Antifungal Activities of Five Commercial Extracts Against Alternaria alternate

The inhibitory effects of five commercial extracts (Allium sativum, Chamaemelum nobile, Thymus vulgaris, Zingiber officinale and Ricinus communis ) against Alternaria alternata were tested three concentrations (2.5, 5, 12.5 µl/ml) in vitro. T. vulgaris and R.communis extracts both exhibited the most effective antifungal activity against A. alternate with diameter of inhibition zones of 54 mm. The C. nobile extract exhibited a lower degree of inhibition 24.5 to 45 mm at three concentrations. The antifungal indices of T. vulgaris and R.communis extracts at three concentrations against A. alternata were all 98.14%, while this of C. nobile extract was 43.52% at 12.5 µl/ml. The results show that the five commercial extracts have potential for the development of natural antifungal agents, which could be an alternative to chemicals for control of phytopathogenic fungi on fruits or vegetables.


Introduction
Plant pathogenic fungi negatively affect a large number of important fruits and vegetables, and limit crop production worldwide, especially in developing countries [1,2]. The use of chemical control to these pathogens increase the productivity of the crop but it is inappropriate and nondiscriminatory use for human and animal health, as well as contaminating the environment [3,4]. The search of new fungicides effective, biodegradable and with greater selectivity is necessary to face chemicals' related problems [5]. Within this context is the utilization of plant extracts which are natural sources of antimicrobial substances, regarded as safe and degraded by natural soil microbes; they do not pose any health residual or environmental problems at any concentration which they are used [6,7]. Antifungal potential of plant extracts has been demonstrated in several works [1,8,9]. For example, there are studies evaluating the inhibitory activity of extract on plant pathogenic fungi; Arora and Kaushik [10] investigated ginger with 40 different plant extracts for their activity against soybean fungal pathogens as F. oxysporum and they published that ginger inhibit their mycelial growth. Saha, et al. [11], tested ethanol and aqueous extracts of 30 plants toward the development of ecofriendly antifungal compounds for controlling pathogens responsible fungal diseases of tea ( Pestalotiopsis theae. (Saw.) Stey., Colletotrichum camelliae. Mess., Curvularia eragrostidis. (P. Hennings) Meyer, and Botryodiplodia theobromae). Results of this study showed that ethanol and aqueous extracts of Allium sativum. L., Datura metel. L., Dryopteris filix-mas. (L.) Schott, Zingiber officinale. Rosc., Smilax zeylanica. L., Azadirachta indica., A. Joss. and Curcuma longa. L. recorded 100% inhibition of spore germination. Al-Rahmah, et al.
[3] evaluated fungal activity of five methanolic plant extracts from Lantana camara, Salvadora persica, T. vulgaris, Z. officinale and Ziziphus spina-christi on tomato phytopathogenic fungi, F. oxysporum, Pythium aphanidermatum and Rhizoctonia solani. They found that methanolic extracts from T.vulgaris and Z. officinales were strongly active on these phytopathogenic fungi. Alemu, et al. [12] tested antifungal effects of 20 plants against Colletotrichum gloeosporioides. They showed that Datura stramonium L. methanol extract exhibited a very good antifungal effect on the tested fungus, Datura stramonium L. and E. globulus Labill. of leaf extracts inhibited the pathogenic spore germination more than the other extracts (14.7% and 15.7%, respectively).
Alternaria alternata is one of the most common saprophytes found throughout the world [13]. A. alternata cause a range of diseases with economic impact on a large variety of important agronomic host plants and fruits including potatoes, pomegranate, almond, kiwi, aloe vera, tomato, ginseng , citrus, banana, pepper , water hyacinth, Lantana camara, and Amaranthus spp. Dube [14]. This species has been clinically associated with asthma, allergic rhinosinusitis, hypersensitivity, oculomycosis, onychomycosis, skin infections, and allergic bronchopulmonary mycosis [15]. Alternaria alternata is also one of the most important species of Alternia that produces AAL toxins, causing many problems for humans and animals and endangering their health [16]. In addition, Resistance to fungicide has been shown in this species [17][18][19][20].The aim of this work was to evaluate in vitro the potential antifungal activity of five commercial extracts (Allium sativum, Chamaemelum nobile, Thymus vulgaris, Zingiber officinale and Ricinus communis ) against Alternaria alternata, in order to verify possible inhibition activity.

Commercial Extracts
The following five tested extracts were produced by the Egyptian company El Capitaine (CAPPHARM) for the extraction of oils from natural and cosmetic plants: Allium sativum, Chamaemelum nobile, Thymus vulgaris, Zingiber officinale and Ricinus communis.

Fungal Isolates
The fungi Alternaria alternata was isolated from lentil seed (Syria R3) and was cultured on to Sabouraud Dextrose Agar at 28° C for 7 days, in experimental farm of nature and life sciences faculty of Mascara University.

