Advances in Biological Research 7 (5): 163-168, 2013 ISSN 1992-0067 © IDOSI Publications, 2013 DOI: 10.5829/idosi.abr.2013.7.5.7497

Comparative Analysis of Antifungal Properties of Zataria multiflora Boiss, Eucalyptus spp Essence and Malachite Green on Eggs of Kutum (Rutilus frisii Kutum)

Maryam Najafi and Abbas Ali Zamini

Islamic Azad university- Lahijan Branch, Faculty of Natural Resource, Department of Fishery and Aquaculture, Lahijan, Iran. P.O.Box: 1616

Abstract: The objective of this study was to assay the effectiveness of Malachite green, Zataria multiflora and Eucalyptus Spp essence on the fungal CFU (in eggs and water) and hatching success of Rutilus frisii kutum eggs. Eggs were artificially fertilized and transferred to a static bath treatment in different concentrations of Eucalyptus (50 and 100 mg/l),Z. multiflora (100 and 150 mg/l) essence and Malachite green, for 1 h daily, during 3 days. Treatment efficacy was investigated by comparing the percentage of hatched eggs in the treatment group to the untreated control group1–3 days after fertilization. All treatments demonstrated decreasing fungal CFU and lowest CFU was recorded in eggs treated with Eucalyptus essence (100 mg/l) (p<0.05). But there were no remarkable effect of different antifungal on water. Eggs treated with Eucalyptus essence at 100 mg/l recorded greater mean percentage of hatching compared to the control (p<0.05). In conclusion Eucalyptus Spp was more effective than other antifungal for the treatment of fungus -infected kutum eggs.

Key words: Egg Eucalyptus Spp Zataria Multiflora Malachite Green Rutilus Frisii Kutum

INTRODUCTION chemical drugs have been replaced by herbal medicine in aquaculture [6, 16]. Also, numerous studies have Recently, more efficient drugs, derived from herbal demonstrated stimulating role of plant extracts in fish resources, against microbial infections have been immune system challenged with bacterial and fungal recommended by investigators [1]. Use of chemical agent's [16- 20]. substances such as malachite green, formalin and sodium Since, plant extracts are considered as a chloride to control diseases and aquatic pests has been prominentantimicrobial agent, eucalyptus and Zataria banned worldwide because of high residue levels in the multiflora with medicalproperties have been used in the animals [2, 3]. Unlike chemical materials, plant treatment of infectious diseases [21-23]. Zataria immunostimulants have no residuals in the environment multiflora with the common Persian name “Avishan-e and more attention has been payedto these plants for Shirazi”belongs to the family of Labiatae which is a their immune stimulating functions in fish culture [4-10]. native plant of Iran, Pakistan and Afghanistan. Water mold infections cause casualties of fish and their Furthermore, it is one of the plants with characterized eggs in both nature and commercial fish farms [11]. antimicrobial properties [22]. Moreover, there are reports Hence, use of plant extracts can play a role in supporting indicating that Z. multiflora hasimmunostimulatory effects both performance and health status of the animals in common carps [24]. In this regard, Z. multiflora could [12--14]. Unfertilized eggs are susceptible to fungal be safely used to protect the fish as an antimicrobial agent infection during incubation; myceliumoffungisoon [25]. Eucalyptus (Family: Myrtaceae) all around the world spreads to nearby, healthy eggs causing mortality [15]. have more than 700 species, among them at least 500 There are many reports from all over the world that species have essence [28]. Essence of some species of

Corresponding Author: Maryam Najafi,Islamic Azad university- Lahijan Branch, Faculty of Natural Resource, Department of Fishery and Aquaculture, Lahijan, Iran. P.O.Box: 1616,

