**3.4 Results** 

Plant species

*Thymus* 

*Mentha* 

concentrations.

under strict aseptic conditions (Zhang et al., 2006).

*mastichina* \_\_ \_\_\_ \_\_ 19.1 \_\_ \_\_

aAll Values are mean of three replicates.

0.05 were used to detect significant differences in inhibition fungi.

of *Aspergillus fumigatus* was observed with extracts of *Thymus* and *Mentha*.

humans and plants. Several molecules obtained from the natural environment are studied and described in bibliography with antimycotic activity. Several extracts are tested for antifungal activities like crude extracts or isolated constituents like, essential oils, terpenoids, saponins, phenolic compounds, alkaloids, peptides and proteins (Abad et al., 2007).

Aromatic plants have been widely used in folk medicine. About three quarter of the world's population relies on plants and plant extracts for healthcare (Parekh & Chanda, 2007). Several plants have been used in folklore medicine in Portugal (Pina-Vaz et al., 2004; Figueiredo et al., 2008). Spices have been used with primary purpose of enhancing the flavor of foods rather than their medicinal and antioxidant properties (Souza et al., 2005).

Plants generally produce many secondary metabolites with antifungal and microbicide activity (Bobbarala et al., 2009). According to the WHO (World Health Organization), plants would be the best source for obtaining a variety of drugs and a possible way to treat diseases caused by multidrug resistant microorganisms (Bhattacharjee et al., 2006). The medicinal value of plants lies in some chemical substances that produce a definite physiological action on the human body. The most important of these bioactive compounds of plants are alkaloids, flavanoids, tannins and phenolic compounds (Edeoga et al., 2005; Panghal et al., 2011). Some medicinal plants exert strong antifungal properties and could be conveniently used as a promising alternative source for presently problematic antifungal treatment in many areas with respect to their natural origin (Zabka et al., 2011).

Many commercially drugs used in modern medicine was initially used in crude form in traditional or folk healing practices. Benefits of using plant extracts are that they are relatively safer than synthetic alternatives, offering profound therapeutic benefits and more affordable treatment (Panghal et al., 2011).

The genus *Thymus* (Lamiaceae), widely distributed on the Iberian Peninsula, is a taxonomically complex group of aromatic plants traditionally used for medicinal purposes because of their antiseptic, antispasmodic and antitussive properties (Pina-Vaz et al., 2004). Previous studies on the antimicrobial activity of the essential oils of some *Thymus* spp., most of them possessing a large amount of phenolic monoterpenes, showed activity against fungi (Pina-Vaz et al., 2004).

Screening of medicinal plants for antimicrobial activities and phytochemicals is important for finding potential new compounds for therapeutic use (Duraipandiyan et al., 2006).

The main objective of this study was to investigate the inhibitory effects of *Thymus* and *Mentha* extracts against *Aspergillus fumigatus*.

#### **3.3 Methodology**

Fungal strain was obtained from the collection of pathogenic fungi maintained in the University of Trás-os-Montes and Alto Douro, Vila Real, Portugal. Subcultivations on Petri dishes and other manipulations with the strain were carried out in the Bio Security Level 2 (BSL 2) laboratory. The evaluation of antifungal capacity was done by the method of mycelial growth (Zhang et al., 2006). The fungus used in the assays was the fungi *Aspergillus fumigatus*. The mold was grown on potato dextrose agar (PDA). The solution was mixed with PDA culture media respectively to give a series of 5, 10, 20, and 25 mg/mL concentrations of culture media containing the compounds described above. The media were dispensed into sterilized Petri dishes (9 cm). After solidification, a mycelial disk of 4 mm diameter of the test *Aspergillus fumigatus* taken from 4 days -old fungi culture, was placed at the center of the medium.

The mycelial disks on PDA without any test constituents were performed in the same way and used as control. Radial growth of colonies was measured at two points along the diameter of the plate and the mean of these two readings was taken as the diameter of the fungal colony. After incubation at 25°C in darkness, growth zones were measured at the third, fifth and the seventh day. The growth of the colonies in control sets was compared with that of various treatments and the difference was converted into percent inhibition [(*C* - *T*) x 100/*C*] where *C* and *T* are the radial diameters of the colony in control and treatment, respectively. The percentage of *A. fumigatus* growth inhibition is expressed as a mean of three replicate tests for each treatment. The complete antifungal analysis was carried out under strict aseptic conditions (Zhang et al., 2006).

The analyses were performed using SPSS® (Statistical Package for the Social Sciences) version 19.0. The one-way analysis of variance (ANOVA) followed by Tukey's Test with P = 0.05 were used to detect significant differences in inhibition fungi.