Anti-Fungal Assay
The method of Luan, et al. [21] and Hasnaoui, et al. [22] with modifications was employed for antifungal evaluation of the selected extracts, which were tested at 2,5 ; 5 et 12,5 μl/ml concentrations against A. alternata added to 20 ml of sterilized potato dextrose agar in 9 cm Petri dishes. After the mixture was cooled in the plate (6.0 cm diameter), 5.0 mm diameter of fungi mycelium was transferred to the test plate and incubated at 28 ± 4°C for 3-7 days. When fungi mycelium reached the edges of control plate (without the presence of extracts), the antifungal index was calculated as follows: Antifungal index (%) = (1−D a /D b ) × 100 where, D a =the diameter of growth zone in the experimental plate (mm), D b = the diameter of growth zone in the control plate (mm).

Antifungal Activity
The antifungal activity of the selected extracts by direct contact against A. alternata was qualitatively assessed by the presence or absence of the inhibition zone. The antifungal activity is summarized in Table 1. The results revealed that the selected extracts showed a higher antifungal activity with diameter of inhibition zones ranged between 24.5 to 54 mm. Among extracts, T. vulgaris and R.communis exhibited the most effective antifungal activity with diameter of inhibition zones of 54 mm in the three concentrations of extracts, followed by A. sativum (47 to 52 mm), Z. officinale (30 to 48 mm) and C. nobile (24.5 to 45 mm) which is the weakly active on A. alternata in comparison to other extracts.
The antifungal indexes of the five selected extracts against A. alternata were shown in Table 2. Results showed that T. vulgaris and R.communis extract induced 98.14% antifungal index of A. alternata with three concentrations (2.5, 5 and 12.5 µl/ml). Similarly, A. sativum extract exhibited strong antifungal action with antifungal indices of 94.44  at concentration of 2.5µl/ml and decrease to 90.74 and 85.18 at concentration of 5 and 12.5µl/ml, respectively.
C. nobile extract which exhibited the lowest antifungal activity induced 81.48 % antifungal index at concentration of 2.5µl/ml and decrease to 75.93 and 43.52 at concentration of 5 and 12.5 µl/ml respectively. were evaluated for their antifungal activities on A. alternata. T. vulgaris and R. communis extract were most active against this phytopathogenic fungi. Thyme oil is one of the 10 most commercial oils worldwide, since it is used as a natural food preservative, has considerable antioxidant, antibacterial, and antifungal effects, and is used as an aromatic additive to a variety of foods and drinks, as well as in personal care products (perfumes, cosmetics, soaps, oral solutions) [23]. Several works reported the antifungal effects of Thymus vulgaris extracts against A.alternata. Indeed, Segvić Klarić, et al. [24] compare the antifungal activities of essential oil of thyme (Thymus vulgaris L.) and pure thymol on different mould species (Aspergillus, Penicillium, Alternaria( include A.alternata) , Ulocladium, Absidia and Mucor, Cladosporium, Trichoderma and Rhizopus, and Chaetomium) isolated from damp dwellings and found that Thymol exhibited approximately three-times stronger inhibition than essential oil of thyme. The MIC and MFC of thyme oil on A.alternata were 4.70 µg /ml and 9.40 µg /ml, respectively. In other study, Hadizadeh, et al. [25] investigated the antifungal effect of essential oils obtained from some medicinal plants of Iran (Urtica dioica L., Thymus vulgaris L., Eucalyptus spp., Ruta graveolens L. and Achillea millefolium L.) against A. alternata. Both U. dioica and T. vulgaris oils exhibited antifungal activity against A. alternata. The thyme oil exhibited a lower degree of inhibition 68.5 and 74.8% at 1500 and 2000 ppm, respectively. In similarly to Segvić Klarić, et al. [24], Perina, et al. [26] reported that thyme essential oil from leaves and its major compound thymol had minimum inhibitory concentrations (MIC s ) of 500 and 250 µg mL -1 respectively against A. alternata.
In other study carried out by Puškárová, et al. [27] on antimicrobial activity of Six essential oils (from oregano, thyme, clove, lavender, clary sage, and arborvitae) against pathogenic (E. coli, S. typhimurium, Y. enterocolitica, S. aureus, L. monocytogenes, and E. faecalis) and environmental bacteria (B. cereus, A. protophormiae, P. fragi) and fungi (C. globosum, P. chrysogenum, C. cladosporoides, A. alternata, and A. fumigatus). The MIC and MFC of thyme oil on A.alternata were 0.025 and 0.05 (% w/v), respectively. Recently, In vitro antifungal activities of vapours of four plant essential oils, cinnamon oil, fennel oil, origanum oil and thyme oil, were investigated by Hong, et al. [28] during in vitro conidial germination and mycelial growth of A. alternata causing the tomato leaf spots to find eco-friendly alternatives for chemical fungicides. The four plant essential oils showed different antifungal activities against in vitro conidial germination of A. alternata in dose-dependent manners. One μl/disc of thyme oil slightly decreased the mycelial growth by ca. 84.3% and increasing to 2 μl/disc led to more decrease in the mycelial growth showing ca. 39.1% compared to that of untreated control. The antimicrobial activities of Thymus vulgaris extract is mostly believed to be related to the thymol and carvacrol contents of the oil [29,30]. Several studies were