163 Advan. Biol. Res., 7 (5): 163-168, 2013 eucalyptus is used in treatment of some diseases [29]. were washed 3-5 times with distilled water and then the Several studies have been documented essence of some obtained suspension composed of water and fungus eucalyptus species as agent which has antimicrobialand was diluted ten-fold with distilled water in sterile glass remedial activities [1, 30-34]. Kutum, Rutilus frisii kutum pipes. After that, 0.5 mL of diluted suspension was belonged to Cyprinidae family with an ecological and incubated in culture mediums i.e. SabroDextrose Agar economic importance [35]. Kutum is in great demand by (SDA) and corn meal containing Chloramphenicol and virtue of its high taste quality and the cuisine customs of Gentamisin. To calculate the fungal colony numbers, the the local residents. Available evidence showed that inoculated plates were incubated for a period of 48-72 h natural reproduction of this species in recent years and 3-5 days at 25°C, respectively. After this period, the declined markedly due to overfishing, increasing colonies were counted and the CFU (Colony-Forming pollution, river body degradation, migration and lack of Unit) values were calculated on the basis of the mL of security area [35-38]. So, the Iranian fisheries organization water and egg. For CFU calculation, the arithmetical release up to 200 million fry (average weight, 1g) into the average of two fungal counting was multiplied at the Caspian Sea annually to restock this valuable species. dilution rate. All data were subjected to a one-way In spite of positive effects of mentioned plants in analysis of variance (ANOVA) after confirmation of controlling some microbial diseases [28, 39-44], there is a normality and homogeneity of variance. Significance of lack of data about their antibacterial effects. Hence, the the differences between means was tested using aim of present study was to evaluate the effect of two Duncan’s multiple range test (P<0.05). All assays were important and traditional medicinal plants (eucalyptus and performed in triplicates and data were shown as mean ± Z. multiflora) on fungal agents in Kutum (Rutilus frisii SE for each treatment. kutum) egg. RESULTS AND DISCUSSION MATHERIALS AND METHODS Fungal CFU in eggs and water for all treatments were This research was carried out in the reproductive shown in Figs. 1 and 2, respectively. Significant season of kutum in Shahid AnsariFish Propagationand difference was observed between control eggs and RearingCenter, Guilan, Iran from March until May 2011. treatment groups in terms of CFU (p<0.05). Significant In this study Eucdlyptus Spp and Zataria multiflora decrease of fungal CFU for Eucdlyptus Spp was measured extracts were purchased from Zardband Pharmaceutical in eggs treated with 100 mg/l (Fig. 1) and the Company, Tehran, Iran. For this purpose 60 mature highestfungal CFU was significantly observed in control females (average body weight: 960±142.5 g and length: group (p<0.05). 44.4±3.9 cm) were caught in the Sefidroud River estuary The obtained results showed that the fungal by seine nets. The eggs were collected and poured into growth in water treated with different concentrations of plastic jars with pressure on the abdominal. Then stripped Z. multiflora, Eucalyptus Spp essence and Malachite eggs were mixed with sperm that obtained from three greenhad not significant difference compared with males. Following fertilization, the eggs were stirred for 1 h control group (p>0.05) (Fig. 2). and then eggs rinsed with hatchery water and placed into the incubator.Hatching occurred between 1 – 3 days after fertilization. The mean water temperature was maintained within 10±2°C, dissolved oxygen concentration and pH were calculated approximately 7 ppm and 7.8- 8.2 during the experiment, respectively. The egg batchs were randomly assigned in triplicate to static bath treatments of given concentrations of either Eucalyptus Spp essence (50 and 100 mg/l), Zataria multiflora essence (100 and 150 mg/l), Malachite green and a control (0 ppm) for 1 h daily during 3 days exposure before being transferred to Fig. 1: Fungal CFU (ml 1) of Kutum eggs after exposure randomized compartments of the incubation tank. to different treatments. Data are expressed as The fungal infection of egg was estimated as follows: mean ± SE. Mean values bearing same A total of 150 infected eggs were sampled and placed in superscriptare not statistically significant sterile glass bottle with closed plug. Afterwards, the eggs (p>0.05).

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flavus. Similar results were also obtained by Mousavi et al., [15], they demonstrated the inhibitory effects of Eucalyptus essence in control of fungal growth and increasing the hatching rate of rainbow trout (Oncorhynchus mykiss) eggs. Data analysis revealed no significant difference between control group and treatment (p>0.05) in terms of infected eggs by fungi (abundance) (Fig. 3). In this experiment, there was a significant effect on hatching rate based on Eucalyptus treatment (p<0.05) (Fig. 4). Several studies have investigated using natural Fig. 2: Fungal CFU (ml 1) of incubation water of Kutum products such as Z. multiflora and Eucalyptus Spp as after exposure to different treatments. Data are fungicidal agents on fish eggs [46- 49] and they have expressed as mean ± SE. Mean values bearing proved antifungal activity against different fungal same superscriptare not statistically significant agents such as Aspergillus and Fusarium species [50, 51]. (p<0.05) In interaction effect, the highest hatching rate was significantly observed in group treated with 100 mg/l of Eucalyptus. Also, Ahmadi et al. [52] reported that Eucalyptus essence in dose of 200 ppm is effective in control of Saprolegniasis and resulted in higher hatching rate in Persian sturgeon, Acipenser Persicus. The control of eggs infected with Saprolegnia by dosage of 100 ppm Eucalyptus essence has also been recorded for Kutum by Najafi et al. [53]. Similar results were reported in other literature, Macchioni et al. [54] and Rai et al. [47] found that extract of some plants such as Artemisia verlotorum, Antolina etrusca, Guizotia abyssynica and Tagetes Fig. 3: Infected eggs by fungi (abundance) in Kutum erecta could be used as antifungal agents. Use of after exposure to different treatments. Data are chemical materials such as malachite green has routinely expressed as mean ± SE. Mean values bearing been used in Iran for controlling of eggs infection against same superscriptare not statistically significant fungi for several years [52]. According to literature, (p>0.05). malachite green has proved to be an excellent antifungal agent in fish eggs at given concentrations [56]. On the other hand, from the results of some studies, different dosages of malachite green decreased hatching rates of Chinese sucker (Myxocyprinus asiaticus) eggs [57] and in another study by Marking et al. [58], presented delayed hatching of rainbow trout eggs. In surveying of disinfection material, the lowest percentage of hatch was recorded in malachite green treatment (Fig. 3). In spite of antifungal effects of malachite green, it causes cancer and environment pollution [52]. Meanwhile, higher Fig. 4: Hatching rate in different treatments (%) in Kutum concentrations of malachite green are toxic to the eggs after exposure to different treatments. Data are [59] and damage egg membrane and chorion [60]. In this expressed as mean ± SE. Mean values bearing regard, Ahmadi et al. [52] reported that the highest same superscriptare not statistically significant unhatched eggs treated with malachite Green, as we (p>0.05). showed in the present experiment. As a conclusion, essence of plants has greater In line with these results a study done by others antimicrobial activity than chemical components and [45], reported that eucalyptus essence the most several experiments have shown that chemical drugs in effective against some fungi such as Aspegilus aquaculture system have been replaced by herbal

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