reported antimicrobial effects of thymol alone or in combination with carvacrol [31][32][33][34]. They cause structural and functional disturbances in the cellular membrane [35]. Thymol is lipophilic compound, that alone or with carvacrol, can change the cell membrane fluidity and permeability [36]. In addition to this, the compound can changes the cell membrane in fungi by the affect the function of the cell membrane enzymes that catalyzes the synthesis of the cell wall polysaccharide compounds such as  -glucan and inhibit the growth of cells [37,38]. R.communis extracts exhibited also similar and stronger antifungal effect on A. alternata as T. vulgaris extracts. Comparing our results with other researchers, they are in good agreement with those of Jassim [39] who tested antifungal activity of Conocarpus lancifolius, Ricinus communis, Nerium oleandra and Clerodendron inerme extracts on the fungus A. alternata the causal agent of leaves spots of Date Palm. Regarding the treatment of R. communis extract 10 and 15% concentrations the inhibition percentage of mycelial growth were 72 and 74.44% respectively. The antifungal activity of R. communis extracts is mainly attributed to the presence of camphor [40], which reported to have antimicrobial properties [41,42].
A. sativum extracts exhibited also remarkable antifungal effects on A. alternata near to those of two precedent extracts (T. vulgaris and R. communis). The results obtained in our screen are in agreement with published results for Taskeen, et al. [43], Şesan, et al. [44], Alseeni, et al. [45] which reported that the extract of A. sativum possessed antifungal activity on A. alternata. The antimicrobial activity of garlic is believed to be due to the effect of allicin (diallyl thiosulfinate), ajoene, and other sulfite compounds [46].
For Z. officinale extracts, it exhibited less antifungal activity than the precedent tested extracts. The results of our study are in line with the report by many researchers [47]; Sharma and Tiwari [48]; Osman, et al. [49]; Ahmad and Qureshi [50]; Alseeni, et al. [45], which showed the extracts from ginger possess 1antibacterial activity against A. alternata. The gingerol and shogaol are the major components of ginger, which 1could be responsible agents for the antimicrobial properties of ginger. They are phenolic compounds causing 1rupture of the bacterial cell membrane and loss of their properties [51] and greater loss of cell contents 1or critical output of molecules and ions can lead to cell death [52,53].
Comparatively, A. alternata were less sensitive to the inhibitory activity of the C. nobile extracts than other four tested extracts. To our best knowledge, therefore, this is the first study to determine the antifungal activity of C. nobile extract on A. alternata. In contrary to our find, [54] tested sixty nine for its antimicrobial activity against three bacteria (S. aureus, S. enteritidis, P. aeruginosa) and three fungi (A. alternata, A. niger, P.digitatum), and among these tested oils, Anthemis nobilis (currently C. nobile) don't has activity on A. alternata . Some studies have been performed concerning the antifungal activity of essential oils or extracts of other Chamaemelum species or on Alternaria sp. Rizwana, et al. [55] have screened in-vitro the antimicrobial activity of five organic solvents extracts(Chloroform, ethyl acetate, acetone, ethanol and methanol) of Matricaria aurea (golden chamomile) on S. aureus, B. subtilis, S.pyogenes , E. faecalis, E. coli, P.aeruginosa, K. pneumonia, F. oxysporum, F. solani, A. alternata, A. niger, A. flavus and C. gleosporoides and found that all extracts inhibit the mycelial growth of A. alternata in a percentage ranged between 50.74-100. In other study, the volatile oil of Anthemis nobilis (currently C. nobile) inhibited the growth of dermatophytons, Alternaria sp., Aspergillus fumigatus and A. parasiticus [56]. The antimicrobial properties of chamomile have been well documented in some studies [57][58][59]. Compounds in the essential oil of chamomile were effective against Staphylococcus and Candida [60]. Of chamomile's essential oil components, -bisabolol had the strongest activity against Gram-positive and Gram-negative bacteria. Chamazulene also had strong antimicrobial activity. Spiroethers had weak activity against Gram-positive bacteria but were inactive against Gram-negative bacteria [61]. German chamomile esters and lactones showed activity against Mycobacterium tuberculosis and M. avium [62]. Chamazulene, α-bisabolol, flavonoids and umbelliferone displayed antifungal properties against Trichophyton mentagrophytes, T. rubrum and Candida albicans [61,[63][64][65].

Conclusion
In this study, we investigated the antifungal activities of five plant extracts (A. sativum, C. nobile, T. vulgaris, Z. officinale and R. communis) against A. alternata. Our study demonstrated that the five plant extracts showed good antifungal activities against this fungi. In particular, T. vulgaris and R.communi offer effective inhibition activity to the growth of the A.alternata. Even at low concentrations, these extracts showed strong antifungal activity. Thus, the results show that the five plant extracts have potential for the development of natural preservatives as alternative to antibiotics and artificial preservatives both of which can be toxic at certain concentrations.